Desktop-stereolithography 3D printing of a decellularized extracellular matrix/mesenchymal stem cell exosome bioink for vaginal reconstruction

Desktop-stereolithography 3D printing of a decellularized extracellular matrix/mesenchymal stem cell exosome bioink for vaginal reconstruction | 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 Research Article Desktop-stereolithography 3D printing of a decellularized extracellular matrix/mesenchymal stem cell exosome bioink for vaginal reconstruction Wenxin Shi, Jiahua Zheng, Jingkun Zhang, Xiaoli Dong, Zhongkang Li, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3203552/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Jun, 2024 Read the published version in Tissue Engineering and Regenerative Medicine → Version 1 posted You are reading this latest preprint version Abstract Abstarct Background 3D-printing is widely used in regenerative medicine and is expected to achieve vaginal morphological restoration and true functional reconstruction.Mesenchymal stem cells-derived exosomes (MSCs-Exos) were applyed in the regeneration of various tissues.The current study aimed to explore the effctive of MSCs-Exos in vaginal reconstruction. Results In this work, hydrogel was designed using decellularized extracellular matrix (dECM) and gelatin methacrylate (GelMA) and silk fibroin (SF).The biological scaffolds was constructed using desktop-stereolithography.The physicochemical properties of the hydrogels were evaluated.It was observed that the sustained release property of exosomes in the hydrogel both in vitro and in vitro.The results revealed that 3D scaffold encapsulating exosomes expressed significant effects on the vascularization and musule regeneration of the regenerative vagina tissue.Also, MSCs-Exos strongly promoted vascularization in the vaginal reconstruction of rats,which may through the PI3K/AKT signaling pathway. Conclusions Our results indicated that the 3D-printed, lumenal scaffold encapsulating exosomes might be used as a cell-free alternative treatment strategy for vaginal reconstruction. Vaginal reconstruction 3D printing Vascularization Decellularized extracellular matrix PI3K/AKT signaling pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Congenital vaginal agenesis is a birth defect that shows a morbidity rate of 1/4000–1/10000[ 1 ]. Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome accounts for the most frequently recorded form, which refers to Mullerian duct aplasia or dysplasia, and causes partial vaginal agenesis[ 2 ]. Vaginoplasty is a traditional way to generate a functional vagina, but it has limitations, such as contracture, necrosis, prolapse, intestinal obstruction, and malignancy. In clinical practice, traditional approaches cannot attain satisfactory results, and thus, novel management strategies need to be developed for vaginal regeneration and reconstruction for treating congenital vaginal aplasia or acquired disorders, such as male-to-female sex surgery, trauma, and gynecological tumor[ 3 ]. Thus, optimal vaginal reconstruction methods need to be developed. In recent years, mesenchymal stem/stromal cells (MSCs) have been used as an alternative to regenerative medicine, as they have several advantages, such as pro-angiogenic, anti-apoptotic, immunomodulatory, and lesser ethical issues. However, several studies have shown that the benefits derived from MSCs are associated with the paracrine action due to the release of extracellular vehicles (EVs), but not with cell transplantation or response at the site of injury[ 4 , 5 , 6 ]. Exosomes can be generated from MSCs through paracrine signaling and can be used for encapsulating proteins, liposomes, miRNAs, and mRNAs. Exosomes have various activities,including cell signaling modulation, immunity,and intercellular communication. As regenerative nano-conveyors, MSC-derived exosomes were previously suggested to exert an effect similar to the corresponding source cells on enhancing angiogenesis, proliferation, and immunomodulation[ 7 , 8 , 9 ]. Additionally, compared to original MSCs, exosomes are easy to store, have high biological stability, can easily perfuse in tissues, and have an immune-privileged status[ 10 ]. Thus, exosomes are better for tissue repair and regeneration. In this study, we determined whether MSC-exosomes might be used as a cell-free-based treatment for tissue engineering and human organ diseases. However, exosomes are easily removed from the body[ 11 ]. Therefore, a good carrier substance is required to load exosomes and continuously release exosomes to achieve the desired tissue regeneration effect. Three-dimensional printed