Multiplexed PLGA scaffolds with nitric oxide-releasing zinc oxide and melatonin-modulated extracellular vesicles for severe chronic kidney disease

Multiplexed PLGA scaffolds with nitric oxide-releasing zinc oxide and melatonin-modulated extracellular vesicles for severe chronic kidney disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article Multiplexed PLGA scaffolds with nitric oxide-releasing zinc oxide and melatonin-modulated extracellular vesicles for severe chronic kidney disease Dong Keun Han*, Won-Kyu Rhim, Jiwon Woo, Jun Yong Kim, Eun Hye Lee, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2815340/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract With prevalence of chronic kidney disease (CKD) in worldwide, the strategies to recover renal function via tissue regeneration could provide alternatives to kidney replacement therapies. However, due to relatively low reproducibility of renal basal cells and limited bioactivities of implanted biomaterials along with the high probability of substance-inducible inflammation and immunogenicity, kidney tissue regeneration could be challenging. To exclude various side effects from cell transplantations, in this study, we have designed cell-free hybrid PMEZ scaffolds incorporating essential bioactive components, such as ricinoleic acid grafted Mg(OH) 2 (M), extracellular matrix (E), and alpha lipoic acid-conjugated ZnO (Z) based on biodegradable porous PLGA (P) platform. Consecutively, for functional improvements, melatonin-modulated extracellular vesicles (mEVs), derived from the human umbilical cord MSCs in chemically defined media without serum impurities, were also attached onto PMEZ scaffolds to construct the multiplexed PMEZ/mEV scaffold. The continuous nitric oxide-releasing property of modified ZnO and remarkably upregulated regenerative functionalities of mEVs showed significantly enhanced kidney regenerative activities. Based on these, the structural and functional restoration has been practically achieved in 5/6 nephrectomy mouse models that mimicked severe human CKD. Our innovative implantations aim at kidney tissue recovery with functional restoration and could be a promising therapeutic alternative for CKD treatment. Biological sciences/Biotechnology/Regenerative medicine Biological sciences/Biotechnology/Biomaterials/Biomedical materials PME scaffold ZnO melatonin-modulated extracellular vesicles (mEVs) Chemically defined media kidney regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Chronic kidney disease (CKD) is a global public health problem with a gradual and irreversible structural and functional abnormalities of the kidney, which results in reduced glomerular filtration rate and increased urinary albumin secretion. 1,2 In end-stage kidney disease (ESKD), the therapeutic strategies are limited to renal replacement therapies, such as dialysis and transplantation. Two types of dialysis- hemodialysis and peritoneal dialysis, remove the wastes and excess fluids from the blood to maintain homeostasis in the body. 3 However, dialysis treatment cannot replace the role of the kidney and various risks remain due to extracorporeal circulations. 4 Although the kidney transplant is a fundamental approach to restoring kidney functions but has severe limitations like extremely limited donors, and long-lasting use of immunosuppressive drugs after transplantation. 5–7 The limited current therapies for ESKD have led researchers to focus on tissue engineering and regenerative medicine approaches for restoring and replacing the partial or total function of the kidney. To provide mechanical stability and contain biochemical signals through combination with various bioactive components, the scaffold-based strategies have been explored as structural and functional platforms to give a proper microenvironment for the regeneration and engineering of the injured tissues. Among diverse scaffold types, composed of metals, ceramics, polymers, and composites, biodegradable polymer-based scaffolds have started looking attractive for biomedical applications. With qualities like degradation property, ease of processing, close biological properties with many tissues and extracellular matrix (ECM), and the absence of the need for additional surgery to remove the scaffolds, the scaffold-based platform looks very promising. 8 Biodegradable polymer-based scaffolds have been utilized for various clinical applications such as to restore the function of injured tissues, like bone regeneration, cartilage repair, skin wound healing, and nerve conduits. 9,10 Design of appropriate biodegradable scaffolds for specific tissue regeneration with optimum mechanical, chemical, and biological properties is crucial for accurate and active tissue engineering processes. Biodegradable polymer-engineered porous scaffolds support mechanical stability and spatial flexibility which plays a pivotal role in regeneration and tissue engineering through interaction with surrounding environments. However, there are some limitations to be addressed, mainly shortcomings derived from the hydrolysis of a biodegradable polyester polymer such as poly(lactic- co -glycolic) acid (PLGA). The acidic byproducts induce a pH decrease in the tissue environment, resulting in cellular necrosis, inflammation, and fibrosis of closed tissues. 11 The lactic acid and glycolic acid derived from PLGA degradation induce the classical complement pathway which is amplified by an alternative pathway and stimulates pro-inflammatory cytokine via activating monocytes and macrophages. 12 Many studies have been reported to solve these problems occurring from the byproducts of biodegradable polymers. The incorporation of alkaline hyaluronic acid or drugs partially neutralizes the acidity, but they cannot completely avoid the inflammation in the peripheral tissues. 13,14 Therefore, strategies to improve the bioactivity of the materials inducing tissue regeneration are essential. The kidney is a very complex organ composed of various cells and ECM components with lots of growth factors and proteins. As a result, many types of ECMs have been studied to functionalize polymer-based scaffold bioactivity and their interaction with cells surrounding the implanted scaffolds. 15,16 To neutralize acidification by the byproducts during PLGA degradation and to enable enhancement of bioactivity of the surrounding cells, we previously reported the use of PME scaffold composites with ricinoleic acid grafted-magnesium hydroxide (Mg(OH) 2 -RA; MH-RA; M) and porcine kidney-derived extracellular matrix (ECM; E) incorporated in porous PLGA (P) scaffold. 17 Magnesium hydroxide was utilized as an antacid component via partially dissolving magnesium and hydroxide ions, where hydroxide ion combines with acidic byproducts of PLGA inhibiting inflammation and fibrotic pathway of kidney tissues. In addition, one of the biocompatible fatty acids, ricinoleic acid was grafted to the surface of MH to enhance the dispersity in organic solvents and have more anti-inflammatory effects. 18 By mimicking the microenvironment of kidney tissues using ECM, the properties of kidney tissue regeneration could be improved. Despite successful glomerulus regeneration for restoration of kidney function, the demand for bioactive components to promote the biophysical properties of scaffolds has increased. For kidney tissue regeneration and function restoration, integrated bioactive scaffolds, containing polydeoxyribonucleotide (PDRN) and TNF-α/IFN-γ-primed MSC-derived extracellular vesicles (TI-EVs) into PME scaffolds, have been developed. 19 By mimicking the microenvironment of kidney tissues using PME scaffolds, and incorporating bioactive components- PDRN and TI-EVs, the properties of kidney tissue regeneration could be facilitated. Despite successful glomerulus regeneration in mouse models with 3/4 nephrectomy, the improved scaffolds with multifunctional bioactivities are required for the complete recovery in severely injured (5/6 nephrectomy) CKD mouse models to simulate application to human CKD patients. For the success of tissue engineering with biomaterials, angiogenesis is one of the most prominent processes. Many types of pro-angiogenic biochemical molecules have been utilized to give an angiogenic property, but their biological activities are limited to in vivo systems due to short half-lives and low bioactivities. 20 Nitric oxide (NO) can be clinically applied to regulate angiogenesis at optimum concentrations. 21 NO is a soluble gas involved in many regulatory functions in various tissues, which is synthesized in vascular endothelium with various types of nitric oxide synthases. 22 NO facilitates the proliferation of cells, immune regulations, and anti-apoptotic effects with the appropriate concentrations. Although a lot of functionalities of NO have been actively utilized in vitro , there have been limitations with the bioactivities in vivo , due to constraints of exposure area and short half-lives with rapid thermal and photochemical decompositions. 23 To overcome these limitations, various types of metals, metal oxides, and nonmetallic particles have been introduced as a prodrug that releases NO by reacting with endogenous NO donors. 24 The zinc oxide (ZnO) particle, as one of the promising NO-releasing components that exhibit a proper combination with innate NO donors, achieved a long-lasting release of NO with excellent stability against biological degradation. 25 However, the hydrophilic accelerating fast release of ZnO from the scaffolds needs to be surmounted. Several studies have indicated the regenerative and therapeutic effects of mesenchymal stem cells (MSC) for injured tissues. 26–28 The transplantation of MSCs has potent regenerative activity using lineage reprogramming of stem cells, cell fusion, and mitochondrial transfer, however, it also has the potential to cause tumorigenesis, immunogenicity, and limited cell survival simultaneously. 29 This led researchers to focus on the paracrine factors that have been considered as the key mechanism of MSC-based therapeutics and stimulate peripheral MSCs to differentiate for tissue regeneration. Especially, extracellular vesicles (EVs) i.e., small vesicular particles secreted from cells, play a pivotal role to direct the paracrine effects of MSCs by mediating intercellular communications. 30 MSC-derived EVs show biological functional similarities, including tissue regeneration-related activities, to the parent cells by containing and delivering active biocomponents to the targeted cells or tissues. 31 Isolation of EVs from the cells of interest with high purity and bioactivity has become significant as it improved their therapeutic potential in clinical use. 32,33 In this regard, serum-depleted conditions (starvation) have been known to exclude unknown side effects derived from the serum during the EVs isolation process. However, starvation resulted in a diminished proliferation rate and changes in cell characteristics which were overcome by using chemically defined media (CDM), as published previously. The CDM maintained healthy MSCs during EV isolation without serum components, supporting the isolation of highly purified EVs with enhanced bioactivities. Moreover, the bioactivities of EVs can be modulated using growth factors, transcription factors, and biochemical and/or biophysical stimulations during cell culturing. 34 Especially, melatonin has been utilized to promote the therapeutic activities of EVs for kidney tissue regeneration in starvation conditions by modulating inflammation and fibrosis in a CKD model. 35,36 Extensive comparative analysis has been studied for the optimization of cell modulation in CDM to modify starved conditions during cell stimulations and EV isolation, and to maximize the regeneration ability of melatonin-modulated MSCs with high purity. 37 In this study, we designed porous hybrid PMEZ scaffolds based on PLGA (P) with ricinoleic acid grafted magnesium hydroxide (MH-RA; M) to enhance the pH neutralization ability of MH, and extracellular matrix (ECM; E) to mimic kidney tissue environment. Alpha lipoic acid-conjugated zinc oxide nanoparticles (ZnO-ALA; Z) were used to promote angiogenic properties with continuous dual NO release in kidney tissues. Additionally, the upregulation of internal miRNA was evaluated using bioinformatics analysis for melatonin-modulated hUCMSC-derived extracellular vesicles (mEV), preconditioned in chemically defined media (CDM). Various bioactivities related to kidney regeneration have been proved using mEV-incorporated multiplexed PMEZ/mEV scaffolds (Fig. 1). Finally, structural, and functional recovery have been confirmed using PMEZ/mEV scaffolds in the 5/6 nephrectomy mouse model to simulate progressive renal failure in humans without cell transplantation. Figure 1 2. Results And Discussion 2.1. Characterization of melatonin-modulated EVs derived from hUCMSC cultured in chemically defined media (CDM) To maximize the production and purity of extracellular vesicles (EVs), serum-free chemically defined media, CellCor™ CD MSC (CDM; Xcell Therapeutics, Seoul, Korea) was utilized to culture human umbilical cord mesenchymal stem cells (hUCMSCs). In our previous study, we proved that CDM promoted EV release from hUCMSC with high purity and bioactivity and cells maintained an excellent proliferation rate without animal-derived proteins and other impurities. 34 Furthermore, cells primed with pro-inflammatory factors have been shown to improve the functions of EVs and keep cells healthy in CDM. Contrary to cell priming approaches with pro-inflammatory cytokines, which torment cells to secrete substances to overcome inflammatory situations, melatonin plays a critical role in immune regulation by reducing apoptotic and necrotic alterations, inflammatory cell infiltration, and tissue fibrosis after tissue injury and thus maintains healthy cells. 38 Numerous reports have suggested that melatonin improved the therapeutic efficacy of stem cells in treating several disorders, including hindlimb ischemia 39 , hepatic ischemia 40 , and kidney ischemia 41 . To maximize the regeneration ability of EVs without serum components for future translational applications, we isolated EVs from melatonin-treated hUCMSC cultured in CDM using tangential flow filtration system. In accordance with MISEV 2018 guidelines for characterization of EVs 42 , the size and number of melatonin-modulated EVs (mEVs) were analyzed with the MONO ZetaView® (Fig. 2a). Moreover, the representative surface markers of EVs (CD63, CD81, and CD9) were obtained by Western blot analysis (Fig. 2b), and double-layered spherical structures were observed using transmission electron microscopy (Fig. 2c) to express the characteristics of EVs. 2.2. Comparative analysis of miRNA profiling for CDM EV and CDM mEV We performed small RNA sequencing to confirm the difference between EVs derived from melatonin-modulated hUCMSC in CDM (CDM mEV) and hUCMSC in CDM without melatonin stimulations (CDM EV). According to the heatmap analysis for the highest expressed miRNAs in EVs, a clear separation of miRNA expressions was shown between two different types of EVs (Fig. 2d). As large parts of miRNAs overlapped with the types of EV-derived miRNA classified in Vesiclepedia and ExoCarta libraries, it could be indirectly confirmed that the corresponding miRNA is derived from EVs (Fig. 2e and S1). To predict the roles of the identified miRNAs in the biological systems, bioinformatic analysis was performed as an alternative approach. A large number of miRNAs were associated with angiogenesis, apoptosis, cell migration, cell proliferation, and inflammatory responses, which are strongly related to regulatory bioactivities for tissue regenerations (Fig. 2f). With a volcano plot analysis, the distribution of miRNAs was distinguished in CDM EV and CDM mEV, and five types of highly expressed miRNAs in CDM mEV were selected for further analysis (Fig. 2g). The red dots (hsa-miR-3195, hsa-miR-1301-3p, hsa-miR-181d-5p, hsa-miR-30c-5p, and hsa-miR-30e-3p) on the volcano plot indicate the miRNAs that are very abundant in CDM mEV compared to CDM EV. The targeting genes of these five miRNAs were identified with miRWalk. Additionally, gene ontology (GO), Kyoto encyclopedia genes, and genomes (KEGG) analysis were performed using DAVID (Fig. 2h). The GO-biological process (GO-BP) showed “Positive regulation of cell proliferation,” “Positive regulation of cell migration,” “Negative regulation of the apoptotic process,” “Wnt signaling pathway,” “Cell migration,” and “Positive regulation of angiogenesis,” meaning the functionalities related to cell proliferation, migration, angiogenesis, and anti-apoptosis of CDM mEV. The analysis of GO-cellular components (GO-CC) revealed that miRNA of CDM mEV appeared to target subcellular regions, such as cytosol, nucleoplasm, and cytoplasm. In GO-molecular functions (GO-MF) terms, “Protein binding” displayed the largest p values. The association of CDM mEV with the activity of intracellular processes was inferred via the QuickGO database ( https://www.ebi.ac.uk/QuickGO/ ). The protein binding implies “cell adhesion molecule binding,” “cytokine binding,” and “cytoskeletal protein binding.” Finally, KEGG analysis revealed various signaling pathways, including “The Ras signaling pathway,” “The Rap1 signaling pathway,” “The ErbB signaling pathway,” “The MAPK signaling pathway,” “The PI3K-Akt signaling pathway,” and “The Wnt signaling pathway.” The Ras signaling pathway is known to be associated with cell proliferation and differentiation. 43 The Rap1 signaling pathway is well-known to be involved in cell adhesion and angiogenesis. 44,45 The ErbB signaling pathway can promote regenerative proliferation and migration, and it may also enhance renal tubular cell regeneration and repair after renal ischemia in vivo . 46 The MAPK signaling pathway is involved in wound healing, whereas the PI3K-Akt signaling pathway is implicated in a variety of biological functions such as cell division, autophagy, survival, differentiation, and bone formation. 47,48 Lastly, the Wnt signaling pathway is known to play a role in both tissue regeneration by promoting cell differentiation and activity in numerous tissues, 49 and M2 polarization of macrophages. 50–52 Taken together, these results suggest that CDM mEV would show more closely related bioactivities during regeneration as compared to CDM EV. Figure 2 2.3. Physicochemical properties of the scaffolds Although various polymer-based synthetic scaffolds, approved by FDA, have been utilized for tissue engineering, the limited biomimetic properties suggested the need for the development of polymer-based scaffolds with improved functionality. In our previous study, the PME scaffold, based on poly(lactide- co -glycolide) (PLGA; P) scaffold with ricinoleic acid grafted-magnesium hydroxide (Mg(OH) 2 -RA; MH-RA; M) and porcine kidney-derived extracellular matrix (kECM; E), was shown to facilitate kidney tissue regeneration compared to the native PLGA scaffold in 3/4 nephrectomy mouse model. More recently, the integrated bioactive scaffolds incorporated with two types of bioactive components, polydeoxyribonucleotide (PDRN) and TNF-α/IFN-γ-primed MSC-derived extracellular vesicles (TI-EVs), facilitated regenerative activities for injured kidney tissues. 19 To verify the structural and functional recovery ability in the 5/6 nephrectomy mouse models that mimic severe CKD in humans, in this study, intense bioactive components were applied to functionalize PME scaffolds. The multifunctional porous PMEZ scaffolds were engineered with modified PME scaffolds using alpha lipoic acid-conjugated zinc oxide (ZnO-ALA; Z) by the ice particle leaching method. Additionally, extracellular vesicles derived from melatonin-preconditioned hUCMSC cultured in CDM (mEV) were immobilized onto the PMEZ scaffold after coating with positively charged polyethylenimine (PEI) to capture EVs and maximize therapeutic activities with sustained release of EVs in biological condition. 