materials are suitable carriers of exosomes. Hence, a protective hydrogel might be used to carry exosomes as they can prevent the degradation of exosomes and act as a persistent reservoir, thus enhancing its clinical efficacy. Tissue engineering technologies and biomaterials are used for vaginal reconstruction, such as reconstruction with decellularized matrices[ 1 ],small intestinal submucosa (SIS)[ 12 ], and the acellular vaginal matrix (AVM)[ 13 ]. The AVM is a novel decellularized matrix that might be used for vaginal reconstruction. It is derived from porcine vaginal tissue and processed to remove antigenic and cellular components for decreasing immune response while retaining the natural ECM skeleton[ 14 ]. There are many peptides in the decellularized ECM, including proteoglycans, fibrin, and collagen. Such peptide-exposed matricryptic sites can enhance cell migration and adhesion[ 15 ]. Gelatin methacryloyl (GelMA) hydrogels have properties similar to those of the native ECM, which facilitate cell proliferation and spread. They are extensively used in different biomedical applications because they have suitable bio-activities and adjustable physical features. To enhance the mechanical properties of the hydrogel, we added silk fibroin. Specifically, we added dECM to GelMA/silk fibroin hydrogels for bioprinting and tested whether this combination might be the ideal procedure for the continuous release of exosomes. In recent years, 3D bioprinting has been used in regenerative medicine for developing tissue models with high specificity based on traditional tissue engineering technologies. The use of 3D bioprinting in tissue regeneration can enhance the properties of regenerated tissue features, such as native morphological and anatomical properties, and porosity[ 16 ]. Three-dimensional printing technology has been widely and successfully used in organ reconstruction and plastic surgery. Typically, 3D-bioprinted tissue implants have been used in various tissues, including the heart[ 17 ], blood vessels[ 18 ], ovarian cells[ 19 ], bladder[ 20 ], ears[ 20 ], bones[ 21 ], skin[ 21 ], and cornea[ 22 ]. Additionally, the effective role of exosomes in regenerative medicine has also been confirmed by many studies, including the regeneration of nerves, heart tissue, bones, cartilage, kidneys, liver, and muscles, and wound healing[ 23 ]. However, studies on the effects of exosomes in vaginal regeneration are limited. Therefore, we developed a degradable biological scaffold, which was composed of dECM and gelatin methcryloyl/silk fibroin (dECM/GS) or dECM and gelatin methcryloyl/silk fibroin/exosomes (dECM/GS-exos). Our findings showed that this 3D printing system might be ideal for vaginal reconstruction. Results Identification of hUCMSCs and hUCMSC-Exos The inoculated MSCs were separated from the human umbilical cord and cultured to form long fusiform cells whose morphological characteristics were similar to fibroblasts. After reaching the 4th − 8th passage, the cells appeared spindle-like (Fig. 1 A). Flow cytometry was conducted to characterize the immunophenotypes of the MSCs using suitable biomarkers. We found that the expression of CD73 (99.61%), CD105 (99.70%), CD44 (99.98%), CD29 (99.90%), and CD90 (99.58%) was high, while the expression of CD45 (1.02%) and HLA-DR (1.5%) was low in the hUCMSCs (Fig. 1 B). Additionally, hUCMSCs were induced to develop different cell lines in the culture system. We found that the hUCMSCs stained positively with Alizarin Red and Oil Red O, which suggested that these cells might be differentiated into multiple lineages (Fig. 1 C and 1 D). The above characterizations met the standards of human MSCs. Next, hUCMSC-Exos were identified following the minimal experimental requirements for defining extracellular vesicles[ 25 ]. We found that the exosomes were cup-shaped (Fig. 1 E), 50–100 nm in size, and had a concentration of 8.69 × 10 10 particles/mL (Fig. 1 F). Additionally, surface markers, including the CD63, ALIX, and TSG101 proteins, were positively stained in exosomes, as determined via western blotting assays (Fig. 1 G). To summarize, exosomes were successfully isolated from hUCMSCs. Characterization of the physicochemical structure of the hydrogel The hydrogel had a typical 3D porous structure, as determined via cryo-scanning electron microscopy (Fig. 2 A). The hydrogel was based on silk fibroin (SF) and gelatin methacrylate (GelMA) generated through sequential polymerization. The structure and biological properties of the hydrogel were tunable. We applied this prepared photocrosslinkable interpenetrating polymer network (IPN) hydrogel to an