53 The formation of highly porous structures to enable cell migration and diffusion of bioactive components between scaffolds and peripheral tissues was monitored using cross-sectional images from scanning electron microscopy. All types of scaffolds showed similar porosity with the addition of MH-RA, ECM, and ZnO-ALA due to the use of an equal ratio of ice particles for controlling scaffold porosity (Fig. 3a). To prove the neutralizing property of MH-RA, pH changes depending on the scaffold degradation were monitored in a phosphate-buffered saline (PBS) solution at 37°C for 56 days. Although the pH of native PLGA started to decrease after 28 days to around pH 6 along with slow degradation of high molecular weight PLGA (MW; 110 kDa, 50:50 of LA:GA), indicating auto-accelerated formation of an acidic environment by hydrolysis of PLGA scaffolds. The scaffolds containing MH-RA, PME, PMEZ, and PMEZ/mEV, maintained the pH around 7 with a slight increase initially due to critical neutralizing effects of MH-RA during the 56 days (Fig. 3b and 3c). MH-RA effectively neutralized PME, PMEZ, and PMEZ/mEV as compared to PLGA scaffold, in spite of relatively fast degradation rate with addition of bioactive components. Zinc oxide (ZnO) particles, known to generate nitric oxide (NO), reacted with innate glutathione peroxidase and glycosidase which allows decomposing donors to release NO in physiological conditions. 24 To address the shortcomings derived from the hydrophilicity of ZnO, lipophilic alpha lipoic acid (ALA) was conjugated onto the surfaces of ZnO to enhance the dispersity of ZnO in organic solvent and thus enable sustained release from the PLGA scaffold. Furthermore, ALA is utilized to treat oxidative stress-associated diseases and the disulfide bonding group of ALA continuously generates NO by reacting with glutathione (GSH) to form RSSG that reacts with s-nitroso-N-acetylpenicillamine (SNAP) in the body. 54 Consecutively, RSSR compounds, byproducts of the reaction are transferred to RSSG by reacting with GSH, where the continuous release of NO is available. Based on this information, long-lasting NO release with sustained release of ZnO-ALA and multiple NO releases with disulfide bonding groups of ALA has been achieved. The NO-releasing property of ZO-ALA (Z) incorporated PMEZ scaffold was monitored using a fluorescence-based DAF-FM assay (Fig. 3d). DAF-FM is a highly sensitive, photo-stable fluorescence probe to quantify NO release. 55 In the presence of only GSH and SNAP as NO donors in the PBS solution, very weak fluorescence signals were obtained due to the natural decomposition of NO donors, while significantly intense signals were generated with PMEZ scaffolds. In the group containing PME scaffolds, moderate fluorescence signals were also detected due to the catalytic effects of metal oxides and hydroxides, such as MgO and Mg(OH) 2 , for NO release. 56 The continuous NO-releasing profile derived from ZO-ALA in the PMEZ scaffold with the addition of each 10 µM GSH (G) and SNAP (S) was monitored using a nitric oxide analyzer (NOA) for over 60 min (Figs. 3e and S2). After 40 min, relatively higher amounts of NO started generating, because of the sustained release of ZO-ALA from the PMEZ scaffolds. By infusing mEV onto PEI-coated PMEZ scaffolds, the distribution of DiO-labeled mEV was observed using confocal microscopy. The fluorescence signal was distributed in PMEZ/mEV scaffolds, whereas no signal was detected in PMEZ scaffolds without mEV (Fig. 3f). The proportions of two types of inorganic components, MH-RA and ZnO-ALA, were evaluated using inductively coupled plasma optical emission spectroscopy (ICP-OES; Table S1 ) and thermos gravimetric analysis (TGA; Fig. 3g). As expected, 15 and 5 wt% (ratios to PLGA) of MH-RA and ZnO-ALA were incorporated into PMEZ scaffolds, respectively, and all PLGA composites with different compositions of additives started to thermally decompose slightly earlier than the native PLGA scaffolds. Additionally, the mechanical properties of scaffolds containing MH-RA, ECM, ZnO-ALA, and mEV were investigated with the compressive stress and modulus analysis using a universal testing machine (Fig. 3h). The compressive modulus calculated by slope between 5 ~ 10% of the strain-stress curve improved with the addition of the inorganic components, MH-RA and ZnO-ALA, and slightly decreased in the addition of hydrated mEV (Fig. 3i). The relatively robust property of scaffolds contributed to a sustained release of biochemical components and could inhibit premature degradation of scaffolds after implantations. The changes in water contact angle (WCA) also proved the incorporations of bioactive components in PMEZ/mEV scaffolds (Table S2). The WCA decreased gradually with the addition of MH-RA, ECM, and ZnO-ALA compared to native PLGA scaffolds, and mEV components dropped the WCA to zero (wetting), which means to interact with water molecules strongly. This makes the scaffolds more biocompatible by inhibiting nonspecific interactions with proteins in the body and facilitating the adhesion and penetration of peripheral cells in a biological system. 57 Figure 3 2.4. In vitro analysis of various scaffold bioactivities To investigate the advanced regenerative properties of multiplexed scaffolds, various regeneration-related biological activities of the scaffolds were compared with the incorporation of bioactive components. In regenerative medicine studies, the promotion of vascularization and angiogenesis is a crucial step. 58 To endow angiogenic functionality, ZnO-ALA and mEV in PME scaffolds were fabricated. The expression levels of angiogenesis-related genes, hypoxia-inducible factor 1-a (HIF-1α), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF), were observed to be higher with the addition of ZnO-ALA, and further upregulated with mEV in human proximal tubular epithelial cell line (HK2) (Fig. 4a). ZnO originally increases HIF-1α and stimulates the secretion of VEGF and HGF via NO release. 59 Similar to VEGF, HGF has angiogenic properties and the potential to stimulate cell invasion and motility for the promotion of cell proliferation. 60 Furthermore, in bioinformatics analysis using the DAVID tool, mEVs are expected to exhibit superior angiogenic properties as shown in the results where cells upregulate relevant miRNA in mEVs by stimulating cells with melatonin while culturing cells in serum-free CDM. To demonstrate the strong angiogenic properties of PMEZ/mEV, the angiogenesis-related tube formation assay was conducted with human umbilical vein endothelial cells (HUVECs) (Fig. 4b). The results showed that the ability of tube formation was facilitated in the PMEZ, and significantly accelerated in PMEZ/mEVs, which displayed similar trends to the gene expression. With the NO-based angiogenic property of ZnO, mEV also indicated a synergistic angiogenic activity on scaffolds. All parameters of tube formation, such as total length, number of nodes, number of junctions, and number of master junctions, exhibited a gradual increase with the addition of ZnO-ALA and mEVs in PME scaffolds. Tissue injury leads to a rapid acute inflammatory response, resulting in the loss of regenerative properties in peripheral cells. 61 Moreover, acidic byproducts can induce additional inflammatory stimulations with the hydrolysis of biodegradable PLGA scaffolds. To enhance the anti-inflammatory potentials of PLGA scaffolds, we incorporated MH-RA for neutralizing acidities from the byproducts of PLGA decomposition and additionally fabricated with ZnO-ALA and mEVs. The changes in the expression level of a representative inflammatory factor, NF-κB, were evaluated with immunocytochemistry (ICC) in TNF-α pretreated HK2 cells (Fig. 4c). The expression level of NF-κB was downregulated gradually as the bioactive components were introduced. The NF-κB is widely known as a major contributor to inflammation-related disorders and stimulates the release of the pro-inflammatory cytokine, including IL-1β and IL-8. The quantitative analysis of gene expression levels related to pro-inflammatory cytokines also revealed that MH-RA slightly downregulated the release of pro-inflammatory cytokines (NF-κB, IL-1β, and IL-8) and incorporations of ZnO-ALA and mEV led to significant regulation of inflammation (Fig. 4d). Figure 4 The inhibition of epithelial-mesenchymal transition (EMT) of tubular epithelial cells was expected to enable the reduction of fibrosis for kidney regeneration. 62 With the excellent inhibition property of NF-κB to improve anti-fibrotic pathway involving PMEZ/mEV and results of bioinformatics analysis of miRNA in CDM mEVs, synergistic effects of PMEZ and mEV for anti-fibrosis were anticipated. From the ICC images with fibronectin staining in HK2 cells, the PMEZ/mEV scaffolds showed inhibitory effects on the representative fibrotic marker, fibronectin, induced by transforming growth factor β (TGF-β) (Fig. 5a). Significant inhibition of protein expression level for fibronectin was also demonstrated using Western blot analysis (Fig. 5b). Furthermore, the imbalance between the production of ROS and defense activity with antioxidants induces cell dysfunction and tissue injury. 63 Scavenging ROS is a strategy to modulate the apoptosis of injured tissues. Correlated with the expected anti-apoptotic activity of miRNA derived from CDM mEVs, PMEZ/mEV demonstrated the highest ROS scavenging activity in apoptosis induced HK2 cells pretreated with hydrogen peroxide (H 2 O 2 ) (Fig. 5c). The quantitative results also indicated a superior anti-apoptotic property of PMEZ/mEV, resulting in a similar ROS level as the control group (Fig. 5d). The macrophages have emerged as a therapeutic target for tissue regeneration due to their critical role in regulating multiple stages of tissue repair by fine-tuning the phenotypic shifts. 64 The imbalance in macrophage phenotypes, M1 and M2, causes unresolved inflammation and limits regeneration, especially, since the activation of the M2 macrophage regulates immune modulations and facilitates tissue regeneration. 65 Transformation of macrophage phenotype could be a promising strategy for tissue regeneration in addition to tuning the properties of the biomaterials. The combinatorial properties of ZnO-ALA and mEV in PMEZ/mEV scaffolds for macrophage polarization from M1 to M2 were investigated with the differences in ratios of M1 (INOS) and M2 (Arg-1) markers in murine macrophage cell line, RAW264.7 when incubated with various types of scaffolds. The RAW264.7 cell incubated with PMEZ/mEV showed the lowest INOS signal, whereas exhibited the highest Arg-1 expressions (Fig. 5e). Similar trends were obtained quantitatively using flow cytometry analysis (Figs. 5f and g). With multi-functionalities of intense bioactive components, PMEZ/mEV can be expected as a promising regenerative scaffold with immunomodulation properties. Figure 5 2.5. In vivo evaluation of the scaffolds for regeneration-related bioactivities Three types of scaffolds, PLGA, PMEZ, and PMEZ/mEV, were implanted into a nephrectomy mouse model. In this study, we utilized 5/6 nephrectomy mice as the animal models for severe chronic kidney disease to demonstrate superior bioactivities of our scaffold system (PMEZ-mEV) in the regeneration and restoration of injured kidney tissues. All parameters related to regenerative bioactivities were evaluated at 2- and 8-weeks intervals after scaffold implantations in injured kidney tissues (Fig. 6a). The expression levels of angiogenesis-related genes were upregulated in PMEZ groups at 2 weeks with the initial release of ZO-ALA, and further improved in PMEZ/mEV at 8 weeks due to sustained release of mEV from PEI-coated PMEZ scaffolds (Fig. 6b and S3). In addition, the intensity of a representative pro-inflammatory factor, TNF-α, displayed the highest in the native PLGA scaffold at 2 and 8 weeks after implantation, which was a more intense signal than the sham group, mainly due to acidic byproducts from the degradation of PLGA, and diminished in PMEZ and PMEZ/mEV groups as correlated with the results from in vitro assays (Fig. 6c and S4a). In parallel, gene expression levels of pro-inflammatory cytokines, IL-1β, IL-6, and TNF-α, significantly decreased with the incorporations of ZnO-ALA and mEV in PME scaffolds, whereas anti-inflammatory cytokines, IL-1Ra, increased in these groups (Fig. 6d and S4b). With slow degradation properties of high molecular weight PLGA scaffold (MW; 110 kDa, 50:50 of LA:GA), used in this study, the expression levels of PLGA-derived induction of pro-inflammatory cytokines were maintained even at 8 weeks after implantations unlike the early inflammations observed in previous reports where low molecular weight PLGA scaffolds were used (MW; 40 K, 50:50 of LA:GA) [ 17 ] and were prohibited with the synergistic effects of MH-RA, ECM, ZnO-ALA, and mEVs. The higher expression level of α-SMA, an EMT-related fibrotic marker, incubated with PLGA scaffolds significantly dropped when incubated with PMEZ/mEV scaffolds (Fig. 6e and S5a). All fibrosis-related genes induced by EMT were also dramatically downregulated with synergistic effects of MH-RA, ECM, and ZnO-ALA, and further regulated within PMEZ/mEV scaffolds (Fig. 6f and S5b). Increased ROS production leads to tissue damage associated with inflammation. Superoxide dismutases (SODs) convert superoxide to hydrogen peroxide, being removed by catalases, and prevent the formation of highly active ROS components. 66 And, the level of malondialdehyde (MDA) reflects oxygen radical activity during inflammation. 67 With evaluations of SOD and MDA levels in scaffold implanted tissues using enzyme-linked immunosorbent analysis (ELISA), ROS levels of tissues could be predicted indirectly. As expected, PMEZ/mEV scavenged ROS efficiently in injured tissue with scaffold implantations (Fig. 6g). The population of CD206-positive M2 macrophage was enhanced in the PMEZ group, and significantly upregulated in PMEZ/mEV as monitored using immunohistochemical (IHC) analysis from tissues with scaffold implantations (Fig. 6h and S6). Figure 6 2.6. In vivo regeneration and functional restoration of kidney tissues To analyze kidney tissue regeneration with implantation of scaffolds in 5/6 nephrectomy mouse models, the total number of glomeruli was counted in kidney tissues with histological staining. Podocytes are neighboring cells of the Bowman’s capsule that wraps around the capillaries. Since lost podocytes cannot be replaced by proliferating podocytes in adult tissues, other mechanisms of compensation must be implemented in the glomerulus. Especially, while depletion of up to 20% of the podocytes can be compensated with the aid of implanted cells, a loss of more than 60% of podocytes leads to glomerulosclerosis. 68 With our PMEZ/mEV scaffolds, glomeruli were regenerated at most in 5/6 resected kidney tissues without additional cell treatments. After 2 weeks of implantation, the number of glomeruli was similar in the PMEZ and PMEZ/mEV groups, mainly due to the regenerative effects of MH-RA, ECM, and ZnO-ALA in the early stage. After 8 weeks, glomerular regeneration was shown to be improved due to the sustained release of mEV, with maximized regeneration-related paracrine effects of melatonin-modulated hUCMSCs cultured in CDM, from PMEZ/mEV (Fig. 7a). With the increased expression levels of kidney development-related factors- Pax2, Wt1, and Emx2, the recruitment and infiltration of the host renal stem/progenitor cells are believed to facilitate regeneration of glomeruli with various physiologically active components in the scaffolds, although further studies are needed to elucidate more precise mechanisms (Figure S7). 69 Mast cells have a pivotal role in the exacerbation of CKD, and the number of mast cells is correlated with the loss of kidney functions via a fibrotic pathway. 70 With implantations of PLGA scaffolds, critical mast cell infiltration was observed, which was dramatically inhibited in the PMEZ and PMEZ/mEV scaffolds (Fig. 7b). In this regard, a level of c-reactive protein (CRP), representing acute inflammation due to serious infection, injury, and/or chronic disease, was significantly lowered in a PMEZ/mEV group (Fig. 7c). Finally, the restoration of kidney functions was comparatively analyzed using biochemical evaluation for levels of serum blood urea nitrogen (BUN) and creatinine (Fig. 7d). With the incorporation of bioactive components, both factors showed significantly lowered levels in PMEZ/mEV scaffolds, and in particular, the creatinine level was similar to that of the normal mouse model (a control group) at 8 weeks after scaffold implantations. These results successfully proved that the structural regeneration and functional restoration of kidney tissues were achieved with implantations of PMEZ/mEV scaffolds due to synergistic bioactivities of MH-RA, ECM, ZnO-ALA, and mEV in PLGA scaffolds, even in 5/6 nephrectomy mouse model. Figure 7 3. Conclusion Our study has proved the synergistic bioactivities of the biodegradable PLGA-based multiplexed scaffold for kidney tissue regeneration in 5/6 nephrectomy mouse representing a severe chronic kidney diseases (CKD) model. We, firstly, designed hybrid PMEZ scaffolds with the incorporation of multifunctional bioactive components, such as antacid MH-RA (M), biomimetic acellular ECM (E), nitric oxide (NO) generating ZnO-ALA (Z), based on porous PLGA (P) scaffolds. Additionally, melatonin-modulated extracellular vesicles (mEVs), derived from hUCMSC in chemically defined media (CDM), were functionalized into PMEZ scaffolds to facilitate regeneration activities via sustained release into the injured tissues. The synergistic bioactivities of distinguished components of PMEZ/mEV, such as MH-RA, ECM, ZO-ALA, and mEV that were grafted on the porous PLGA scaffolds, provided optimal microenvironments for the morphogenetic formations of renal tissues and functional restoration. Especially, the continuous NO-releasing property of ZO-ALA and augmented regeneration ability of mEV, accelerated angiogenesis, and anti-inflammation activities, resulting in the recovery from renal fibrosis and apoptotic damages. Based on these results, our multiplexed scaffold system could be a highly successful strategy for kidney tissue engineering and regeneration. 4. Experimental Section 4.1. Cell culture The human proximal tubular epithelial cell line, HK2 and the murine macrophage cell line, RAW264.7 were purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were maintained in RPMI 1640 media (GIBCO, NY, USA) and DMEM high glucose media (HyClone laboratories, UT, USA), which were supplemented with 10% fetal bovine serum (FBS; HyClone laboratories, UT, USA) and 1% antibiotic-antimycotic solution (GIBCO, NY, USA). All types of cells were incubated in a humid environment with 5% CO 2 at 37°C. 4.2. The isolation and characterizations of extracellular vesicle (EV) To isolate EV, the hUCMSC were cultured using CellCor™ CD MSC media (CDM; Xcell Therapeutics, Seoul, Korea). The melatonin (1 µM) was added to CDM during cell culture to isolate EVs derived from melatonin-preconditioned hUCMSC in CDM (CDM mEV). The conditioned cell culture media were collected every 24 h for 4 days. The collected cell culture media were centrifuged at 1300 rpm for 3 min and filtered through a 0.22 µm Vacuum Filter/Storage Bottle System to remove non-exosomal large particles, including cells, cell debris, microvesicles, and apoptotic bodies. Finally, the EVs were isolated using a tangential flow filtration (TFF; KR2i, Repligen, MA, USA) system with a 500 kDa molecular weight cut-off filter. The size and number of isolated EVs were determined using MONO ZetaView® (PMX-120, Particle Metrix, Meerbusch, Germany) with 488 nm scatter mode. The parameters of sensitivity, shutter, minimum trace length, and cell temperature were set at 75, 100, 15, and 25°C, respectively, for all samples to achieve reliable analysis. The morphology of EV was elucidated using transmission electron microscopy (TEM; Hitachi, H-7600, 80 kV, Tokyo, Japan). The EV solution was dried on a 150-meshed formvar/carbon supported copper grid (FCF150-CU, Electron Microscopy Sciences, USA) and stained using an EM stain 336 solution (R1260D; Agar Scientific, Stansted, UK) for negative staining. For EV characterization with miRNA components, the miRNAs contained in EVs were compared to databases of Vesiclepedia ( http://microvesicles.org ) and ExoCarta ( http://www.exocarta.org ). 4.3. The miRNA extraction in EVs The TRIzol™ LS reagent (Ambion, Life Technology, CA, USA) was used to isolate the exosomal miRNA in accordance with the manufacturer's instructions. The quality of miRNA was evaluated using the RNA 6000 Pico Chip (Agilent Technologies, CA, USA) by an Agilent 2100 bioanalyzer, and quantity was determined using a NanoDrop 2000 Spectrophotometer system (Thermo Fisher Scientific, OH, USA). 4.4. The miRNA sequencing of EVs and bioinformatics analysis Using the NEBNext Multiplex Small RNA Library Prep kit (New England BioLabs, MA, USA) and following the manufacturer's instructions, libraries were built for the various types of RNAs. Briefly, for developing libraries, total RNAs were used to ligate the adaptors and then cDNA was synthesized using reverse-transcriptase with adaptor-specific primers. For library amplification, polymerase chain reaction (PCR) was used, and libraries were cleaned up using a polyacrylamide gel electrophoresis (PAGE) and a QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). The yield and size distributions of the small RNA libraries were assessed by the Agilent 2100 Bioanalyzer instrument for the High-sensitivity DNA Assay (Agilent Technologies, CA, USA). High-throughput sequences were produced by the NextSeq550 system as a way of single-end 75 sequencing (Illumina, CA, USA). The public algorithms, miRwalk online prediction software ( http://mirwalk.umm.uni-heidelberg.de/ ) and TargetScan 8.0 ( www.targetscan.org ), were used to predict the targeting genes of miRNA in EVs. The DAVID (Database for annotation, visualization and integrated discovery; https://david.ncifcrf.gov/ ) was used to assess the gene ontology (GO) and kyoto encyclopedia genes and genomes (KEGG) pathway for prediction of functionalities. 4.5. Fabrication and characterizations of scaffolds All scaffolds (PLGA, PME, and PMEZ) were fabricated with an ice particle leaching method. The deionized water was sprayed into liquid nitrogen to create the ice particles (300–500 µm) for controlling porosity of scaffold. The 0.25 g of PLGA (P; MW 110 kDa, LA:GA = 50:50, Evonik Ind.) used as the backbone was blended with a 15 wt% magnesium hydroxide-ricinoleic acid (MH-RA; M), 20 wt% extracellular matrix (ECM: E), and 5 wt% zinc oxide-alpha lipoic acid (ZnO-ALA; Z) in a 0.3 M dichloromethane (DCM) solution (All wt% refer to ratio to PLGA). The blended materials and ice particles were placed in 7 x 2 mm circular PTFE mold. The filled molds were freeze-dried for two days to remove the residual ice particles and organic solvent. To maximize biological activities of released mEVs from the PLGA-based scaffold, polyethylenimine (PEI) was coated onto the surfaces of scaffolds as already established protocol from our Lab. 53 Continuously, mEVs were loaded onto the hydrated PEI-coated PMEZ scaffolds. The cross sectioned morphology of the scaffolds was observed using scanning electron microscopy (SEM; GENESIS-1000, Emcraft, Gwangju, Korea). The thermal properties of the scaffolds were examined with the thermal gravimetric analyzer (TGA 4000, PerkinElmer, MA, USA). The compression test for determining the mechanical properties of scaffolds was performed with the universal testing machine (UTM; Instron 4464, MA, USA). The linear coefficient of the strain-stress curve's slope between 5 ~ 10% was used to calculate the compressive modulus of the scaffolds. The pH changes by scaffolds were measured by a pH meter (Mettler Toledo, OH, USA) in 5 mL of PBS solution at 37°C for 56 days. At the same time, the weight changes of the scaffolds were also evaluated to monitor the degradation rate. Using a contact angle analyzer (Phoenix 300, Surface Electro Optics, Suwon, Korea), the water contact angle (WCA) was evaluated to identify the hydrophilicity for surface of scaffolds. The nitric oxide (NO) releasing profile from the scaffolds was detected using GE Sievers 280i Nitric Oxide Analyzer (NOA; GE Analytical Instruments, CO, USA) over 60 min. 4.6. Tube formation assay To assess the angiogenic effects, 200 µL of Matrigel matrix (Corning, NY, USA) was added to pre-chilled 24-well plate and then incubated at 37°C for 1 h. The human umbilical vein endothelial cells (HUVECs) were seeded onto Matrigel coated wells at the density of 1×10 5 cells/well, then added 1 mL of four types of scaffolds incubated in EBM-2 (Lonza, Basel, Switzerland) media with containing 1% FBS with indirect co-cultured system using the trans-well inserts (SPLInsert™, SPL, Korea). The cells were stained with calcein AM (C1430, Thermo Scientific, MS, USA) after 16 h. The images were obtained using a fluorescence microscopy (CKX53, OLYMPUS, Japan). The angiogenesis analyzer plugin for Image J (Wayne Rasband, NIH, USA) was used to evaluate angiogenesis-related properties. 4.7. Fibrosis assay The HK2 cells were seeded at a 6-well plate (1.5×10 5 cells/well). After 24 h of seeding, the cells were damaged with TGF-β (2 ng/ml, 100 − 21, Peprotech, NJ, USA) for 2 h and PLGA, PME, PMEZ, and PMEZ/mEV (1×10 8 EVs/mL) scaffolds were treated using trans-well inserts system after 24 h. Cell nucleus and fibronectin were stained using Hoechst (1µg/ml, 62249, Thermo Fisher Scientific, OH, USA) and fibronectin primary antibody (Abcam, MA, USA) for ICC, and proteins were extracted to compare inhibition activity of scaffolds for fibrosis markers, N-cadherin and fibronectin, using Western blot analysis. 4.8. DCF-DA The HK2 cells were seeded at a 6-well plate (5×10 5 cells/well). After 24 h of seeding, PLGA, PME, PMEZ, and PMEZ/mEV (1×10 8 EVs/mL) scaffolds were treated using trans-well inserts system and cells were damaged with H 2 O 2 (0.5 mM, Sigma-Aldrich, MO, USA) for 2 h as already established from our Lab. 71 DCF-DA (ab113851; Abcam, MA, USA) was treated as a working concentration with colorless media, and 30 min later, Hoechst was treated for 10 min to counter-staining. 4.9. Macrophage polarization The RAW264.7 cells were seeded on a 6-well plate with 5x10 5 cells per well. After 24 h, cells were stimulated with 50 ng/mL IFN-γ (R&D systems, MN, USA) and 10 ng/mL lipopolysaccharide (LPS, Sigma-Aldrich, MO, USA), followed by being treated with PLGA, PME, PMEZ, and PMEZ/mEV (1×10 8 EVs/mL) scaffolds using trans-well inserts system, respectively. Accutase™ (Stemcell Technologies, WA, USA) was used to detach the cells after 24 h. The same number of detached cells was fixed with 4% paraformaldehyde for 15 min, blocked with Fc blocking solution (553142; BD bioscience, CA, USA) for 15 min, incubated with antibody solution for, 20 min at 4°C, and measured with flow cytometer (CytoFLEX, Beckman coulter, CA, USA). The primary antibody has been used as follows: PE anti-mouse CD86 (Invitrogen, MA, USA) or FITC anti-mouse CD206 (Invitrogen, MA, USA). The cells were washed to remove excess antibodies, and resuspended in 300 µL of stain buffer (BD Pharmingen, CA, USA), followed by flow cytometer analysis. 4.10. Immunocytochemistry (ICC) Cells were immobilized at room temperature with 4% paraformaldehyde for 15 min and then permeabilized with 0.2% Triton X-100 dissolved in PBS solutions. The fixed cells were immediately incubated overnight with the primary antibodies containing 1% BSA. The antibodies were used as follows: NF-kB (Santa Cruz, TX, USA), fibronectin (Abcam, MA, USA), arginase-1 (Santa Cruz, TX, USA), iNOS (Invitrogen, MA, USA), CD206 (Santa Cruz, Dallas, TX), donkey anti-rabbit Alexa Fluor 488 (Invitrogen, MA, USA), and goat anti-mouse Alexa Fluor 555 (Invitrogen, MA, USA). The nucleus was stained with Hoechst (62249, 1µg/ml, Thermo Fisher Scientific, OH, USA). The ICC images were visualized using a confocal microscopy (LSM880, Carl Zeiss, Jena, Germany). 4.11. Western blot analysis The same quantity of samples (proteins and EVs) was loaded onto nitrocellulose (NC) membranes for parallel comparisons after being loaded with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A TBST solution diluted in 5% skim milk was used to block the NC membrane. The protein-transferred NC membranes were incubated with primary antibodies against CD81 (Santa Cruz Biotechnology, CA, USA), CD63 (Abcam, MA, USA), and CD9 (Abcam, MA, USA) to confirm EV surface markers and primary antibodies against N-cadherin (13116S, 1000:1, Cell Signaling Technology, MA, USA), fibronectin (ab2413, 500:1, Abcam, MA, USA), and GAPDH (5174T, 1000:1, Cell Signaling Technology, MA, USA) to confirm fibrosis related markers, respectively. After incubation with primary antibodies, HRP-linked secondary antibodies (Cell Signaling Technology, MA, USA) were applied. The enhanced chemiluminescence solution (GE Healthcare, WI, USA) was used to prepare the membrane before being visualized using ChemiDoc™ XRS + and ImageLab software (Bio-Rad, CA, USA). 4.12. Real-Time quantitative PCR (RT-qPCR) of in vitro samples Total cellular RNA was extracted using AccuPrep® Universal RNA Extraction Kit (Bioneer, Daejeon, Korea). The PrimeScript™ RT reagent kit (Takara, Shiga, Japan) was utilized to perform reverse transcription for extracted RNA to cDNA. A mixture of SYBR green PCR reagents (Applied Biosystems, CA, USA) was used for RT-qPCR. Reactions with the primers (Table S3) were conducted using QuantStudio 3 (Applied Biosystems, CA, USA). The 2 −ΔΔCt method with 18s rRNA as a reference was used for data quantification. 4.13. The design for in vivo model All animal study protocols were approved by the institutional animal ethics committee of Yeungnam University, College of Medicine (YUMC-AEC2021-024). For in vivo model, 6-week-old male ICR mouse (total = 50) was purchased from Orient Bio (Seongnam, Korea) and randomly divided into five groups (control = 3, sham = 4, PLGA = 6, PMEZ = 6, PMEZ/mEV = 6). Animals were anesthetized with 16 mg/kg of rompun and 0.04 mg/kg zoletil by intramuscular injection. For the 5/6 nephrectomy mouse model, the left kidney was completely removed and 2/3 of the right kidney was partially resected. The scaffolds were then implanted in the partially resected right kidney. At 2 and 8 weeks after scaffold implantations, animals were sacrificed and kidney tissues were retrieved for subsequent analyzes. 4.14. Histological analysis The collected kidney tissues were fixed in 10% formalin and embedded in paraffin. Tissue paraffin blocks were sectioned into 3–4 um thickness. Tissue slides were stained with hematoxylin and eosin (H&E; Abcam, MA, USA) for general histopathology, Massons’s trichrome (Abcam, MA, USA) for collagen fiber, toluidine blue (VitroView™, MD, USA) for mast cells, and periodic acid Schiff (Sigma-Aldrich) stain for glomerulus visualization using commercial kits following the manufacturer’s instruction. For immunohistochemistry (IHC), the slides were deparaffinized and hydrated prior to antigen retrieval using Triton-X solution. After blocking, primary antibodies (1:100) were applied for 18 h at 4˚C. FITC conjugated secondary antibody was applied for 2 h at room temperature. The slides were mounted with DAPI staining media (Vector Laboratories, Burlingame, CA, USA) and examined under fluorescence microscopy. 4.15. Real-Time quantitative PCR (RT-qPCR) of in vivo samples RNA was isolated from the collected kidney tissues by using Maxwell® RSC simply RNA cell kit using Maxwell™ 16 instrument (Promega Corporation, Madison, WI, USA). Isolated RNA was used in synthesis of cDNA with GoScript TM Reverse Tanscription Mix (Promega Corporation, Madison, WI, USA) according to the product protocols. Real-time PCR was performed in StepOnePlus™ Real-Time PCR System (Applied Biosystems® Inc., Foster City, CA, USA). Primers for in vivo analyzes were listed in Table S4. 4.16. Statistical analysis All statistical analyzes were assessed using the GraphPad Prism 7 program. To determine group differences, unpaired t tests or one-way analysis of variance (ANOVA) with Tukey's multiple comparison post-test were examined. Statistical significance was determined for p values below 0.05 (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). Declarations Author contribution All authors listed meet the authorship criteria and significantly contributed to the completion of this work as follows and W.-K.Rhim, J.Woo, and J.Y.Kim equally contributed as co-first authors; W.-K.Rhim: Conceptualization, Data analysis, Resources, Investigation, Writing original draft, Review and editing, Funding acquisition. J.Woo: Data analysis, Methodology, Visualization. J.Y.Kim: Data analysis, Resources, Methodology, Visualization, Writing original draft. E.H.Lee: Data analysis, Resources, Methodology, Visualization. S.-G.Cha: Data analysis, Resources, Methodology, Visualization. D.-S.Kim: Data analysis, Methodology. S.-W.Baek: Conceptualization, Methodology. C.G.Park: Conceptualization, Investigation. B.S.Kim: Conceptualization, Methodology, Investigation, Supervision, Project administration. T.G.Kwon: Conceptualization, Methodology, Investigation, Supervision, Project administration. D.K.Han: Conceptualization, Methodology, Investigation, Supervision, Project administration, Review and edition, Funding acquisition. Availability of data and material The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Declaration of competing interest The authors declare that they have no competing interests. Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2023R1A2C3003807) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2022R1I1A1A01068486). This research was supported by Korean Fund for Regenerative Medicine funded by Ministry of Science and ICT, and Ministry of Health and Welfare (RS-2023-00215369, Republic of Korea). References Webster, A. C., Nagler, E. V., Morton, R. L. & Masson, P. Chronic Kidney Disease. Lancet 389 , 1238–1252 (2017). Franco, M. L., Beyerstedt, S. & Rangel, É. B. Klotho and mesenchymal stem cells: A review on cell and gene therapy for chronic kidney disease and acute kidney disease. Pharmaceutics 14 , (2022). Liyanage, T. et al. Worldwide access to treatment for end-stage kidney disease: A systematic review. Lancet 385 , 1975–1982 (2015). Polaschegg, H. D. Red blood cell damage from extracorporeal circulation in hemodialysis. Semin. Dial. 22 , 524–531 (2009). Qiang, W., Jianchen, W., MacDonald, R., Monga, M. & Wilt, T. J. Antibiotic prophylaxis for transurethral prostatic resection in men with preoperative urine containing less than 100,000 bacteria per ml: A systematic review. J. Urol. 173 , 1175–1181 (2005). Do, A. V., Khorsand, B., Geary, S. M. & Salem, A. K. 3D Printing of Scaffolds for Tissue Regeneration Applications. Adv. Healthc. Mater. 4 , 1742–1762 (2015). Goodarzi, H., Hashemi-Najafabadi, S., Baheiraei, N. & Bagheri, F. Preparation and Characterization of Nanocomposite Scaffolds (Collagen/β-TCP/SrO) for Bone Tissue Engineering. Tissue Eng. Regen. Med. 16 , 237–251 (2019). Gwon, K., Choi, W. Il, Lee, S., Lee, J. S. & Shin, J. H. Biodegradable hyaluronic acid-based, nitric oxide-releasing nanofibers for potential wound healing applications. Biomater. Sci. 9 , 8160–8170 (2021). Kim, M. S. et al. Polymeric scaffolds for regenerative medicine. Polym. Rev. 51 , 23–52 (2011). Ali, M. & Payne, S. L. Biomaterial-based cell delivery strategies to promote liver regeneration. Biomater. Res. 25 , 1–21 (2021). Park, K. S. et al. Versatile effects of magnesium hydroxide nanoparticles in PLGA scaffold–mediated chondrogenesis. Acta Biomater. 73 , 204–216 (2018). Ma, S. et al. The pro-inflammatory response of macrophages regulated by acid degradation products of poly(lactide-co-glycolide) nanoparticles. Eng. Life Sci. 21 , 709–720 (2021). Lih, E. et al. Modified Magnesium Hydroxide Nanoparticles Inhibit the Inflammatory Response to Biodegradable Poly(lactide- co-glycolide) Implants. ACS Nano 12 , 6917–6925 (2018). Cha, M. et al. Three-dimensional printed polylactic acid scaffold integrated with BMP-2 laden hydrogel for precise bone regeneration. Biomater. Res. 25 , 1–11 (2021). Liao, J., Guo, X., Grande-Allen, K. J., Kasper, F. K. & Mikos, A. G. Bioactive polymer/extracellular matrix scaffolds fabricated with a flow perfusion bioreactor for cartilage tissue engineering. Biomaterials 31 , 8911–8920 (2010). Lih, E. et al. Biomimetic Porous PLGA Scaffolds Incorporating Decellularized Extracellular Matrix for Kidney Tissue Regeneration. ACS Appl. Mater. Interfaces 8 , 21145–21154 (2016). Lih, E. et al. A Bioinspired Scaffold with Anti-Inflammatory Magnesium Hydroxide and Decellularized Extracellular Matrix for Renal Tissue Regeneration. ACS Cent. Sci. 5 , 458–467 (2019). Kim, J. K. et al. PLGA Microspheres Containing Hydrophobically Modified Magnesium Hydroxide Particles for Acid Neutralization-Mediated Anti-Inflammation. Tissue Eng. Regen. Med. 18 , 613–622 (2021). Ko, K.-W. et al. Integrated Bioactive Scaffold with Polydeoxyribonucleotide and Stem-Cell-Derived Extracellular Vesicles for Kidney Regeneration. ACS Nano 15 , 7575–7585 (2021). Guo, X. et al. Bone regeneration with active angiogenesis by basic fibroblast growth factor gene transfected mesenchymal stem cells seeded on porous β-TCP ceramic scaffolds. Biomed. Mater. 1 , 93–99 (2006). Cooke, J. P. & Losordo, D. W. Nitric oxide and angiogenesis. Circulation 105 , 2133–2135 (2002). Fukumura, D. et al. Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability. Proc. Natl. Acad. Sci. U. S. A. 98 , 2604–2609 (2001). Sadrearhami, Z. et al. Recent advances in nitric oxide delivery for antimicrobial applications using polymer-based systems. J. Mater. Chem. B 6 , 2945–2959 (2018). Yang, T., Fruergaard, A. S., Winther, A. K., Zelikin, A. N. & Chandrawati, R. Zinc Oxide Particles Catalytically Generate Nitric Oxide from Endogenous and Exogenous Prodrugs. Small 16 , (2020). Chug, M. K., Bachtiar, E., Narwold, N., Gall, K. & Brisbois, E. J. Tailoring nitric oxide release with additive manufacturing to create antimicrobial surfaces. Biomater. Sci. 9 , 3100–3111 (2021). Le Blanc, K. Mesenchymal stromal cells: Tissue repair and immune modulation. Cytotherapy 8 , 559–561 (2006). Choi, S. et al. The role of mesenchymal stem cells in the functional improvement of chronic renal failure. Stem Cells Dev. 18 , 521–529 (2009). Peired, A. J., Sisti, A. & Romagnani, P. Mesenchymal Stem Cell-Based Therapy for Kidney Disease: A Review of Clinical Evidence. Stem Cells Int. 2016 , (2016). Wei, L., Fraser, J. L., Lu, Z. Y., Hu, X. & Yu, S. P. Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells enhances angiogenesis and neurogenesis after cerebral ischemia in rats. Neurobiol. Dis. 46 , 635–645 (2012). Phinney, D. G. & Pittenger, M. F. Concise review: MSC-derived exosomes for cell-free therapy. Stem Cells 35 , 851–858 (2017). Nikfarjam, S., Rezaie, J., Zolbanin, N. M. & Jafari, R. Mesenchymal stem cell derived-exosomes: a modern approach in translational medicine. J. Transl. Med. 18 , 1–21 (2020). Wiklander, O. P. B., Brennan, M., Lötvall, J., Breakefield, X. O. & Andaloussi, S. E. L. Advances in therapeutic applications of extracellular vesicles. Sci. Transl. Med. 11 , 1–16 (2019). Kim, J. Y. et al. Defined MSC exosome with high yield and purity to improve regenerative activity. J. Tissue Eng. 12 , (2021). Kim, J. Y. et al. Comparative Analysis of MSC-Derived Exosomes Depending on Cell Culture Media for Regenerative Bioactivity. Tissue Eng. Regen. Med. 18 , 355–367 (2021). Yea, J. H., Yoon, Y. M., Lee, J. H., Yun, C. W. & Lee, S. H. Exosomes isolated from melatonin-stimulated mesenchymal stem cells improve kidney function by regulating inflammation and fibrosis in a chronic kidney disease mouse model. J. Tissue Eng. 12 , (2021). Aouichat, S. et al. Melatonin improves endoplasmic reticulum stress-mediated ire1α pathway in zücker diabetic fatty rat. Pharmaceuticals 14 , 1–14 (2021). Kim, J. Y. et al. The Upregulation of Regenerative Activity for Extracellular Vesicles with Melatonin Modulation in Chemically Defined Media. (2022). Han, Y. S., Yoon, Y. M., Go, G., Lee, J. H. & Lee, S. H. Melatonin protects human renal proximal tubule epithelial cells against high glucose-mediated fibrosis via the cellular prion protein-tgf-β-smad signaling axis. Int. J. Med. Sci. 17 , 1235–1245 (2020). Takhtfooladi, H., Takhtfooladi, M., Moayer, F. & Mobarakeh, S. Melatonin attenuates lung injury in a hind limb ischemia-reperfusion rat model. Rev. Port. Pneumol. 