animal model. The hydrogel maintained the open 3D structure with high porosity, which allowed for nutrient transfer and facilitated continuous reconstruction. It had a low swelling ratio and degradation rate and a high compressive modulus[ 26 ]. The pre-print and post-print bioink morphology were shown in the Fig. 2B1 and 2B2; The appearance of the biostaffold of pre-transplant was shown in Fig. 2B3;The adhesion of the biological scaffold was reflected in Fig. 2 C.The hydrogel rheological properties are shown in Fig. 2 D. The G′ value of the hydrogel was higher than that of G″, and hence, it showed solid-like properties; otherwise, it would exhibit liquid-like properties. After characterizing the hydrogel, we also performed an FT-IR analysis to assess the chemical structure of the hydrogel (Fig. 2 E ). To observe the release of exosomes in the 3D printing scaffold, we placed the scaffold loaded with exosomes in a 24-well plate, added 2 mL of PBS, and then, placed it in an incubator at 37°C. We collected the PBS solution after every two days and added fresh PBS solution. Then, all sample solutions collected on D1, D3, D5, D7, D9, and D11 were analyzed to determine the CD63 contents using a suitable ELISA kit to estimate the content of exosomes at different time points. The release of exosomes in vitro was a continuous process along with the degradation of the scaffold, which required a minimum of 11 days (Fig. 2 F). Additionally, we added the DIR-labeled exosomes to the scaffold and implanted them into the animals. The biological scaffold was removed on days 3, 7, and 10 after implantation. We found that the exosomes could persist in vivo for a long time, as determined by fluorescence imaging (Fig. 2 G, 2 H and 2 G). Exosomes promoted vaginal reconstruction in rats For the histological analysis, H&E and Masson’s trichrome staining were conducted to assess tissue regeneration. We found that hUCMSC-Exos promoted epithelialization, angiogenesis, and muscle regeneration of the vagina, as determined by H&E staining (Fig. 3 ) and Masson’s trichrome staining (Fig. 4 ). Compared to normal vaginal tissue (Fig. 3 A and Fig. 4 A), two weeks after the implantation of the scaffold in the dEGS-Exos group, the regenerated vaginal epithelium was thicker and had more papillary protrusions (Fig. 3 B and 3 C, Fig. 4 B and 4 C). However, four weeks after implantation, the epithelium gradually became thinner and more regular, which was closest to the tissue morphology of the normal vagina. The regenerated vaginal epithelium of the dEGS group showed a slow rate of epithelial regeneration, thin epithelium, and fewer papillary epithelial protrusions.The regenerative vagina in the dEGS-Exos group had more smooth muscle cells, which indicated smooth muscle regeneration, four weeks after implantation, compared to that in the dEGS group (Fig. 3 D and 3 E, Fig. 4 D and 4 E). Eight weeks after implantation, the smooth muscle was thicker and more regular in the dEGS-Exos group, but muscle regeneration was irregular and discontinuous in the other group (Fig. 3 F and 3 G, Fig. 4 F and 4 G). The smooth muscle area was significantly greater in the dEGS-Exos group than in the dEGS group after four weeks and eight weeks of transplantation. Overall, these results indicated that hUCMSC-Exos encapsulated in hydrogel promoted vaginal reconstruction in rats. The dEGS-Exos promoted angiogenesis and muscle regeneration during vaginal reconstruction in rats Four weeks after transplantation, the morphology and thickness of the regenerated vaginal epithelium in the dEGS-Exos group were closer to that of normal vaginal tissue, while the regenerated vagina in the dEGS group showed different thickness and an irregular morphology (Fig. 5,A2-A3). Eight weeks after transplantation, the regenerated epithelial tissue in the dEGS-Exos group was more regular, while the other presented haphazardly arranged epithelial cells (Fig. 5,A6-A7). Angiogenesis plays a key role in tissue regeneration[ 27 ]. The number of blood vessels in the vaginal regenerative tissue of the dEGS-Exos group was significantly higher than that in the vaginal regenerative tissue of the dEGS group (Fig. 5,B2-B5), two weeks and four weeks after transplantation. The number of blood vessels in the regenerated tissue was not significantly different between the groups eight weeks after transplantation(Fig. 5, B6-B7). The new smooth muscle area of the dEGS-Exos group increased considerably four weeks and eight weeks after transplantation, as determined by examining α-SMA via histochemical staining, which indicated the regeneration of smooth muscles. However, muscle regeneration