21 , 30–35 (2015). Zhou, L. et al. Melatonin prevents lung injury induced by hepatic ischemia-reperfusion through anti-inflammatory and anti-apoptosis effects. Int. Immunopharmacol. 29 , 462–467 (2015). Li, Z. et al. Melatonin protects kidney grafts from ischemia/reperfusion injury through inhibition of NF-kB and apoptosis after experimental kidney transplantation. J. Pineal Res. 46 , 365–372 (2009). Théry, C. et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 7 , (2018). Xie, L., Overbeek, P. A. & Reneker, L. W. Ras signaling is essential for lens cell proliferation and lens growth during development. Dev. Biol. 298 , 403–414 (2006). Boettner, B. & Van Aelst, L. Control of cell adhesion dynamics by Rap1 signaling. Curr. Opin. Cell Biol. 21 , 684–693 (2009). Gaonac’h-Lovejoy, V., Boscher, C., Delisle, C. & Gratton, J. P. Rap1 is Involved in Angiopoietin-1-Induced Cell-Cell Junction Stabilization and Endothelial Cell Sprouting. Cells 9 , 1–15 (2020). Melenhorst, W. B. W. H. et al. Epidermal growth factor receptor signaling in the kidney key roles in physiology and disease. Hypertension 52 , 987–993 (2008). Escuin-Ordinas, H. et al. Cutaneous wound healing through paradoxical MAPK activation by BRAF inhibitors. Nat. Commun. 7 , 12348 (2016). Miao, S. et al. Materials Today Bio A 3D bioprinted nano-laponite hydrogel construct promotes osteogenesis by activating PI3K / AKT signaling pathway. Mater. Today Bio 16 , 100342 (2022). Clevers, H., Loh, K. M. & Nusse, R. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control. Science (80-. ). 346 , (2014). Vergadi, E., Ieronymaki, E., Lyroni, K., Vaporidi, K. & Tsatsanis, C. Akt Signaling Pathway in Macrophage Activation and M1/M2 Polarization. J. Immunol. 198 , 1006–1014 (2017). Yang, Y. et al. Crosstalk between hepatic tumor cells and macrophages via Wnt/β-catenin signaling promotes M2-like macrophage polarization and reinforces tumor malignant behaviors. Cell Death Dis. 9 , (2018). Tian, X. et al. Long noncoding RNA LINC00662 promotes M2 macrophage polarization and hepatocellular carcinoma progression via activating Wnt/β-catenin signaling. Mol. Oncol. 14 , 462–483 (2020). Woo, J., Ko, K. W., Cha, S. G., Heo, Y. & Han, D. K. Comparison of surface functionalization of PLGA composite to immobilize extracellular vesicles. Polymers (Basel). 13 , (2021). Wolhuter, K. & Eaton, P. How widespread is stable protein S-nitrosylation as an end-effector of protein regulation? Free Radic. Biol. Med. 109 , 156–166 (2017). Guo, R.-Y. et al. Bioinspired Design of Reversible Fluorescent Probes for Tracking Nitric Oxide Dynamics in Live Cells. CCS Chem. 3 , 116–128 (2021). Melvin, A. C., Jones, W. M., Lutzke, A., Allison, C. L. & Reynolds, M. M. S-Nitrosoglutathione exhibits greater stability than S-nitroso-N-acetylpenicillamine under common laboratory conditions: A comparative stability study. Nitric Oxide - Biol. Chem. 92 , 18–25 (2019). Kim, D. S. et al. Promotion of bone regeneration using bioinspired PLGA/MH/ECM scaffold combined with bioactive PDRN. Materials (Basel). 14 , 1–12 (2021). Jahani, M. et al. Regenerative medicine and angiogenesis; Challenges and Opportunities. Adv. Pharm. Bull. 10 , 490–501 (2020). Yamamoto, N. et al. VEGF and bFGF induction by nitric oxide is associated with hyperbaric oxygen-induced angiogenesis and muscle regeneration. Sci. Rep. 10 , 1–13 (2020). Tomita, N. et al. Angiogenic property of hepatocyte growth factor is dependent on upregulation of essential transcription factor for angiogenesis, ets-1. Circulation 107 , 1411–1417 (2003). Galliot, B., Crescenzi, M., Jacinto, A. & Tajbakhsh, S. Trends in tissue repair and regeneration. Dev. 144 , 357–364 (2017). Sheng, L. & Zhuang, S. New Insights Into the Role and Mechanism of Partial Epithelial-Mesenchymal Transition in Kidney Fibrosis. Front. Physiol. 11 , 1–11 (2020). Redza-Dutordoir, M. & Averill-Bates, D. A. Activation of apoptosis signalling pathways by reactive oxygen species. Biochim. Biophys. Acta - Mol. Cell Res. 1863 , 2977–2992 (2016). Novak, M. L. & Koh, T. J. Macrophage phenotypes during tissue repair. J. Leukoc. Biol. 93 , 875–881 (2013). Hou, Y., Li, J., Guan, S. & Witte, F. The therapeutic potential of MSC-EVs as a bioactive material for wound healing. Eng. Regen. 2 , 182–194 (2021). Wang, Y., Branicky, R., Noë, A. & Hekimi, S. Superoxide dismutases: Dual roles in controlling ROS damage and regulating ROS signaling. J. Cell Biol. 217 , 1915–1928 (2018). Sori, R. K. et al. Evaluation of analgesic activity of sodium valproate and ethanolic extract of Vitex negundo in experimental analgesic models in wistar rats. Int. J. Basic Clin. Pharmacol. 7 , 2036 (2018). Hagmann, H. & Brinkkoetter, P. T. Experimental models to study podocyte biology: Stock-taking the toolbox of glomerular research. Front. Pediatr. 6 , 1–9 (2018). Yu, N. hee et al. In Vivo Safety and Regeneration of Long-Term Transported Amniotic Fluid Stem Cells for Renal Regeneration. Tissue Eng. Regen. Med. 16 , 81–92 (2019). Lee, S. B. & Kalluri, R. Mechanistic connection between inflammation and fibrosis. Kidney Int. 78 , S22–S26 (2010). Kim, J. Y. et al. Bolstering the secretion and bioactivities of umbilical cord MSC‑ derived extracellular vesicles with 3D culture and priming in chemically defined media. Nano Converg. (2022) doi:10.1186/s40580-022-00349-z. Additional Declarations (Not answered) Supplementary Files 20230414NPJKidneySIDKHanF.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2815340","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":194537451,"identity":"bb4fc3fc-152f-4f08-b9d7-bdf9b056c677","order_by":0,"name":"Dong Keun 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University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chun","middleName":"Gwon","lastName":"Park","suffix":""},{"id":194537460,"identity":"e3818234-c5bc-4560-a76a-191762aef469","order_by":9,"name":"Bum Soo Kim","email":"","orcid":"","institution":"Kyungpook National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bum","middleName":"Soo","lastName":"Kim","suffix":""},{"id":194537461,"identity":"f756446d-e86c-439a-999f-3d1ae258d461","order_by":10,"name":"Tae Gyun Kwon","email":"","orcid":"","institution":"Kyungpook National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tae","middleName":"Gyun","lastName":"Kwon","suffix":""}],"badges":[],"createdAt":"2023-04-14 04:40:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2815340/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2815340/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":36339869,"identity":"5ae757bd-11da-48de-a28d-1cec36035254","added_by":"auto","created_at":"2023-04-26 17:59:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1210952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of kidney regeneration with multiplexed PMEZ/mEV scaffolds.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2815340/v1/6170f900b27b2e514a4300bc.png"},{"id":36339522,"identity":"b13d187c-2972-42cd-9334-58c0fcc69888","added_by":"auto","created_at":"2023-04-26 17:51:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":461463,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterizations of melatonin-modulated extracellular vesicles.\u003c/strong\u003e (a) The size and number of particles from ZetaView\u003csup\u003e®\u003c/sup\u003e analysis. (b) The Western blot analysis of mEV for representative EV-specific surface markers (CD63, CD81, and CD9). (c) The morphologies of EVs are characterized by TEM. Scale bars equal to 100 nm. (d) Heatmap analysis of the top 100 significant expressed miRNAs in the CDM EV and CDM mEV. (e) Identification of canonical EV markers. Red circle indicates miRNA of CDM mEV, blue indicates the top 100 miRNA markers in EVs from Vesiclepedia, and purple indicates those from ExoCarta. (f) The related biological categories of miRNAs that CDM mEV expresses twice more than CDM EV. (g) The volcano plot of the miRNAs between in the CDM EV and CDM mEV. The red dots indicate the most significantly expressed five miRNAs in CDM mEV compared to CDM EV. (h) The DAVID analysis for Gene Ontology (GO)-BP, GO-CC, GO-MF, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway for the target genes from significantly expressed miRNAs in CDM-mEV compared to CDM EV.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2815340/v1/81b800681d497be5f8ca740d.png"},{"id":36339870,"identity":"88b217ef-9325-44e8-9ddb-45a3f6583b7b","added_by":"auto","created_at":"2023-04-26 17:59:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":649592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhysicochemical properties of the scaffolds. \u003c/strong\u003e(a) Representative SEM images of microporous structures for PLGA, PME, PMEZ, and PMEZ/mEV scaffolds. Scale bars equal to 60 μm in upper images and 10 μm in lower magnified images, respectively. (b) Changes of pH value and (c) degradation behavior of scaffolds during \u003cem\u003ein vitro \u003c/em\u003edegradation at 37 ºC during 56 days. (d) DAF-FM assays for monitoring NO release of PMEZ scaffolds in the presence of GSH (G) and SNAP (S). (e) Nitric oxide releasing profiles of PMEZ scaffolds using the nitric oxide analyzer (NOA).\u003cstrong\u003e \u003c/strong\u003e(f)\u003cstrong\u003e \u003c/strong\u003eRepresentative confocal images showing distribution of DiO-labeled mEV in the PMEZ scaffold. Scale bars equal to 50 μm. (g) Thermal gravimetric analysis (TGA) thermograms of PMEZ and PMEZ/mEV scaffolds. (h) Compressive stress-strain curve and (i) compressive modulus at 5 to 10% of stress-strain curve. (Values are presented as mean ± SD (n = 3) and statistical significance was obtained with unpaired \u003cem\u003et\u003c/em\u003e tests or one-way analysis of variance (ANOVA) with Tukey's multiple comparison post-test (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001)).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2815340/v1/73e3b3168f53351160cac7f1.png"},{"id":36339871,"identity":"eae350fa-2460-4509-a904-f4169d7cbfae","added_by":"auto","created_at":"2023-04-26 17:59:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":576600,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e analyzes for effects of the scaffolds on angiogenesis and anti-inflammation.\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e(a)\u003cstrong\u003e \u003c/strong\u003eThe gene expression levels for representative angiogenic markers (HIP-1α, VEGF, and HGF). (b) Representative images of angiogenesis effects of scaffolds by tube formation assay and analysis for total length, number of nodes, number of junctions, and number of master junction from tube formation assay. Scale bars equal to 200 μm. (c) Representative immunofluorescence images of TNF-α pretreated HK2 cells with incubating scaffolds for pro-inflammatory cytokine, NF-κB. (d) Gene expression levels of inflammation-related factors (NF-κB, IL-1β, and IL-8). (Values are presented as mean ± SD (n = 3) and statistical significance was obtained with unpaired \u003cem\u003et\u003c/em\u003e tests or one-way analysis of variance (ANOVA) with Tukey's multiple comparison post-test (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001; ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001)).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2815340/v1/361f76446fe3aa22445f17ef.png"},{"id":36339526,"identity":"039405d6-83e8-433a-bdf9-265c1c289da1","added_by":"auto","created_at":"2023-04-26 17:51:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1121354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e analyzes for bioactivities of the scaffolds on fibrosis, ROS, and macrophage polarization. \u003c/strong\u003e(a) Representative fibronectin immunofluorescence images and (b) Western blot analysis showing the protein expression of fibronectin for TGF-b pretreated HK2 cell with incubating scaffolds. (c) Representative images and (d) quantitative analysis of DCF-DA for cellular ROS level. (e) Representative INOS and Arg-1 immunofluorescence images of IFN-γ/LPS-stimulated RAW264.7 cells. (f) Flow cytometry of the M1 (CD86) and M2 (CD206) macrophage markers in IFN- \u0026nbsp;/LPS stimulated RAW264.7 cells and (g) Quantitative analysis for the expression levels of CD86 and CD206. (Values are presented as mean ± SD (n = 3) and statistical significance was obtained with unpaired \u003cem\u003et\u003c/em\u003e tests or one-way analysis of variance (ANOVA) with Tukey's multiple comparison post-test (*\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001)).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2815340/v1/dc076246770560c167e9c1a5.png"},{"id":36339525,"identity":"df36f0a0-4492-4316-a403-b27dff117197","added_by":"auto","created_at":"2023-04-26 17:51:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1733673,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e evaluations for regeneration-related bioactivities of the scaffolds. \u003c/strong\u003e(a) A timetable for scaffold implantations and \u003cem\u003ein vivo\u003c/em\u003e analysis for 5/6 nephrectomy mouse models. (b) Gene expression levels of angiogenic markers (HIP-1α, VEGF, and HGF) at 8 weeks after implantations. (c) Representative images for immunohistochemical analysis of TNF-α in the scaffold implanted areas at 8 weeks after implantations. (d) Gene expression levels of inflammatory-related factors at 8 weeks after scaffold implantations. (Pro-inflammatory cytokines; IL-1β, IL-6, and TNF-α, Anti-inflammatory cytokine; IL-1Ra). (e) Representative images for immunohistochemical analysis of α-SMA in the scaffold implanted areas at 8 weeks after implantations. (f) Gene expression levels of fibrosis markers (TGF-β, vimentin, collagen type 1, α-SMA, fibronectin, and E-cadherin) using qRT-RCR at 8 weeks after scaffold implantations and (g) protein expression levels of ROS-related factors (SOD, MDA) with ELISA at 2 and 8 weeks after scaffold implantations. (h) Representative images for immunohistochemical analysis of CD206 in the scaffold implanted area at 8 weeks after implantations. (Control: No surgical treatment, Sham: 5/6 nephrectomy model without scaffold implantations, PLGA: PLGA scaffold implantations into 5/6 nephrectomy model, PMEZ: PMEZ scaffold implantations into 5/6 nephrectomy model, PMEZ/mEV: PMEZ/mEV scaffold implantations into 5/6 nephrectomy model, Values are presented as mean ± SD (n = 3) and statistical significance was obtained with unpaired \u003cem\u003et\u003c/em\u003e tests or one-way analysis of variance (ANOVA) with Tukey's multiple comparison post-test (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001; ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001)).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2815340/v1/9af80aa646c84e259aa38906.png"},{"id":36339528,"identity":"e2b5e37b-388b-4d4f-96e2-8e27645f8d79","added_by":"auto","created_at":"2023-04-26 17:51:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1315897,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eevaluations for kidney tissue regeneration and kidney function restoration properties of the scaffolds. \u003c/strong\u003e(a) Representative H\u0026amp;E staining and quantitative analysis for glomeruli formations in the scaffold implanted area at 2 and 8 weeks after implantations. The red arrows indicate regenerated glomeruli. Scale bars equal to 100 μm. (b) Number of mast cells in scaffold implanted area at 2 and 8 weeks after implantations. (c) The level of CRP at 2 and 8 weeks after implantation. (d) The levels of blood urea nitrogen (BUN) and creatinine in the serum at 2 and 8 weeks after implantations. (Control: No surgical treatment, Sham: 5/6 nephrectomy model without scaffold implantations, PLGA: PLGA scaffold implantations into 5/6 nephrectomy model, PMEZ: PMEZ scaffold implantations into 5/6 nephrectomy model, PMEZ/mEV: PMEZ/mEV scaffold implantations into 5/6 nephrectomy model, Values are presented as mean ± SD (n = 3) and statistical significance was obtained with unpaired \u003cem\u003et\u003c/em\u003e tests or one-way analysis of variance (ANOVA) with Tukey's multiple comparison post-test (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001)).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-2815340/v1/aeac6c0ccef9947f2e12698a.png"},{"id":39435718,"identity":"ffde7451-e6a5-47c2-9e3c-808282ca19e9","added_by":"auto","created_at":"2023-07-03 04:21:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5069115,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2815340/v1/4885cc94-b00e-41c1-9a9b-2ea6531c15f7.pdf"},{"id":36339529,"identity":"854a1ed8-325c-4f76-8461-a5cb096b5ee3","added_by":"auto","created_at":"2023-04-26 17:51:08","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":13990838,"visible":true,"origin":"","legend":"","description":"","filename":"20230414NPJKidneySIDKHanF.docx","url":"https://assets-eu.researchsquare.com/files/rs-2815340/v1/41df45d4e1c100619fdc55e5.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Multiplexed PLGA scaffolds with nitric oxide-releasing zinc oxide and melatonin-modulated extracellular vesicles for severe chronic kidney disease","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eChronic kidney disease (CKD) is a global public health problem with a gradual and irreversible structural and functional abnormalities of the kidney, which results in reduced glomerular filtration rate and increased urinary albumin secretion.\u003csup\u003e1,2\u003c/sup\u003e In end-stage kidney disease (ESKD), the therapeutic strategies are limited to renal replacement therapies, such as dialysis and transplantation. Two types of dialysis- hemodialysis and peritoneal dialysis, remove the wastes and excess fluids from the blood to maintain homeostasis in the body.\u003csup\u003e3\u003c/sup\u003e However, dialysis treatment cannot replace the role of the kidney and various risks remain due to extracorporeal circulations.\u003csup\u003e4\u003c/sup\u003e Although the kidney transplant is a fundamental approach to restoring kidney functions but has severe limitations like extremely limited donors, and long-lasting use of immunosuppressive drugs after transplantation.\u003csup\u003e5\u0026ndash;7\u003c/sup\u003e The limited current therapies for ESKD have led researchers to focus on tissue engineering and regenerative medicine approaches for restoring and replacing the partial or total function of the kidney.\u003c/p\u003e \u003cp\u003eTo provide mechanical stability and contain biochemical signals through combination with various bioactive components, the scaffold-based strategies have been explored as structural and functional platforms to give a proper microenvironment for the regeneration and engineering of the injured tissues. Among diverse scaffold types, composed of metals, ceramics, polymers, and composites, biodegradable polymer-based scaffolds have started looking attractive for biomedical applications. With qualities like degradation property, ease of processing, close biological properties with many tissues and extracellular matrix (ECM), and the absence of the need for additional surgery to remove the scaffolds, the scaffold-based platform looks very promising.\u003csup\u003e8\u003c/sup\u003e Biodegradable polymer-based scaffolds have been utilized for various clinical applications such as to restore the function of injured tissues, like bone regeneration, cartilage repair, skin wound healing, and nerve conduits.\u003csup\u003e9,10\u003c/sup\u003e Design of appropriate biodegradable scaffolds for specific tissue regeneration with optimum mechanical, chemical, and biological properties is crucial for accurate and active tissue engineering processes. Biodegradable polymer-engineered porous scaffolds support mechanical stability and spatial flexibility which plays a pivotal role in regeneration and tissue engineering through interaction with surrounding environments. However, there are some limitations to be addressed, mainly shortcomings derived from the hydrolysis of a biodegradable polyester polymer such as poly(lactic-\u003cem\u003eco\u003c/em\u003e-glycolic) acid (PLGA). The acidic byproducts induce a pH decrease in the tissue environment, resulting in cellular necrosis, inflammation, and fibrosis of closed tissues.\u003csup\u003e11\u003c/sup\u003e The lactic acid and glycolic acid derived from PLGA degradation induce the classical complement pathway which is amplified by an alternative pathway and stimulates pro-inflammatory cytokine \u003cem\u003evia\u003c/em\u003e activating monocytes and macrophages.\u003csup\u003e12\u003c/sup\u003e Many studies have been reported to solve these problems occurring from the byproducts of biodegradable polymers. The incorporation of alkaline hyaluronic acid or drugs partially neutralizes the acidity, but they cannot completely avoid the inflammation in the peripheral tissues.\u003csup\u003e13,14\u003c/sup\u003e Therefore, strategies to improve the bioactivity of the materials inducing tissue regeneration are essential. The kidney is a very complex organ composed of various cells and ECM components with lots of growth factors and proteins. As a result, many types of ECMs have been studied to functionalize polymer-based scaffold bioactivity and their interaction with cells surrounding the implanted scaffolds.\u003csup\u003e15,16\u003c/sup\u003e To neutralize acidification by the byproducts during PLGA degradation and to enable enhancement of bioactivity of the surrounding cells, we previously reported the use of PME scaffold composites with ricinoleic acid grafted-magnesium hydroxide (Mg(OH)\u003csub\u003e2\u003c/sub\u003e-RA; MH-RA; M) and porcine kidney-derived extracellular matrix (ECM; E) incorporated in porous PLGA (P) scaffold.\u003csup\u003e17\u003c/sup\u003e Magnesium hydroxide was utilized as an antacid component \u003cem\u003evia\u003c/em\u003e partially dissolving magnesium and hydroxide ions, where hydroxide ion combines with acidic byproducts of PLGA inhibiting inflammation and fibrotic pathway of kidney tissues. In addition, one of the biocompatible fatty acids, ricinoleic acid was grafted to the surface of MH to enhance the dispersity in organic solvents and have more anti-inflammatory effects.\u003csup\u003e18\u003c/sup\u003e By mimicking the microenvironment of kidney tissues using ECM, the properties of kidney tissue regeneration could be improved. Despite successful glomerulus regeneration for restoration of kidney function, the demand for bioactive components to promote the biophysical properties of scaffolds has increased. For kidney tissue regeneration and function restoration, integrated bioactive scaffolds, containing polydeoxyribonucleotide (PDRN) and TNF-α/IFN-γ-primed MSC-derived extracellular vesicles (TI-EVs) into PME scaffolds, have been developed.\u003csup\u003e19\u003c/sup\u003e By mimicking the microenvironment of kidney tissues using PME scaffolds, and incorporating bioactive components- PDRN and TI-EVs, the properties of kidney tissue regeneration could be facilitated. Despite successful glomerulus regeneration in mouse models with 3/4 nephrectomy, the improved scaffolds with multifunctional bioactivities are required for the complete recovery in severely injured (5/6 nephrectomy) CKD mouse models to simulate application to human CKD patients.\u003c/p\u003e \u003cp\u003eFor the success of tissue engineering with biomaterials, angiogenesis is one of the most prominent processes. Many types of pro-angiogenic biochemical molecules have been utilized to give an angiogenic property, but their biological activities are limited to \u003cem\u003ein vivo\u003c/em\u003e systems due to short half-lives and low bioactivities.\u003csup\u003e20\u003c/sup\u003e Nitric oxide (NO) can be clinically applied to regulate angiogenesis at optimum concentrations.\u003csup\u003e21\u003c/sup\u003e NO is a soluble gas involved in many regulatory functions in various tissues, which is synthesized in vascular endothelium with various types of nitric oxide synthases.\u003csup\u003e22\u003c/sup\u003e NO facilitates the proliferation of cells, immune regulations, and anti-apoptotic effects with the appropriate concentrations. Although a lot of functionalities of NO have been actively utilized \u003cem\u003ein vitro\u003c/em\u003e, there have been limitations with the bioactivities \u003cem\u003ein vivo\u003c/em\u003e, due to constraints of exposure area and short half-lives with rapid thermal and photochemical decompositions.\u003csup\u003e23\u003c/sup\u003e To overcome these limitations, various types of metals, metal oxides, and nonmetallic particles have been introduced as a prodrug that releases NO by reacting with endogenous NO donors.\u003csup\u003e24\u003c/sup\u003e The zinc oxide (ZnO) particle, as one of the promising NO-releasing components that exhibit a proper combination with innate NO donors, achieved a long-lasting release of NO with excellent stability against biological degradation.\u003csup\u003e25\u003c/sup\u003e However, the hydrophilic accelerating fast release of ZnO from the scaffolds needs to be surmounted.\u003c/p\u003e \u003cp\u003eSeveral studies have indicated the regenerative and therapeutic effects of mesenchymal stem cells (MSC) for injured tissues.\u003csup\u003e26\u0026ndash;28\u003c/sup\u003e The transplantation of MSCs has potent regenerative activity using lineage reprogramming of stem cells, cell fusion, and mitochondrial transfer, however, it also has the potential to cause tumorigenesis, immunogenicity, and limited cell survival simultaneously.\u003csup\u003e29\u003c/sup\u003e This led researchers to focus on the paracrine factors that have been considered as the key mechanism of MSC-based therapeutics and stimulate peripheral MSCs to differentiate for tissue regeneration. Especially, extracellular vesicles (EVs) i.e., small vesicular particles secreted from cells, play a pivotal role to direct the paracrine effects of MSCs by mediating intercellular communications.\u003csup\u003e30\u003c/sup\u003e MSC-derived EVs show biological functional similarities, including tissue regeneration-related activities, to the parent cells by containing and delivering active biocomponents to the targeted cells or tissues.\u003csup\u003e31\u003c/sup\u003e Isolation of EVs from the cells of interest with high purity and bioactivity has become significant as it improved their therapeutic potential in clinical use.\u003csup\u003e32,33\u003c/sup\u003e In this regard, serum-depleted conditions (starvation) have been known to exclude unknown side effects derived from the serum during the EVs isolation process. However, starvation resulted in a diminished proliferation rate and changes in cell characteristics which were overcome by using chemically defined media (CDM), as published previously. The CDM maintained healthy MSCs during EV isolation without serum components, supporting the isolation of highly purified EVs with enhanced bioactivities. Moreover, the bioactivities of EVs can be modulated using growth factors, transcription factors, and biochemical and/or biophysical stimulations during cell culturing.\u003csup\u003e34\u003c/sup\u003e Especially, melatonin has been utilized to promote the therapeutic activities of EVs for kidney tissue regeneration in starvation conditions by modulating inflammation and fibrosis in a CKD model.\u003csup\u003e35,36\u003c/sup\u003e Extensive comparative analysis has been studied for the optimization of cell modulation in CDM to modify starved conditions during cell stimulations and EV isolation, and to maximize the regeneration ability of melatonin-modulated MSCs with high purity.\u003csup\u003e37\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn this study, we designed porous hybrid PMEZ scaffolds based on PLGA (P) with ricinoleic acid grafted magnesium hydroxide (MH-RA; M) to enhance the pH neutralization ability of MH, and extracellular matrix (ECM; E) to mimic kidney tissue environment. Alpha lipoic acid-conjugated zinc oxide nanoparticles (ZnO-ALA; Z) were used to promote angiogenic properties with continuous dual NO release in kidney tissues. Additionally, the upregulation of internal miRNA was evaluated using bioinformatics analysis for melatonin-modulated hUCMSC-derived extracellular vesicles (mEV), preconditioned in chemically defined media (CDM). Various bioactivities related to kidney regeneration have been proved using mEV-incorporated multiplexed PMEZ/mEV scaffolds (Fig.\u0026nbsp;1). Finally, structural, and functional recovery have been confirmed using PMEZ/mEV scaffolds in the 5/6 nephrectomy mouse model to simulate progressive renal failure in humans without cell transplantation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 1\u003c/b\u003e \u003c/p\u003e"},{"header":"2. Results And Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Characterization of melatonin-modulated EVs derived from hUCMSC cultured in chemically defined media (CDM)\u003c/h2\u003e \u003cp\u003eTo maximize the production and purity of extracellular vesicles (EVs), serum-free chemically defined media, CellCor\u0026trade; CD MSC (CDM; Xcell Therapeutics, Seoul, Korea) was utilized to culture human umbilical cord mesenchymal stem cells (hUCMSCs). In our previous study, we proved that CDM promoted EV release from hUCMSC with high purity and bioactivity and cells maintained an excellent proliferation rate without animal-derived proteins and other impurities.\u003csup\u003e34\u003c/sup\u003e Furthermore, cells primed with pro-inflammatory factors have been shown to improve the functions of EVs and keep cells healthy in CDM. Contrary to cell priming approaches with pro-inflammatory cytokines, which torment cells to secrete substances to overcome inflammatory situations, melatonin plays a critical role in immune regulation by reducing apoptotic and necrotic alterations, inflammatory cell infiltration, and tissue fibrosis after tissue injury and thus maintains healthy cells.\u003csup\u003e38\u003c/sup\u003e Numerous reports have suggested that melatonin improved the therapeutic efficacy of stem cells in treating several disorders, including hindlimb ischemia\u003csup\u003e39\u003c/sup\u003e, hepatic ischemia\u003csup\u003e40\u003c/sup\u003e, and kidney ischemia\u003csup\u003e41\u003c/sup\u003e. To maximize the regeneration ability of EVs without serum components for future translational applications, we isolated EVs from melatonin-treated hUCMSC cultured in CDM using tangential flow filtration system. In accordance with MISEV 2018 guidelines for characterization of EVs\u003csup\u003e42\u003c/sup\u003e, the size and number of melatonin-modulated EVs (mEVs) were analyzed with the MONO ZetaView\u0026reg; (Fig.\u0026nbsp;2a). Moreover, the representative surface markers of EVs (CD63, CD81, and CD9) were obtained by Western blot analysis (Fig.\u0026nbsp;2b), and double-layered spherical structures were observed using transmission electron microscopy (Fig.\u0026nbsp;2c) to express the characteristics of EVs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Comparative analysis of miRNA profiling for CDM EV and CDM mEV\u003c/h2\u003e \u003cp\u003eWe performed small RNA sequencing to confirm the difference between EVs derived from melatonin-modulated hUCMSC in CDM (CDM mEV) and hUCMSC in CDM without melatonin stimulations (CDM EV). According to the heatmap analysis for the highest expressed miRNAs in EVs, a clear separation of miRNA expressions was shown between two different types of EVs (Fig.\u0026nbsp;2d). As large parts of miRNAs overlapped with the types of EV-derived miRNA classified in Vesiclepedia and ExoCarta libraries, it could be indirectly confirmed that the corresponding miRNA is derived from EVs (Fig.\u0026nbsp;2e and S1).\u003c/p\u003e \u003cp\u003eTo predict the roles of the identified miRNAs in the biological systems, bioinformatic analysis was performed as an alternative approach. A large number of miRNAs were associated with angiogenesis, apoptosis, cell migration, cell proliferation, and inflammatory responses, which are strongly related to regulatory bioactivities for tissue regenerations (Fig.\u0026nbsp;2f). With a volcano plot analysis, the distribution of miRNAs was distinguished in CDM EV and CDM mEV, and five types of highly expressed miRNAs in CDM mEV were selected for further analysis (Fig.\u0026nbsp;2g). The red dots (hsa-miR-3195, hsa-miR-1301-3p, hsa-miR-181d-5p, hsa-miR-30c-5p, and hsa-miR-30e-3p) on the volcano plot indicate the miRNAs that are very abundant in CDM mEV compared to CDM EV. The targeting genes of these five miRNAs were identified with miRWalk. Additionally, gene ontology (GO), Kyoto encyclopedia genes, and genomes (KEGG) analysis were performed using DAVID (Fig.\u0026nbsp;2h). The GO-biological process (GO-BP) showed \u0026ldquo;Positive regulation of cell proliferation,\u0026rdquo; \u0026ldquo;Positive regulation of cell migration,\u0026rdquo; \u0026ldquo;Negative regulation of the apoptotic process,\u0026rdquo; \u0026ldquo;Wnt signaling pathway,\u0026rdquo; \u0026ldquo;Cell migration,\u0026rdquo; and \u0026ldquo;Positive regulation of angiogenesis,\u0026rdquo; meaning the functionalities related to cell proliferation, migration, angiogenesis, and anti-apoptosis of CDM mEV. The analysis of GO-cellular components (GO-CC) revealed that miRNA of CDM mEV appeared to target subcellular regions, such as cytosol, nucleoplasm, and cytoplasm. In GO-molecular functions (GO-MF) terms, \u0026ldquo;Protein binding\u0026rdquo; displayed the largest \u003cem\u003ep\u003c/em\u003e values. The association of CDM mEV with the activity of intracellular processes was inferred \u003cem\u003evia\u003c/em\u003e the QuickGO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/QuickGO/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/QuickGO/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The protein binding implies \u0026ldquo;cell adhesion molecule binding,\u0026rdquo; \u0026ldquo;cytokine binding,\u0026rdquo; and \u0026ldquo;cytoskeletal protein binding.\u0026rdquo; Finally, KEGG analysis revealed various signaling pathways, including \u0026ldquo;The Ras signaling pathway,\u0026rdquo; \u0026ldquo;The Rap1 signaling pathway,\u0026rdquo; \u0026ldquo;The ErbB signaling pathway,\u0026rdquo; \u0026ldquo;The MAPK signaling pathway,\u0026rdquo; \u0026ldquo;The PI3K-Akt signaling pathway,\u0026rdquo; and \u0026ldquo;The Wnt signaling pathway.\u0026rdquo; The Ras signaling pathway is known to be associated with cell proliferation and differentiation.\u003csup\u003e43\u003c/sup\u003e The Rap1 signaling pathway is well-known to be involved in cell adhesion and angiogenesis.\u003csup\u003e44,45\u003c/sup\u003e The ErbB signaling pathway can promote regenerative proliferation and migration, and it may also enhance renal tubular cell regeneration and repair after renal ischemia \u003cem\u003ein vivo\u003c/em\u003e.\u003csup\u003e46\u003c/sup\u003e The MAPK signaling pathway is involved in wound healing, whereas the PI3K-Akt signaling pathway is implicated in a variety of biological functions such as cell division, autophagy, survival, differentiation, and bone formation.\u003csup\u003e47,48\u003c/sup\u003e Lastly, the Wnt signaling pathway is known to play a role in both tissue regeneration by promoting cell differentiation and activity in numerous tissues,\u003csup\u003e49\u003c/sup\u003e and M2 polarization of macrophages.\u003csup\u003e50\u0026ndash;52\u003c/sup\u003e Taken together, these results suggest that CDM mEV would show more closely related bioactivities during regeneration as compared to CDM EV.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 2\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Physicochemical properties of the scaffolds\u003c/h2\u003e \u003cp\u003eAlthough various polymer-based synthetic scaffolds, approved by FDA, have been utilized for tissue engineering, the limited biomimetic properties suggested the need for the development of polymer-based scaffolds with improved functionality. In our previous study, the PME scaffold, based on poly(lactide-\u003cem\u003eco\u003c/em\u003e-glycolide) (PLGA; P) scaffold with ricinoleic acid grafted-magnesium hydroxide (Mg(OH)\u003csub\u003e2\u003c/sub\u003e-RA; MH-RA; M) and porcine kidney-derived extracellular matrix (kECM; E), was shown to facilitate kidney tissue regeneration compared to the native PLGA scaffold in 3/4 nephrectomy mouse model. More recently, the integrated bioactive scaffolds incorporated with two types of bioactive components, polydeoxyribonucleotide (PDRN) and TNF-α/IFN-γ-primed MSC-derived extracellular vesicles (TI-EVs), facilitated regenerative activities for injured kidney tissues.\u003csup\u003e19\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTo verify the structural and functional recovery ability in the 5/6 nephrectomy mouse models that mimic severe CKD in humans, in this study, intense bioactive components were applied to functionalize PME scaffolds. The multifunctional porous PMEZ scaffolds were engineered with modified PME scaffolds using alpha lipoic acid-conjugated zinc oxide (ZnO-ALA; Z) by the ice particle leaching method. Additionally, extracellular vesicles derived from melatonin-preconditioned hUCMSC cultured in CDM (mEV) were immobilized onto the PMEZ scaffold after coating with positively charged polyethylenimine (PEI) to capture EVs and maximize therapeutic activities with sustained release of EVs in biological condition.\u003csup\u003e53\u003c/sup\u003e The formation of highly porous structures to enable cell migration and diffusion of bioactive components between scaffolds and peripheral tissues was monitored using cross-sectional images from scanning electron microscopy. All types of scaffolds showed similar porosity with the addition of MH-RA, ECM, and ZnO-ALA due to the use of an equal ratio of ice particles for controlling scaffold porosity (Fig.\u0026nbsp;3a). To prove the neutralizing property of MH-RA, pH changes depending on the scaffold degradation were monitored in a phosphate-buffered saline (PBS) solution at 37\u0026deg;C for 56 days. Although the pH of native PLGA started to decrease after 28 days to around pH 6 along with slow degradation of high molecular weight PLGA (MW; 110 kDa, 50:50 of LA:GA), indicating auto-accelerated formation of an acidic environment by hydrolysis of PLGA scaffolds. The scaffolds containing MH-RA, PME, PMEZ, and PMEZ/mEV, maintained the pH around 7 with a slight increase initially due to critical neutralizing effects of MH-RA during the 56 days (Fig.\u0026nbsp;3b and 3c). MH-RA effectively neutralized PME, PMEZ, and PMEZ/mEV as compared to PLGA scaffold, in spite of relatively fast degradation rate with addition of bioactive components.\u003c/p\u003e \u003cp\u003eZinc oxide (ZnO) particles, known to generate nitric oxide (NO), reacted with innate glutathione peroxidase and glycosidase which allows decomposing donors to release NO in physiological conditions.\u003csup\u003e24\u003c/sup\u003e To address the shortcomings derived from the hydrophilicity of ZnO, lipophilic alpha lipoic acid (ALA) was conjugated onto the surfaces of ZnO to enhance the dispersity of ZnO in organic solvent and thus enable sustained release from the PLGA scaffold. Furthermore, ALA is utilized to treat oxidative stress-associated diseases and the disulfide bonding group of ALA continuously generates NO by reacting with glutathione (GSH) to form RSSG that reacts with s-nitroso-N-acetylpenicillamine (SNAP) in the body.