was not significantly different between the groups two weeks after transplantation (Fig. 5, C2-C3).However, there was a significant difference between the two groups at 4 weeks and 8 weeks post-transplantation (Fig. 5, C4-C7). Next, fluorescent double staining of CD31 and α-SMA was performed to evaluate angiogenesis after treatment with dEGS-Exos. The results were similar to those of the histochemistry experiments (Fig. 6 ). We also quantified the regenerated epithelial tissue, angiogenesis, and smooth muscle tissue of different groups at different regeneration periods by performing western blotting assays (Fig. 7 ), (P < 0.05). Activation of the PI3K/AKT pathway for promoting angiogenesis To better understand the mechanism underlying dEGS-Exos group in enhancing graft angiogenesis, pro-angiogenic proteins involved in the PI3K/AKT pathway were analyzed. The levels of p-AKT, p-PI3K, and VEGF were upregulated in the dEGS-Exos group, relative to that in the dEGS group, two weeks and four weeks after transplantation, whereas the levels of total AKT and total PI3K were not significantly different between the groups (Fig. 8 ),(P < 0.05). Additionally, VEGF, p-AKT, and p-PI3K (Fig. 8 ),(P < 0.05) were not significantly different eight weeks after transplantation between the dEGS-Exos and dEGS groups. Overall, we found that exosomes strongly promoted angiogenesis, probably by activating the PI3K/AKT pathway. Discussion In this study, we fabricated a novel 3D-bioprinted vaginal scaffold, using dECM and “bioink” of dECM/Gelma/silk fibroin, and showed that exosomes can promote the reconstruction of the vagina. Our 3D scaffold extended exosome retention and promoted blood flow through neovascularization which facilitated tissue regeneration. We also showed that the scaffold with exosomes was more effective at various stages of the tissue regeneration phase compared to those without exosomes. Finally, our results suggested that exosomes might regulate the PI3K/AKT pathway to facilitate angiogenesis. Exosomes belong to a nano-sized subclass of EVs (40–150 nm). They can be derived from paracrine effects and have been investigated by researchers in the fields of regenerative medicine, nanomedicine, and pharmacology because they are generated by cells [ 28 , 29 ]. They have multiple functions and also influence tissue regeneration. Many researchers have investigated the functions of MSCs and MSC-derived exosomes for vaginal tissue repair[ 30 , 31 ].The transplantation of UC-MSC can have multiple reparative benefits, such as normal vaginal epithelial appearance, decreased fibrosis, and higher neovascularization [ 30 ]. In another study, exosomes derived from stem cells were shown to efficiently enhance angiogenesis and epithelization in rabbit vaginal mucosal defects[ 31 ]. However, studies on the therapeutic efficacy of hUCMSC-derived exosomes (hUCMSC-exos) in vaginal regeneration, especially vaginal reconstruction, are limited. Three-dimensional bioprinting has been successful to different degrees because of its various advantages, such as great control and reproducibility, rapid fabrication, personalized series of products, inexpensive, and easy product modifications at the designated level without any restriction on the spatial arrangement [ 32 ]. By using 3D printing technology, native tissue mimics can be developed using living cells and biomaterials or active factors. Tissue models with high specificity have been prepared in regenerative medicine for improvement based on traditional tissue engineering approaches, using 3D bioprinting methods. Few studies have investigated 3D printing for vaginal tissue reconstruction. Based on the above-mentioned results, we used exosomes along with 3D bioprinting technology for vaginal tissue regeneration and found that the exosomes improved vaginal reconstruction in rats and promoted the revascularization of grafts, which in turn facilitated tissue reconstruction. Our findings confirmed the therapeutic effect of exosomes in vaginal reconstruction and expanded their application in regenerative medicine. Good biomaterials are a key element of 3D printing technology. Biomaterials are natural or synthesized substances or their combinations, which can autonomously substitute and restore tissues in the body if used as a part of the functional system[ 33 ]. The application of biomaterials for vaginal regeneration, such as SIS and hydrogels, was the candidate method for delivering and maintaining the effective acting factor in the target organ[ 30 , 31 ]. However, such biomaterials might not be able to completely mimic the complicated extracellular ecological