\u003csup\u003e54\u003c/sup\u003e Consecutively, RSSR compounds, byproducts of the reaction are transferred to RSSG by reacting with GSH, where the continuous release of NO is available. Based on this information, long-lasting NO release with sustained release of ZnO-ALA and multiple NO releases with disulfide bonding groups of ALA has been achieved. The NO-releasing property of ZO-ALA (Z) incorporated PMEZ scaffold was monitored using a fluorescence-based DAF-FM assay (Fig.\u0026nbsp;3d). DAF-FM is a highly sensitive, photo-stable fluorescence probe to quantify NO release.\u003csup\u003e55\u003c/sup\u003e In the presence of only GSH and SNAP as NO donors in the PBS solution, very weak fluorescence signals were obtained due to the natural decomposition of NO donors, while significantly intense signals were generated with PMEZ scaffolds. In the group containing PME scaffolds, moderate fluorescence signals were also detected due to the catalytic effects of metal oxides and hydroxides, such as MgO and Mg(OH)\u003csub\u003e2\u003c/sub\u003e, for NO release.\u003csup\u003e56\u003c/sup\u003e The continuous NO-releasing profile derived from ZO-ALA in the PMEZ scaffold with the addition of each 10 \u0026micro;M GSH (G) and SNAP (S) was monitored using a nitric oxide analyzer (NOA) for over 60 min (Figs.\u0026nbsp;3e and S2). After 40 min, relatively higher amounts of NO started generating, because of the sustained release of ZO-ALA from the PMEZ scaffolds. By infusing mEV onto PEI-coated PMEZ scaffolds, the distribution of DiO-labeled mEV was observed using confocal microscopy. The fluorescence signal was distributed in PMEZ/mEV scaffolds, whereas no signal was detected in PMEZ scaffolds without mEV (Fig.\u0026nbsp;3f).\u003c/p\u003e \u003cp\u003eThe proportions of two types of inorganic components, MH-RA and ZnO-ALA, were evaluated using inductively coupled plasma optical emission spectroscopy (ICP-OES; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and thermos gravimetric analysis (TGA; Fig.\u0026nbsp;3g). As expected, 15 and 5 wt% (ratios to PLGA) of MH-RA and ZnO-ALA were incorporated into PMEZ scaffolds, respectively, and all PLGA composites with different compositions of additives started to thermally decompose slightly earlier than the native PLGA scaffolds. Additionally, the mechanical properties of scaffolds containing MH-RA, ECM, ZnO-ALA, and mEV were investigated with the compressive stress and modulus analysis using a universal testing machine (Fig.\u0026nbsp;3h). The compressive modulus calculated by slope between 5\u0026thinsp;~\u0026thinsp;10% of the strain-stress curve improved with the addition of the inorganic components, MH-RA and ZnO-ALA, and slightly decreased in the addition of hydrated mEV (Fig.\u0026nbsp;3i). The relatively robust property of scaffolds contributed to a sustained release of biochemical components and could inhibit premature degradation of scaffolds after implantations. The changes in water contact angle (WCA) also proved the incorporations of bioactive components in PMEZ/mEV scaffolds (Table S2). The WCA decreased gradually with the addition of MH-RA, ECM, and ZnO-ALA compared to native PLGA scaffolds, and mEV components dropped the WCA to zero (wetting), which means to interact with water molecules strongly. This makes the scaffolds more biocompatible by inhibiting nonspecific interactions with proteins in the body and facilitating the adhesion and penetration of peripheral cells in a biological system.\u003csup\u003e57\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 3\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. \u003cem\u003eIn vitro\u003c/em\u003e analysis of various scaffold bioactivities\u003c/h2\u003e \u003cp\u003eTo investigate the advanced regenerative properties of multiplexed scaffolds, various regeneration-related biological activities of the scaffolds were compared with the incorporation of bioactive components. In regenerative medicine studies, the promotion of vascularization and angiogenesis is a crucial step.\u003csup\u003e58\u003c/sup\u003e To endow angiogenic functionality, ZnO-ALA and mEV in PME scaffolds were fabricated. The expression levels of angiogenesis-related genes, hypoxia-inducible factor 1-a (HIF-1α), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF), were observed to be higher with the addition of ZnO-ALA, and further upregulated with mEV in human proximal tubular epithelial cell line (HK2) (Fig.\u0026nbsp;4a). ZnO originally increases HIF-1α and stimulates the secretion of VEGF and HGF via NO release.\u003csup\u003e59\u003c/sup\u003e Similar to VEGF, HGF has angiogenic properties and the potential to stimulate cell invasion and motility for the promotion of cell proliferation.\u003csup\u003e60\u003c/sup\u003e Furthermore, in bioinformatics analysis using the DAVID tool, mEVs are expected to exhibit superior angiogenic properties as shown in the results where cells upregulate relevant miRNA in mEVs by stimulating cells with melatonin while culturing cells in serum-free CDM. To demonstrate the strong angiogenic properties of PMEZ/mEV, the angiogenesis-related tube formation assay was conducted with human umbilical vein endothelial cells (HUVECs) (Fig.\u0026nbsp;4b). The results showed that the ability of tube formation was facilitated in the PMEZ, and significantly accelerated in PMEZ/mEVs, which displayed similar trends to the gene expression. With the NO-based angiogenic property of ZnO, mEV also indicated a synergistic angiogenic activity on scaffolds. All parameters of tube formation, such as total length, number of nodes, number of junctions, and number of master junctions, exhibited a gradual increase with the addition of ZnO-ALA and mEVs in PME scaffolds.\u003c/p\u003e \u003cp\u003eTissue injury leads to a rapid acute inflammatory response, resulting in the loss of regenerative properties in peripheral cells.\u003csup\u003e61\u003c/sup\u003e Moreover, acidic byproducts can induce additional inflammatory stimulations with the hydrolysis of biodegradable PLGA scaffolds. To enhance the anti-inflammatory potentials of PLGA scaffolds, we incorporated MH-RA for neutralizing acidities from the byproducts of PLGA decomposition and additionally fabricated with ZnO-ALA and mEVs. The changes in the expression level of a representative inflammatory factor, NF-κB, were evaluated with immunocytochemistry (ICC) in TNF-α pretreated HK2 cells (Fig.\u0026nbsp;4c). The expression level of NF-κB was downregulated gradually as the bioactive components were introduced. The NF-κB is widely known as a major contributor to inflammation-related disorders and stimulates the release of the pro-inflammatory cytokine, including IL-1β and IL-8. The quantitative analysis of gene expression levels related to pro-inflammatory cytokines also revealed that MH-RA slightly downregulated the release of pro-inflammatory cytokines (NF-κB, IL-1β, and IL-8) and incorporations of ZnO-ALA and mEV led to significant regulation of inflammation (Fig.\u0026nbsp;4d).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 4\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe inhibition of epithelial-mesenchymal transition (EMT) of tubular epithelial cells was expected to enable the reduction of fibrosis for kidney regeneration.\u003csup\u003e62\u003c/sup\u003e With the excellent inhibition property of NF-κB to improve anti-fibrotic pathway involving PMEZ/mEV and results of bioinformatics analysis of miRNA in CDM mEVs, synergistic effects of PMEZ and mEV for anti-fibrosis were anticipated. From the ICC images with fibronectin staining in HK2 cells, the PMEZ/mEV scaffolds showed inhibitory effects on the representative fibrotic marker, fibronectin, induced by transforming growth factor β (TGF-β) (Fig.\u0026nbsp;5a). Significant inhibition of protein expression level for fibronectin was also demonstrated using Western blot analysis (Fig.\u0026nbsp;5b). Furthermore, the imbalance between the production of ROS and defense activity with antioxidants induces cell dysfunction and tissue injury.\u003csup\u003e63\u003c/sup\u003e Scavenging ROS is a strategy to modulate the apoptosis of injured tissues. Correlated with the expected anti-apoptotic activity of miRNA derived from CDM mEVs, PMEZ/mEV demonstrated the highest ROS scavenging activity in apoptosis induced HK2 cells pretreated with hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) (Fig.\u0026nbsp;5c). The quantitative results also indicated a superior anti-apoptotic property of PMEZ/mEV, resulting in a similar ROS level as the control group (Fig.\u0026nbsp;5d). The macrophages have emerged as a therapeutic target for tissue regeneration due to their critical role in regulating multiple stages of tissue repair by fine-tuning the phenotypic shifts.\u003csup\u003e64\u003c/sup\u003e The imbalance in macrophage phenotypes, M1 and M2, causes unresolved inflammation and limits regeneration, especially, since the activation of the M2 macrophage regulates immune modulations and facilitates tissue regeneration.\u003csup\u003e65\u003c/sup\u003e Transformation of macrophage phenotype could be a promising strategy for tissue regeneration in addition to tuning the properties of the biomaterials. The combinatorial properties of ZnO-ALA and mEV in PMEZ/mEV scaffolds for macrophage polarization from M1 to M2 were investigated with the differences in ratios of M1 (INOS) and M2 (Arg-1) markers in murine macrophage cell line, RAW264.7 when incubated with various types of scaffolds. The RAW264.7 cell incubated with PMEZ/mEV showed the lowest INOS signal, whereas exhibited the highest Arg-1 expressions (Fig.\u0026nbsp;5e). Similar trends were obtained quantitatively using flow cytometry analysis (Figs.\u0026nbsp;5f and g). With multi-functionalities of intense bioactive components, PMEZ/mEV can be expected as a promising regenerative scaffold with immunomodulation properties.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 5\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. \u003cem\u003eIn vivo\u003c/em\u003e evaluation of the scaffolds for regeneration-related bioactivities\u003c/h2\u003e \u003cp\u003eThree types of scaffolds, PLGA, PMEZ, and PMEZ/mEV, were implanted into a nephrectomy mouse model. In this study, we utilized 5/6 nephrectomy mice as the animal models for severe chronic kidney disease to demonstrate superior bioactivities of our scaffold system (PMEZ-mEV) in the regeneration and restoration of injured kidney tissues. All parameters related to regenerative bioactivities were evaluated at 2- and 8-weeks intervals after scaffold implantations in injured kidney tissues (Fig.\u0026nbsp;6a). The expression levels of angiogenesis-related genes were upregulated in PMEZ groups at 2 weeks with the initial release of ZO-ALA, and further improved in PMEZ/mEV at 8 weeks due to sustained release of mEV from PEI-coated PMEZ scaffolds (Fig.\u0026nbsp;6b and S3). In addition, the intensity of a representative pro-inflammatory factor, TNF-α, displayed the highest in the native PLGA scaffold at 2 and 8 weeks after implantation, which was a more intense signal than the sham group, mainly due to acidic byproducts from the degradation of PLGA, and diminished in PMEZ and PMEZ/mEV groups as correlated with the results from \u003cem\u003ein vitro\u003c/em\u003e assays (Fig.\u0026nbsp;6c and S4a). In parallel, gene expression levels of pro-inflammatory cytokines, IL-1β, IL-6, and TNF-α, significantly decreased with the incorporations of ZnO-ALA and mEV in PME scaffolds, whereas anti-inflammatory cytokines, IL-1Ra, increased in these groups (Fig.\u0026nbsp;6d and S4b). With slow degradation properties of high molecular weight PLGA scaffold (MW; 110 kDa, 50:50 of LA:GA), used in this study, the expression levels of PLGA-derived induction of pro-inflammatory cytokines were maintained even at 8 weeks after implantations unlike the early inflammations observed in previous reports where low molecular weight PLGA scaffolds were used (MW; 40 K, 50:50 of LA:GA)\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e and were prohibited with the synergistic effects of MH-RA, ECM, ZnO-ALA, and mEVs. The higher expression level of α-SMA, an EMT-related fibrotic marker, incubated with PLGA scaffolds significantly dropped when incubated with PMEZ/mEV scaffolds (Fig.\u0026nbsp;6e and S5a). All fibrosis-related genes induced by EMT were also dramatically downregulated with synergistic effects of MH-RA, ECM, and ZnO-ALA, and further regulated within PMEZ/mEV scaffolds (Fig.\u0026nbsp;6f and S5b). Increased ROS production leads to tissue damage associated with inflammation. Superoxide dismutases (SODs) convert superoxide to hydrogen peroxide, being removed by catalases, and prevent the formation of highly active ROS components.\u003csup\u003e66\u003c/sup\u003e And, the level of malondialdehyde (MDA) reflects oxygen radical activity during inflammation.\u003csup\u003e67\u003c/sup\u003e With evaluations of SOD and MDA levels in scaffold implanted tissues using enzyme-linked immunosorbent analysis (ELISA), ROS levels of tissues could be predicted indirectly. As expected, PMEZ/mEV scavenged ROS efficiently in injured tissue with scaffold implantations (Fig.\u0026nbsp;6g). The population of CD206-positive M2 macrophage was enhanced in the PMEZ group, and significantly upregulated in PMEZ/mEV as monitored using immunohistochemical (IHC) analysis from tissues with scaffold implantations (Fig.\u0026nbsp;6h and S6).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 6\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. \u003cem\u003eIn vivo\u003c/em\u003e regeneration and functional restoration of kidney tissues\u003c/h2\u003e \u003cp\u003eTo analyze kidney tissue regeneration with implantation of scaffolds in 5/6 nephrectomy mouse models, the total number of glomeruli was counted in kidney tissues with histological staining. Podocytes are neighboring cells of the Bowman\u0026rsquo;s capsule that wraps around the capillaries. Since lost podocytes cannot be replaced by proliferating podocytes in adult tissues, other mechanisms of compensation must be implemented in the glomerulus. Especially, while depletion of up to 20% of the podocytes can be compensated with the aid of implanted cells, a loss of more than 60% of podocytes leads to glomerulosclerosis.\u003csup\u003e68\u003c/sup\u003e With our PMEZ/mEV scaffolds, glomeruli were regenerated at most in 5/6 resected kidney tissues without additional cell treatments. After 2 weeks of implantation, the number of glomeruli was similar in the PMEZ and PMEZ/mEV groups, mainly due to the regenerative effects of MH-RA, ECM, and ZnO-ALA in the early stage. After 8 weeks, glomerular regeneration was shown to be improved due to the sustained release of mEV, with maximized regeneration-related paracrine effects of melatonin-modulated hUCMSCs cultured in CDM, from PMEZ/mEV (Fig.\u0026nbsp;7a). With the increased expression levels of kidney development-related factors- Pax2, Wt1, and Emx2, the recruitment and infiltration of the host renal stem/progenitor cells are believed to facilitate regeneration of glomeruli with various physiologically active components in the scaffolds, although further studies are needed to elucidate more precise mechanisms (Figure S7).\u003csup\u003e69\u003c/sup\u003e Mast cells have a pivotal role in the exacerbation of CKD, and the number of mast cells is correlated with the loss of kidney functions via a fibrotic pathway.\u003csup\u003e70\u003c/sup\u003e With implantations of PLGA scaffolds, critical mast cell infiltration was observed, which was dramatically inhibited in the PMEZ and PMEZ/mEV scaffolds (Fig.\u0026nbsp;7b). In this regard, a level of c-reactive protein (CRP), representing acute inflammation due to serious infection, injury, and/or chronic disease, was significantly lowered in a PMEZ/mEV group (Fig.\u0026nbsp;7c). Finally, the restoration of kidney functions was comparatively analyzed using biochemical evaluation for levels of serum blood urea nitrogen (BUN) and creatinine (Fig.\u0026nbsp;7d). With the incorporation of bioactive components, both factors showed significantly lowered levels in PMEZ/mEV scaffolds, and in particular, the creatinine level was similar to that of the normal mouse model (a control group) at 8 weeks after scaffold implantations. These results successfully proved that the structural regeneration and functional restoration of kidney tissues were achieved with implantations of PMEZ/mEV scaffolds due to synergistic bioactivities of MH-RA, ECM, ZnO-ALA, and mEV in PLGA scaffolds, even in 5/6 nephrectomy mouse model.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 7\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003eOur study has proved the synergistic bioactivities of the biodegradable PLGA-based multiplexed scaffold for kidney tissue regeneration in 5/6 nephrectomy mouse representing a severe chronic kidney diseases (CKD) model. We, firstly, designed hybrid PMEZ scaffolds with the incorporation of multifunctional bioactive components, such as antacid MH-RA (M), biomimetic acellular ECM (E), nitric oxide (NO) generating ZnO-ALA (Z), based on porous PLGA (P) scaffolds. Additionally, melatonin-modulated extracellular vesicles (mEVs), derived from hUCMSC in chemically defined media (CDM), were functionalized into PMEZ scaffolds to facilitate regeneration activities \u003cem\u003evia\u003c/em\u003e sustained release into the injured tissues. The synergistic bioactivities of distinguished components of PMEZ/mEV, such as MH-RA, ECM, ZO-ALA, and mEV that were grafted on the porous PLGA scaffolds, provided optimal microenvironments for the morphogenetic formations of renal tissues and functional restoration. Especially, the continuous NO-releasing property of ZO-ALA and augmented regeneration ability of mEV, accelerated angiogenesis, and anti-inflammation activities, resulting in the recovery from renal fibrosis and apoptotic damages. Based on these results, our multiplexed scaffold system could be a highly successful strategy for kidney tissue engineering and regeneration.\u003c/p\u003e"},{"header":"4. Experimental Section","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Cell culture\u003c/h2\u003e \u003cp\u003eThe human proximal tubular epithelial cell line, HK2 and the murine macrophage cell line, RAW264.7 were purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were maintained in RPMI 1640 media (GIBCO, NY, USA) and DMEM high glucose media (HyClone laboratories, UT, USA), which were supplemented with 10% fetal bovine serum (FBS; HyClone laboratories, UT, USA) and 1% antibiotic-antimycotic solution (GIBCO, NY, USA). All types of cells were incubated in a humid environment with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.2. The isolation and characterizations of extracellular vesicle (EV)\u003c/h2\u003e \u003cp\u003eTo isolate EV, the hUCMSC were cultured using CellCor\u0026trade; CD MSC media (CDM; Xcell Therapeutics, Seoul, Korea). The melatonin (1 \u0026micro;M) was added to CDM during cell culture to isolate EVs derived from melatonin-preconditioned hUCMSC in CDM (CDM mEV). The conditioned cell culture media were collected every 24 h for 4 days. The collected cell culture media were centrifuged at 1300 rpm for 3 min and filtered through a 0.22 \u0026micro;m Vacuum Filter/Storage Bottle System to remove non-exosomal large particles, including cells, cell debris, microvesicles, and apoptotic bodies. Finally, the EVs were isolated using a tangential flow filtration (TFF; KR2i, Repligen, MA, USA) system with a 500 kDa molecular weight cut-off filter. The size and number of isolated EVs were determined using MONO ZetaView\u0026reg; (PMX-120, Particle Metrix, Meerbusch, Germany) with 488 nm scatter mode. The parameters of sensitivity, shutter, minimum trace length, and cell temperature were set at 75, 100, 15, and 25\u0026deg;C, respectively, for all samples to achieve reliable analysis. The morphology of EV was elucidated using transmission electron microscopy (TEM; Hitachi, H-7600, 80 kV, Tokyo, Japan). The EV solution was dried on a 150-meshed formvar/carbon supported copper grid (FCF150-CU, Electron Microscopy Sciences, USA) and stained using an EM stain 336 solution (R1260D; Agar Scientific, Stansted, UK) for negative staining. For EV characterization with miRNA components, the miRNAs contained in EVs were compared to databases of Vesiclepedia (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://microvesicles.org\u003c/span\u003e\u003cspan address=\"http://microvesicles.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and ExoCarta (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.exocarta.org\u003c/span\u003e\u003cspan address=\"http://www.exocarta.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.3. The miRNA extraction in EVs\u003c/h2\u003e \u003cp\u003eThe TRIzol\u0026trade; LS reagent (Ambion, Life Technology, CA, USA) was used to isolate the exosomal miRNA in accordance with the manufacturer's instructions. The quality of miRNA was evaluated using the RNA 6000 Pico Chip (Agilent Technologies, CA, USA) by an Agilent 2100 bioanalyzer, and quantity was determined using a NanoDrop 2000 Spectrophotometer system (Thermo Fisher Scientific, OH, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.4. The miRNA sequencing of EVs and bioinformatics analysis\u003c/h2\u003e \u003cp\u003eUsing the NEBNext Multiplex Small RNA Library Prep kit (New England BioLabs, MA, USA) and following the manufacturer's instructions, libraries were built for the various types of RNAs. Briefly, for developing libraries, total RNAs were used to ligate the adaptors and then cDNA was synthesized using reverse-transcriptase with adaptor-specific primers. For library amplification, polymerase chain reaction (PCR) was used, and libraries were cleaned up using a polyacrylamide gel electrophoresis (PAGE) and a QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). The yield and size distributions of the small RNA libraries were assessed by the Agilent 2100 Bioanalyzer instrument for the High-sensitivity DNA Assay (Agilent Technologies, CA, USA). High-throughput sequences were produced by the NextSeq550 system as a way of single-end 75 sequencing (Illumina, CA, USA). The public algorithms, miRwalk online prediction software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mirwalk.umm.uni-heidelberg.de/\u003c/span\u003e\u003cspan address=\"http://mirwalk.umm.uni-heidelberg.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and TargetScan 8.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://www.ebi.ac.uk/QuickGO/\" target=\"_blank\"\u003ewww.targetscan.org\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.targetscan.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), were used to predict the targeting genes of miRNA in EVs. The DAVID (Database for annotation, visualization and integrated discovery; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov/\u003c/span\u003e\u003cspan address=\"https://david.ncifcrf.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to assess the gene ontology (GO) and kyoto encyclopedia genes and genomes (KEGG) pathway for prediction of functionalities.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.5. Fabrication and characterizations of scaffolds\u003c/h2\u003e \u003cp\u003eAll scaffolds (PLGA, PME, and PMEZ) were fabricated with an ice particle leaching method. The deionized water was sprayed into liquid nitrogen to create the ice particles (300\u0026ndash;500 \u0026micro;m) for controlling porosity of scaffold. The 0.25 g of PLGA (P; MW 110 kDa, LA:GA\u0026thinsp;=\u0026thinsp;50:50, Evonik Ind.) used as the backbone was blended with a 15 wt% magnesium hydroxide-ricinoleic acid (MH-RA; M), 20 wt% extracellular matrix (ECM: E), and 5 wt% zinc oxide-alpha lipoic acid (ZnO-ALA; Z) in a 0.3 M dichloromethane (DCM) solution (All wt% refer to ratio to PLGA). The blended materials and ice particles were placed in 7 x 2 mm circular PTFE mold. The filled molds were freeze-dried for two days to remove the residual ice particles and organic solvent. To maximize biological activities of released mEVs from the PLGA-based scaffold, polyethylenimine (PEI) was coated onto the surfaces of scaffolds as already established protocol from our Lab.\u003csup\u003e53\u003c/sup\u003e Continuously, mEVs were loaded onto the hydrated PEI-coated PMEZ scaffolds. The cross sectioned morphology of the scaffolds was observed using scanning electron microscopy (SEM; GENESIS-1000, Emcraft, Gwangju, Korea). The thermal properties of the scaffolds were examined with the thermal gravimetric analyzer (TGA 4000, PerkinElmer, MA, USA). The compression test for determining the mechanical properties of scaffolds was performed with the universal testing machine (UTM; Instron 4464, MA, USA). The linear coefficient of the strain-stress curve's slope between 5\u0026thinsp;~\u0026thinsp;10% was used to calculate the compressive modulus of the scaffolds. The pH changes by scaffolds were measured by a pH meter (Mettler Toledo, OH, USA) in 5 mL of PBS solution at 37\u0026deg;C for 56 days. At the same time, the weight changes of the scaffolds were also evaluated to monitor the degradation rate. Using a contact angle analyzer (Phoenix 300, Surface Electro Optics, Suwon, Korea), the water contact angle (WCA) was evaluated to identify the hydrophilicity for surface of scaffolds. The nitric oxide (NO) releasing profile from the scaffolds was detected using GE Sievers 280i Nitric Oxide Analyzer (NOA; GE Analytical Instruments, CO, USA) over 60 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.6. Tube formation assay\u003c/h2\u003e \u003cp\u003eTo assess the angiogenic effects, 200 \u0026micro;L of Matrigel matrix (Corning, NY, USA) was added to pre-chilled 24-well plate and then incubated at 37\u0026deg;C for 1 h. The human umbilical vein endothelial cells (HUVECs) were seeded onto Matrigel coated wells at the density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well, then added 1 mL of four types of scaffolds incubated in EBM-2 (Lonza, Basel, Switzerland) media with containing 1% FBS with indirect co-cultured system using the trans-well inserts (SPLInsert\u0026trade;, SPL, Korea). The cells were stained with calcein AM (C1430, Thermo Scientific, MS, USA) after 16 h. The images were obtained using a fluorescence microscopy (CKX53, OLYMPUS, Japan). The angiogenesis analyzer plugin for Image J (Wayne Rasband, NIH, USA) was used to evaluate angiogenesis-related properties.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.7. Fibrosis assay\u003c/h2\u003e \u003cp\u003eThe HK2 cells were seeded at a 6-well plate (1.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well). After 24 h of seeding, the cells were damaged with TGF-β (2 ng/ml, 100\u0026thinsp;\u0026minus;\u0026thinsp;21, Peprotech, NJ, USA) for 2 h and PLGA, PME, PMEZ, and PMEZ/mEV (1\u0026times;10\u003csup\u003e8\u003c/sup\u003e EVs/mL) scaffolds were treated using trans-well inserts system after 24 h. Cell nucleus and fibronectin were stained using Hoechst (1\u0026micro;g/ml, 62249, Thermo Fisher Scientific, OH, USA) and fibronectin primary antibody (Abcam, MA, USA) for ICC, and proteins were extracted to compare inhibition activity of scaffolds for fibrosis markers, N-cadherin and fibronectin, using Western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.8. DCF-DA\u003c/h2\u003e \u003cp\u003eThe HK2 cells were seeded at a 6-well plate (5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well). After 24 h of seeding, PLGA, PME, PMEZ, and PMEZ/mEV (1\u0026times;10\u003csup\u003e8\u003c/sup\u003e EVs/mL) scaffolds were treated using trans-well inserts system and cells were damaged with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (0.5 mM, Sigma-Aldrich, MO, USA) for 2 h as already established from our Lab.\u003csup\u003e71\u003c/sup\u003e DCF-DA (ab113851; Abcam, MA, USA) was treated as a working concentration with colorless media, and 30 min later, Hoechst was treated for 10 min to counter-staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.9. Macrophage polarization\u003c/h2\u003e \u003cp\u003eThe RAW264.7 cells were seeded on a 6-well plate with 5x10\u003csup\u003e5\u003c/sup\u003e cells per well. After 24 h, cells were stimulated with 50 ng/mL IFN-γ (R\u0026amp;D systems, MN, USA) and 10 ng/mL lipopolysaccharide (LPS, Sigma-Aldrich, MO, USA), followed by being treated with PLGA, PME, PMEZ, and PMEZ/mEV (1\u0026times;10\u003csup\u003e8\u003c/sup\u003e EVs/mL) scaffolds using trans-well inserts system, respectively. Accutase\u0026trade; (Stemcell Technologies, WA, USA) was used to detach the cells after 24 h. The same number of detached cells was fixed with 4% paraformaldehyde for 15 min, blocked with Fc blocking solution (553142; BD bioscience, CA, USA) for 15 min, incubated with antibody solution for, 20 min at 4\u0026deg;C, and measured with flow cytometer (CytoFLEX, Beckman coulter, CA, USA). The primary antibody has been used as follows: PE anti-mouse CD86 (Invitrogen, MA, USA) or FITC anti-mouse CD206 (Invitrogen, MA, USA). The cells were washed to remove excess antibodies, and resuspended in 300 \u0026micro;L of stain buffer (BD Pharmingen, CA, USA), followed by flow cytometer analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.10. Immunocytochemistry (ICC)\u003c/h2\u003e \u003cp\u003eCells were immobilized at room temperature with 4% paraformaldehyde for 15 min and then permeabilized with 0.2% Triton X-100 dissolved in PBS solutions. The fixed cells were immediately incubated overnight with the primary antibodies containing 1% BSA. The antibodies were used as follows: NF-kB (Santa Cruz, TX, USA), fibronectin (Abcam, MA, USA), arginase-1 (Santa Cruz, TX, USA), iNOS (Invitrogen, MA, USA), CD206 (Santa Cruz, Dallas, TX), donkey anti-rabbit Alexa Fluor 488 (Invitrogen, MA, USA), and goat anti-mouse Alexa Fluor 555 (Invitrogen, MA, USA). The nucleus was stained with Hoechst (62249, 1\u0026micro;g/ml, Thermo Fisher Scientific, OH, USA). The ICC images were visualized using a confocal microscopy (LSM880, Carl Zeiss, Jena, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.11. Western blot analysis\u003c/h2\u003e \u003cp\u003eThe same quantity of samples (proteins and EVs) was loaded onto nitrocellulose (NC) membranes for parallel comparisons after being loaded with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A TBST solution diluted in 5% skim milk was used to block the NC membrane. The protein-transferred NC membranes were incubated with primary antibodies against CD81 (Santa Cruz Biotechnology, CA, USA), CD63 (Abcam, MA, USA), and CD9 (Abcam, MA, USA) to confirm EV surface markers and primary antibodies against N-cadherin (13116S, 1000:1, Cell Signaling Technology, MA, USA), fibronectin (ab2413, 500:1, Abcam, MA, USA), and GAPDH (5174T, 1000:1, Cell Signaling Technology, MA, USA) to confirm fibrosis related markers, respectively. After incubation with primary antibodies, HRP-linked secondary antibodies (Cell Signaling Technology, MA, USA) were applied. The enhanced chemiluminescence solution (GE Healthcare, WI, USA) was used to prepare the membrane before being visualized using ChemiDoc\u0026trade; XRS\u0026thinsp;+\u0026thinsp;and ImageLab software (Bio-Rad, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.12. Real-Time quantitative PCR (RT-qPCR) of \u003cem\u003ein vitro\u003c/em\u003e samples\u003c/h2\u003e \u003cp\u003eTotal cellular RNA was extracted using AccuPrep\u0026reg; Universal RNA Extraction Kit (Bioneer, Daejeon, Korea). The PrimeScript\u0026trade; RT reagent kit (Takara, Shiga, Japan) was utilized to perform reverse transcription for extracted RNA to cDNA. A mixture of SYBR green PCR reagents (Applied Biosystems, CA, USA) was used for RT-qPCR. Reactions with the primers (Table S3) were conducted using QuantStudio 3 (Applied Biosystems, CA, USA). The 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method with 18s rRNA as a reference was used for data quantification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.13. The design for \u003cem\u003ein vivo\u003c/em\u003e model\u003c/h2\u003e \u003cp\u003e All animal study protocols were approved by the institutional animal ethics committee of Yeungnam University, College of Medicine (YUMC-AEC2021-024). For \u003cem\u003ein vivo\u003c/em\u003e model, 6-week-old male ICR mouse (total\u0026thinsp;=\u0026thinsp;50) was purchased from Orient Bio (Seongnam, Korea) and randomly divided into five groups (control\u0026thinsp;=\u0026thinsp;3, sham\u0026thinsp;=\u0026thinsp;4, PLGA\u0026thinsp;=\u0026thinsp;6, PMEZ\u0026thinsp;=\u0026thinsp;6, PMEZ/mEV\u0026thinsp;=\u0026thinsp;6). Animals were anesthetized with 16 mg/kg of rompun and 0.04 mg/kg zoletil by intramuscular injection. For the 5/6 nephrectomy mouse model, the left kidney was completely removed and 2/3 of the right kidney was partially resected. The scaffolds were then implanted in the partially resected right kidney. At 2 and 8 weeks after scaffold implantations, animals were sacrificed and kidney tissues were retrieved for subsequent analyzes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e4.14. Histological analysis\u003c/h2\u003e \u003cp\u003eThe collected kidney tissues were fixed in 10% formalin and embedded in paraffin. Tissue paraffin blocks were sectioned into 3\u0026ndash;4 um thickness. Tissue slides were stained with hematoxylin and eosin (H\u0026amp;E; Abcam, MA, USA) for general histopathology, Massons\u0026rsquo;s trichrome (Abcam, MA, USA) for collagen fiber, toluidine blue (VitroView\u0026trade;, MD, USA) for mast cells, and periodic acid Schiff (Sigma-Aldrich) stain for glomerulus visualization using commercial kits following the manufacturer\u0026rsquo;s instruction.\u003c/p\u003e \u003cp\u003eFor immunohistochemistry (IHC), the slides were deparaffinized and hydrated prior to antigen retrieval using Triton-X solution. After blocking, primary antibodies (1:100) were applied for 18 h at 4˚C. FITC conjugated secondary antibody was applied for 2 h at room temperature. The slides were mounted with DAPI staining media (Vector Laboratories, Burlingame, CA, USA) and examined under fluorescence microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e4.15. Real-Time quantitative PCR (RT-qPCR) of \u003cem\u003ein vivo\u003c/em\u003e samples\u003c/h2\u003e \u003cp\u003eRNA was isolated from the collected kidney tissues by using Maxwell\u0026reg; RSC simply RNA cell kit using Maxwell\u0026trade; 16 instrument (Promega Corporation, Madison, WI, USA). Isolated RNA was used in synthesis of cDNA with GoScript \u003csup\u003eTM\u003c/sup\u003e Reverse Tanscription Mix (Promega Corporation, Madison, WI, USA) according to the product protocols. Real-time PCR was performed in StepOnePlus\u0026trade; Real-Time PCR System (Applied Biosystems\u0026reg; Inc., Foster City, CA, USA). Primers for \u003cem\u003ein vivo\u003c/em\u003e analyzes were listed in Table S4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e4.16. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyzes were assessed using the GraphPad Prism 7 program. To determine group differences, unpaired \u003cem\u003et\u003c/em\u003e tests or one-way analysis of variance (ANOVA) with Tukey's multiple comparison post-test were examined. Statistical significance was determined for \u003cem\u003ep\u003c/em\u003e values below 0.05 (*\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; *** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; **** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors listed meet the authorship criteria and significantly contributed to the completion of this work as follows and W.-K.Rhim, J.Woo, and J.Y.Kim equally contributed as co-first authors; W.-K.Rhim: Conceptualization, Data analysis, Resources, Investigation, Writing original draft, Review and editing, Funding acquisition. J.Woo: Data analysis, Methodology, Visualization. J.Y.Kim: Data analysis, Resources, Methodology, Visualization, Writing original draft. E.H.Lee: Data analysis, Resources, Methodology, Visualization. S.-G.Cha: Data analysis, Resources, Methodology, Visualization. D.-S.Kim: Data analysis, Methodology. S.-W.Baek: Conceptualization, Methodology. C.G.Park: Conceptualization,\u0026nbsp;Investigation. B.S.Kim:\u0026nbsp;Conceptualization, Methodology, Investigation, Supervision, Project administration. T.G.Kwon: Conceptualization, Methodology, Investigation, Supervision, Project administration. D.K.Han: Conceptualization, Methodology, Investigation, Supervision, Project administration, Review and edition, Funding acquisition. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2023R1A2C3003807) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2022R1I1A1A01068486). This research was supported by Korean Fund for Regenerative Medicine funded by Ministry of Science and ICT, and Ministry of Health and Welfare (RS-2023-00215369, Republic of Korea).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWebster, A. C., Nagler, E. V., Morton, R. L. \u0026amp; Masson, P. Chronic Kidney Disease. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e389\u003c/strong\u003e, 1238\u0026ndash;1252 (2017).\u003c/li\u003e\n \u003cli\u003eFranco, M. L., Beyerstedt, S. \u0026amp; Rangel, \u0026Eacute;. B. Klotho and mesenchymal stem cells: A review on cell and gene therapy for chronic kidney disease and acute kidney disease. \u003cem\u003ePharmaceutics\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, (2022).\u003c/li\u003e\n \u003cli\u003eLiyanage, T. \u003cem\u003eet al.\u003c/em\u003e Worldwide access to treatment for end-stage kidney disease: A systematic review. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e385\u003c/strong\u003e, 1975\u0026ndash;1982 (2015).\u003c/li\u003e\n \u003cli\u003ePolaschegg, H. D. Red blood cell damage from extracorporeal circulation in hemodialysis. \u003cem\u003eSemin. Dial.\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 524\u0026ndash;531 (2009).\u003c/li\u003e\n \u003cli\u003eQiang, W., Jianchen, W., MacDonald, R., Monga, M. \u0026amp; Wilt, T. J. Antibiotic prophylaxis for transurethral prostatic resection in men with preoperative urine containing less than 100,000 bacteria per ml: A systematic review. \u003cem\u003eJ. Urol.\u003c/em\u003e \u003cstrong\u003e173\u003c/strong\u003e, 1175\u0026ndash;1181 (2005).\u003c/li\u003e\n \u003cli\u003eDo, A. V., Khorsand, B., Geary, S. M. \u0026amp; Salem, A. K. 3D Printing of Scaffolds for Tissue Regeneration Applications. \u003cem\u003eAdv. Healthc. Mater.\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 1742\u0026ndash;1762 (2015).\u003c/li\u003e\n \u003cli\u003eGoodarzi, H., Hashemi-Najafabadi, S., Baheiraei, N. \u0026amp; Bagheri, F. Preparation and Characterization of Nanocomposite Scaffolds (Collagen/\u0026beta;-TCP/SrO) for Bone Tissue Engineering. \u003cem\u003eTissue Eng. Regen. Med.\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 237\u0026ndash;251 (2019).\u003c/li\u003e\n \u003cli\u003eGwon, K., Choi, W. Il, Lee, S., Lee, J. S. \u0026amp; Shin, J. H. Biodegradable hyaluronic acid-based, nitric oxide-releasing nanofibers for potential wound healing applications. \u003cem\u003eBiomater. Sci.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 8160\u0026ndash;8170 (2021).\u003c/li\u003e\n \u003cli\u003eKim, M. S. \u003cem\u003eet al.\u003c/em\u003e Polymeric scaffolds for regenerative medicine. \u003cem\u003ePolym. Rev.\u003c/em\u003e \u003cstrong\u003e51\u003c/strong\u003e, 23\u0026ndash;52 (2011).\u003c/li\u003e\n \u003cli\u003eAli, M. \u0026amp; Payne, S. L. Biomaterial-based cell delivery strategies to promote liver regeneration. \u003cem\u003eBiomater. Res.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 1\u0026ndash;21 (2021).\u003c/li\u003e\n \u003cli\u003ePark, K. S. \u003cem\u003eet al.\u003c/em\u003e Versatile effects of magnesium hydroxide nanoparticles in PLGA scaffold\u0026ndash;mediated chondrogenesis. \u003cem\u003eActa Biomater.\u003c/em\u003e \u003cstrong\u003e73\u003c/strong\u003e, 204\u0026ndash;216 (2018).\u003c/li\u003e\n \u003cli\u003eMa, S. \u003cem\u003eet al.\u003c/em\u003e The pro-inflammatory response of macrophages regulated by acid degradation products of poly(lactide-co-glycolide) nanoparticles. \u003cem\u003eEng. Life Sci.\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 709\u0026ndash;720 (2021).\u003c/li\u003e\n \u003cli\u003eLih, E. \u003cem\u003eet al.\u003c/em\u003e Modified Magnesium Hydroxide Nanoparticles Inhibit the Inflammatory Response to Biodegradable Poly(lactide- co-glycolide) Implants. \u003cem\u003eACS Nano\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 6917\u0026ndash;6925 (2018).\u003c/li\u003e\n \u003cli\u003eCha, M. \u003cem\u003eet al.\u003c/em\u003e Three-dimensional printed polylactic acid scaffold integrated with BMP-2 laden hydrogel for precise bone regeneration. \u003cem\u003eBiomater. Res.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 1\u0026ndash;11 (2021).\u003c/li\u003e\n \u003cli\u003eLiao, J., Guo, X., Grande-Allen, K. J., Kasper, F. K. \u0026amp; Mikos, A. G. Bioactive polymer/extracellular matrix scaffolds fabricated with a flow perfusion bioreactor for cartilage tissue engineering. \u003cem\u003eBiomaterials\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 8911\u0026ndash;8920 (2010).\u003c/li\u003e\n \u003cli\u003eLih, E. \u003cem\u003eet al.\u003c/em\u003e Biomimetic Porous PLGA Scaffolds Incorporating Decellularized Extracellular Matrix for Kidney Tissue Regeneration. \u003cem\u003eACS Appl. Mater. Interfaces\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 21145\u0026ndash;21154 (2016).\u003c/li\u003e\n \u003cli\u003eLih, E. \u003cem\u003eet al.\u003c/em\u003e A Bioinspired Scaffold with Anti-Inflammatory Magnesium Hydroxide and Decellularized Extracellular Matrix for Renal Tissue Regeneration. \u003cem\u003eACS Cent. Sci.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 458\u0026ndash;467 (2019).\u003c/li\u003e\n \u003cli\u003eKim, J. K. \u003cem\u003eet al.\u003c/em\u003e PLGA Microspheres Containing Hydrophobically Modified Magnesium Hydroxide Particles for Acid Neutralization-Mediated Anti-Inflammation. \u003cem\u003eTissue Eng. Regen. Med.\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 613\u0026ndash;622 (2021).\u003c/li\u003e\n \u003cli\u003eKo, K.-W. \u003cem\u003eet al.\u003c/em\u003e Integrated Bioactive Scaffold with Polydeoxyribonucleotide and Stem-Cell-Derived Extracellular Vesicles for Kidney Regeneration. \u003cem\u003eACS Nano\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 7575\u0026ndash;7585 (2021).\u003c/li\u003e\n \u003cli\u003eGuo, X. \u003cem\u003eet al.\u003c/em\u003e Bone regeneration with active angiogenesis by basic fibroblast growth factor gene transfected mesenchymal stem cells seeded on porous \u0026beta;-TCP ceramic scaffolds. \u003cem\u003eBiomed. Mater.\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 93\u0026ndash;99 (2006).\u003c/li\u003e\n \u003cli\u003eCooke, J. P. \u0026amp; Losordo, D. W. Nitric oxide and angiogenesis. \u003cem\u003eCirculation\u003c/em\u003e \u003cstrong\u003e105\u003c/strong\u003e, 2133\u0026ndash;2135 (2002).\u003c/li\u003e\n \u003cli\u003eFukumura, D. \u003cem\u003eet al.\u003c/em\u003e Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e \u003cstrong\u003e98\u003c/strong\u003e, 2604\u0026ndash;2609 (2001).\u003c/li\u003e\n \u003cli\u003eSadrearhami, Z. \u003cem\u003eet al.\u003c/em\u003e Recent advances in nitric oxide delivery for antimicrobial applications using polymer-based systems. \u003cem\u003eJ. Mater. Chem. B\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 2945\u0026ndash;2959 (2018).\u003c/li\u003e\n \u003cli\u003eYang, T., Fruergaard, A. S., Winther, A. K., Zelikin, A. N. \u0026amp; Chandrawati, R. Zinc Oxide Particles Catalytically Generate Nitric Oxide from Endogenous and Exogenous Prodrugs. \u003cem\u003eSmall\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, (2020).\u003c/li\u003e\n \u003cli\u003eChug, M. K., Bachtiar, E., Narwold, N., Gall, K. \u0026amp; Brisbois, E. J. Tailoring nitric oxide release with additive manufacturing to create antimicrobial surfaces. \u003cem\u003eBiomater. Sci.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 3100\u0026ndash;3111 (2021).\u003c/li\u003e\n \u003cli\u003eLe Blanc, K. Mesenchymal stromal cells: Tissue repair and immune modulation. \u003cem\u003eCytotherapy\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 559\u0026ndash;561 (2006).\u003c/li\u003e\n \u003cli\u003eChoi, S. \u003cem\u003eet al.\u003c/em\u003e The role of mesenchymal stem cells in the functional improvement of chronic renal failure. \u003cem\u003eStem Cells Dev.\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 521\u0026ndash;529 (2009).\u003c/li\u003e\n \u003cli\u003ePeired, A. J., Sisti, A. \u0026amp; Romagnani, P. Mesenchymal Stem Cell-Based Therapy for Kidney Disease: A Review of Clinical Evidence. \u003cem\u003eStem Cells Int.\u003c/em\u003e \u003cstrong\u003e2016\u003c/strong\u003e, (2016).\u003c/li\u003e\n \u003cli\u003eWei, L., Fraser, J. L., Lu, Z. Y., Hu, X. \u0026amp; Yu, S. P. Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells enhances angiogenesis and neurogenesis after cerebral ischemia in rats. \u003cem\u003eNeurobiol. Dis.\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e, 635\u0026ndash;645 (2012).\u003c/li\u003e\n \u003cli\u003ePhinney, D. G. \u0026amp; Pittenger, M. F. Concise review: MSC-derived exosomes for cell-free therapy. \u003cem\u003eStem Cells\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 851\u0026ndash;858 (2017).\u003c/li\u003e\n \u003cli\u003eNikfarjam, S., Rezaie, J., Zolbanin, N. M. \u0026amp; Jafari, R. Mesenchymal stem cell derived-exosomes: a modern approach in translational medicine. \u003cem\u003eJ. Transl. Med.\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 1\u0026ndash;21 (2020).\u003c/li\u003e\n \u003cli\u003eWiklander, O. P. B., Brennan, M., L\u0026ouml;tvall, J., Breakefield, X. O. \u0026amp; Andaloussi, S. E. L. Advances in therapeutic applications of extracellular vesicles. \u003cem\u003eSci. Transl. Med.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 1\u0026ndash;16 (2019).\u003c/li\u003e\n \u003cli\u003eKim, J. Y. \u003cem\u003eet al.\u003c/em\u003e Defined MSC exosome with high yield and purity to improve regenerative activity. \u003cem\u003eJ. Tissue Eng.\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, (2021).\u003c/li\u003e\n \u003cli\u003eKim, J. Y. \u003cem\u003eet al.\u003c/em\u003e Comparative Analysis of MSC-Derived Exosomes Depending on Cell Culture Media for Regenerative Bioactivity. \u003cem\u003eTissue Eng. Regen. Med.\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 355\u0026ndash;367 (2021).\u003c/li\u003e\n \u003cli\u003eYea, J. H., Yoon, Y. M., Lee, J. H., Yun, C. W. \u0026amp; Lee, S. H. Exosomes isolated from melatonin-stimulated mesenchymal stem cells improve kidney function by regulating inflammation and fibrosis in a chronic kidney disease mouse model. \u003cem\u003eJ. Tissue Eng.\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, (2021).\u003c/li\u003e\n \u003cli\u003eAouichat, S. \u003cem\u003eet al.\u003c/em\u003e Melatonin improves endoplasmic reticulum stress-mediated ire1\u0026alpha; pathway in z\u0026uuml;cker diabetic fatty rat. \u003cem\u003ePharmaceuticals\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1\u0026ndash;14 (2021).\u003c/li\u003e\n \u003cli\u003eKim, J. Y. \u003cem\u003eet al.\u003c/em\u003e The Upregulation of Regenerative Activity for Extracellular Vesicles with Melatonin Modulation in Chemically Defined Media. (2022).\u003c/li\u003e\n \u003cli\u003eHan, Y. S., Yoon, Y. M., Go, G., Lee, J. H. \u0026amp; Lee, S. H. Melatonin protects human renal proximal tubule epithelial cells against high glucose-mediated fibrosis via the cellular prion protein-tgf-\u0026beta;-smad signaling axis. \u003cem\u003eInt. J. Med. Sci.\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 1235\u0026ndash;1245 (2020).\u003c/li\u003e\n \u003cli\u003eTakhtfooladi, H., Takhtfooladi, M., Moayer, F. \u0026amp; Mobarakeh, S. Melatonin attenuates lung injury in a hind limb ischemia-reperfusion rat model. \u003cem\u003eRev. Port. Pneumol.\u0026nbsp;\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 30\u0026ndash;35 (2015).\u003c/li\u003e\n \u003cli\u003eZhou, L. \u003cem\u003eet al.\u003c/em\u003e Melatonin prevents lung injury induced by hepatic ischemia-reperfusion through anti-inflammatory and anti-apoptosis effects. \u003cem\u003eInt. Immunopharmacol.\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 462\u0026ndash;467 (2015).\u003c/li\u003e\n \u003cli\u003eLi, Z. \u003cem\u003eet al.\u003c/em\u003e Melatonin protects kidney grafts from ischemia/reperfusion injury through inhibition of NF-kB and apoptosis after experimental kidney transplantation. \u003cem\u003eJ. Pineal Res.\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e, 365\u0026ndash;372 (2009).\u003c/li\u003e\n \u003cli\u003eTh\u0026eacute;ry, C. \u003cem\u003eet al.\u003c/em\u003e Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. \u003cem\u003eJ. Extracell. Vesicles\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, (2018).\u003c/li\u003e\n \u003cli\u003eXie, L., Overbeek, P. A. \u0026amp; Reneker, L. W. Ras signaling is essential for lens cell proliferation and lens growth during development. \u003cem\u003eDev. Biol.\u003c/em\u003e \u003cstrong\u003e298\u003c/strong\u003e, 403\u0026ndash;414 (2006).\u003c/li\u003e\n \u003cli\u003eBoettner, B. \u0026amp; Van Aelst, L. Control of cell adhesion dynamics by Rap1 signaling. \u003cem\u003eCurr. Opin. Cell Biol.\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 684\u0026ndash;693 (2009).\u003c/li\u003e\n \u003cli\u003eGaonac\u0026rsquo;h-Lovejoy, V., Boscher, C., Delisle, C. \u0026amp; Gratton, J. P. Rap1 is Involved in Angiopoietin-1-Induced Cell-Cell Junction Stabilization and Endothelial Cell Sprouting. \u003cem\u003eCells\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1\u0026ndash;15 (2020).\u003c/li\u003e\n \u003cli\u003eMelenhorst, W. B. W. H. \u003cem\u003eet al.\u003c/em\u003e Epidermal growth factor receptor signaling in the kidney key roles in physiology and disease. \u003cem\u003eHypertension\u003c/em\u003e \u003cstrong\u003e52\u003c/strong\u003e, 987\u0026ndash;993 (2008).\u003c/li\u003e\n \u003cli\u003eEscuin-Ordinas, H. \u003cem\u003eet al.\u003c/em\u003e Cutaneous wound healing through paradoxical MAPK activation by BRAF inhibitors. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 12348 (2016).\u003c/li\u003e\n \u003cli\u003eMiao, S. \u003cem\u003eet al.\u003c/em\u003e Materials Today Bio A 3D bioprinted nano-laponite hydrogel construct promotes osteogenesis by activating PI3K / AKT signaling pathway. \u003cem\u003eMater. Today Bio\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 100342 (2022).\u003c/li\u003e\n \u003cli\u003eClevers, H., Loh, K. M. \u0026amp; Nusse, R. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control. \u003cem\u003eScience (80-. ).\u003c/em\u003e \u003cstrong\u003e346\u003c/strong\u003e, (2014).\u003c/li\u003e\n \u003cli\u003eVergadi, E., Ieronymaki, E., Lyroni, K., Vaporidi, K. \u0026amp; Tsatsanis, C. Akt Signaling Pathway in Macrophage Activation and M1/M2 Polarization. \u003cem\u003eJ. Immunol.\u003c/em\u003e \u003cstrong\u003e198\u003c/strong\u003e, 1006\u0026ndash;1014 (2017).\u003c/li\u003e\n \u003cli\u003eYang, Y. \u003cem\u003eet al.\u003c/em\u003e Crosstalk between hepatic tumor cells and macrophages via Wnt/\u0026beta;-catenin signaling promotes M2-like macrophage polarization and reinforces tumor malignant behaviors. \u003cem\u003eCell Death Dis.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, (2018).\u003c/li\u003e\n \u003cli\u003eTian, X. \u003cem\u003eet al.\u003c/em\u003e Long noncoding RNA LINC00662 promotes M2 macrophage polarization and hepatocellular carcinoma progression via activating Wnt/\u0026beta;-catenin signaling. \u003cem\u003eMol. Oncol.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 462\u0026ndash;483 (2020).\u003c/li\u003e\n \u003cli\u003eWoo, J., Ko, K. W., Cha, S. G., Heo, Y. \u0026amp; Han, D. K. Comparison of surface functionalization of PLGA composite to immobilize extracellular vesicles. \u003cem\u003ePolymers (Basel).\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, (2021).\u003c/li\u003e\n \u003cli\u003eWolhuter, K. \u0026amp; Eaton, P. How widespread is stable protein S-nitrosylation as an end-effector of protein regulation? \u003cem\u003eFree Radic. Biol. Med.\u003c/em\u003e \u003cstrong\u003e109\u003c/strong\u003e, 156\u0026ndash;166 (2017).\u003c/li\u003e\n \u003cli\u003eGuo, R.-Y. \u003cem\u003eet al.\u003c/em\u003e Bioinspired Design of Reversible Fluorescent Probes for Tracking Nitric Oxide Dynamics in Live Cells. \u003cem\u003eCCS Chem.\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 116\u0026ndash;128 (2021).\u003c/li\u003e\n \u003cli\u003eMelvin, A. C., Jones, W. M., Lutzke, A., Allison, C. L. \u0026amp; Reynolds, M. M. S-Nitrosoglutathione exhibits greater stability than S-nitroso-N-acetylpenicillamine under common laboratory conditions: A comparative stability study. \u003cem\u003eNitric Oxide - Biol. Chem.\u003c/em\u003e \u003cstrong\u003e92\u003c/strong\u003e, 18\u0026ndash;25 (2019).\u003c/li\u003e\n \u003cli\u003eKim, D. S. \u003cem\u003eet al.\u003c/em\u003e Promotion of bone regeneration using bioinspired PLGA/MH/ECM scaffold combined with bioactive PDRN. \u003cem\u003eMaterials (Basel).\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1\u0026ndash;12 (2021).\u003c/li\u003e\n \u003cli\u003eJahani, M. \u003cem\u003eet al.\u003c/em\u003e Regenerative medicine and angiogenesis; Challenges and Opportunities. \u003cem\u003eAdv. Pharm. Bull.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 490\u0026ndash;501 (2020).\u003c/li\u003e\n \u003cli\u003eYamamoto, N. \u003cem\u003eet al.\u003c/em\u003e VEGF and bFGF induction by nitric oxide is associated with hyperbaric oxygen-induced angiogenesis and muscle regeneration. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 1\u0026ndash;13 (2020).\u003c/li\u003e\n \u003cli\u003eTomita, N. \u003cem\u003eet al.\u003c/em\u003e Angiogenic property of hepatocyte growth factor is dependent on upregulation of essential transcription factor for angiogenesis, ets-1. \u003cem\u003eCirculation\u003c/em\u003e \u003cstrong\u003e107\u003c/strong\u003e, 1411\u0026ndash;1417 (2003).\u003c/li\u003e\n \u003cli\u003eGalliot, B., Crescenzi, M., Jacinto, A. \u0026amp; Tajbakhsh, S. Trends in tissue repair and regeneration. \u003cem\u003eDev.\u003c/em\u003e \u003cstrong\u003e144\u003c/strong\u003e, 357\u0026ndash;364 (2017).\u003c/li\u003e\n \u003cli\u003eSheng, L. \u0026amp; Zhuang, S. New Insights Into the Role and Mechanism of Partial Epithelial-Mesenchymal Transition in Kidney Fibrosis. \u003cem\u003eFront. Physiol.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 1\u0026ndash;11 (2020).\u003c/li\u003e\n \u003cli\u003eRedza-Dutordoir, M. \u0026amp; Averill-Bates, D. A. Activation of apoptosis signalling pathways by reactive oxygen species. \u003cem\u003eBiochim. Biophys. Acta - Mol. Cell Res.\u003c/em\u003e \u003cstrong\u003e1863\u003c/strong\u003e, 2977\u0026ndash;2992 (2016).\u003c/li\u003e\n \u003cli\u003eNovak, M. L. \u0026amp; Koh, T. J. Macrophage phenotypes during tissue repair. \u003cem\u003eJ. Leukoc. Biol.\u003c/em\u003e \u003cstrong\u003e93\u003c/strong\u003e, 875\u0026ndash;881 (2013).\u003c/li\u003e\n \u003cli\u003eHou, Y., Li, J., Guan, S. \u0026amp; Witte, F. The therapeutic potential of MSC-EVs as a bioactive material for wound healing. \u003cem\u003eEng. Regen.\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 182\u0026ndash;194 (2021).\u003c/li\u003e\n \u003cli\u003eWang, Y., Branicky, R., No\u0026euml;, A. \u0026amp; Hekimi, S. Superoxide dismutases: Dual roles in controlling ROS damage and regulating ROS signaling. \u003cem\u003eJ. Cell Biol.\u003c/em\u003e \u003cstrong\u003e217\u003c/strong\u003e, 1915\u0026ndash;1928 (2018).\u003c/li\u003e\n \u003cli\u003eSori, R. K. \u003cem\u003eet al.\u003c/em\u003e Evaluation of analgesic activity of sodium valproate and ethanolic extract of Vitex negundo in experimental analgesic models in wistar rats. \u003cem\u003eInt. J. Basic Clin. Pharmacol.\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 2036 (2018).\u003c/li\u003e\n \u003cli\u003eHagmann, H. \u0026amp; Brinkkoetter, P. T. Experimental models to study podocyte biology: Stock-taking the toolbox of glomerular research. \u003cem\u003eFront. Pediatr.\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 1\u0026ndash;9 (2018).\u003c/li\u003e\n \u003cli\u003eYu, N. hee \u003cem\u003eet al.\u003c/em\u003e In Vivo Safety and Regeneration of Long-Term Transported Amniotic Fluid Stem Cells for Renal Regeneration. \u003cem\u003eTissue Eng. Regen. Med.\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 81\u0026ndash;92 (2019).\u003c/li\u003e\n \u003cli\u003eLee, S. B. \u0026amp; Kalluri, R. Mechanistic connection between inflammation and fibrosis. \u003cem\u003eKidney Int.\u003c/em\u003e \u003cstrong\u003e78\u003c/strong\u003e, S22\u0026ndash;S26 (2010).\u003c/li\u003e\n \u003cli\u003eKim, J. Y. \u003cem\u003eet al.\u003c/em\u003e Bolstering the secretion and bioactivities of umbilical cord MSC‑ derived extracellular vesicles with 3D culture and priming in chemically defined media. \u003cem\u003eNano Converg.\u003c/em\u003e (2022) doi:10.1186/s40580-022-00349-z.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"PME scaffold, ZnO, melatonin-modulated extracellular vesicles (mEVs), Chemically defined media, kidney regeneration","lastPublishedDoi":"10.21203/rs.3.rs-2815340/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2815340/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWith prevalence of chronic kidney disease (CKD) in worldwide, the strategies to recover renal function \u003cem\u003evia\u003c/em\u003e tissue regeneration could provide alternatives to kidney replacement therapies. However, due to relatively low reproducibility of renal basal cells and limited bioactivities of implanted biomaterials along with the high probability of substance-inducible inflammation and immunogenicity, kidney tissue regeneration could be challenging. To exclude various side effects from cell transplantations, in this study, we have designed cell-free hybrid PMEZ scaffolds incorporating essential bioactive components, such as ricinoleic acid grafted Mg(OH)\u003csub\u003e2\u003c/sub\u003e (M), extracellular matrix (E), and alpha lipoic acid-conjugated ZnO (Z) based on biodegradable porous PLGA (P) platform. Consecutively, for functional improvements, melatonin-modulated extracellular vesicles (mEVs), derived from the human umbilical cord MSCs in chemically defined media without serum impurities, were also attached onto PMEZ scaffolds to construct the multiplexed PMEZ/mEV scaffold. The continuous nitric oxide-releasing property of modified ZnO and remarkably upregulated regenerative functionalities of mEVs showed significantly enhanced kidney regenerative activities. Based on these, the structural and functional restoration has been practically achieved in 5/6 nephrectomy mouse models that mimicked severe human CKD. Our innovative implantations aim at kidney tissue recovery with functional restoration and could be a promising therapeutic alternative for CKD treatment.\u003c/p\u003e","manuscriptTitle":"Multiplexed PLGA scaffolds with nitric oxide-releasing zinc oxide and melatonin-modulated extracellular vesicles for severe chronic kidney disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-26 17:51:02","doi":"10.21203/rs.3.rs-2815340/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ac4d1731-012e-42ca-885c-97183fc7878f","owner":[],"postedDate":"April 26th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":20950658,"name":"Biological sciences/Biotechnology/Regenerative medicine"},{"id":20950659,"name":"Biological sciences/Biotechnology/Biomaterials/Biomedical materials"}],"tags":[],"updatedAt":"2023-07-03T04:21:00+00:00","versionOfRecord":[],"versionCreatedAt":"2023-04-26 17:51:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2815340","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2815340","identity":"rs-2815340","version":["v1"]},"buildId":"wLkW0s4AflPzk-lpfg-fK","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}