environment for cell survival[ 34 ]. The ECM can enhance cell differentiation, infiltration, and recruitment with no need for additional growth factors[ 35 ]. Decellularized ECM (dECM), which is found in native tissues and has special and complicated tissue-specific biochemical characteristics, has been extensively investigated in the field of tissue engineering[ 36 ]. The dECM-based bioinks have drawn the attention of researchers for 3D bioprinting [ 37 ], tissue remodeling, and recovery of functions [ 38 , 39 ]. In the dECM synthesized in our study, most cells were removed, and the native tissue composition and structure were preserved [ 13 , 40 ]. The 3D printing scaffold was successfully prepared using the dECM “bioink” that encapsulated bone marrow mesenchymal stem cells (BMSCs). The scaffold prepared in this study was used for vaginal reconstruction [ 40 ]. Additionally, dECM hydrogels promoted cell migration, which matched the results of another study [ 41 ]. Although the bioink showed good biocompatibility, this natural material-derived bioink had poorer mechanical properties than synthesized materials and limited the use of such hydrogels. Adding silk fibroin (SF) improved the composite hydrogel regarding its mechanical performance and thermostability. Additionally, SF combined with gelatin balanced the degradation rate and the mechanical performance for matching the new vaginal tissue. Such a dually-optimized scaffold was suitable for vaginal reconstruction not only for its suitable degradation rate and continuous release of exosomes but also for its desirable mechanical properties for efficient cell recruitment and proliferation. In this study, we applied the dECM-bioink-encapsulating exosomes and printed lumen bioscaffolds, which provided structural support and promoted the recruitment of cells in situ. The 3D scaffold fabricated had a similar structure to that of the body and allowed recreating the complexity of the in-vivo native tissue milieu. Thus, it overcame the limitations related to the 2D culture system and provided structural support between the tissue environment and the cell culture environment [ 42 ]. Several combinations of different biomaterials and exosomes have shown good regenerative effects in the fields of cardiac regeneration, skin regeneration, bone regeneration, cartilage regeneration, and muscle regeneration [ 43 ]. In this study, we analyzed the therapeutic efficacy of hUCMSC-derived exosomes (hUCMSC-exos) combined with biomaterials, such as hydrogels, for vaginal reconstruction. We found that the exosomes promoted vaginal reconstruction, which involved the generation of new vessels and muscles at different periods after scaffold transplantation. Hence, hydrogels with encapsulated MSC-derived exosomes might be used for cell-free treatment and vaginal reconstruction. However, the metabolic pathway of these exosomes is not known, and the mechanisms underlying the role of exosomes in vaginal reconstruction need to be further determined. Vaginal reconstruction is a complicated process that involves various cells, such as epithelial cells, vascular endothelial cells, smooth muscle cells, macrophages, and nerve cells, along with various biochemical factors generated by them. Angiogenesis also promotes regeneration. We found that exosomes promoted the formation of blood vessels in regenerative tissues. This result was similar to that reported by Yang, who found that biomaterial-based exosome therapy can enhance early angiogenesis of diabetic wounds while promoting skin remodeling and wound healing [ 44 ]. The PI3K/AKT pathway promotes angiogenesis. Our results showed that the expression of p-PI3K, p-AKT, and VEGF in the dEGS-Exos group was upregulated, which suggested that the activation of the PI3K/AKT pathway was probably related to the regulation of the effect of exosomes on angiogenesis. We analyzed angiogenesis and its signaling pathways, and we aim to further study the mechanism by which exosomes promote vascularization. The smooth muscle regeneration in the dEGS-Exos group also needs to be further investigated. More studies on effective muscle regeneration and the anatomical structure of vaginal regeneration tissue need to be conducted to fully understand the regeneration of vaginal tissue. Conclusion To summarize, we synthesized hUCMSC-exos and dECM/GS hydrogels via stereolithography for vaginal reconstruction. The dECM/GS hydrogels persistently released exosomes, thus promoting angiogenesis and smooth muscle regeneration. In vivo, these complexes promoted the biological activities of exosomes and, thus, enhanced vaginal tissue regeneration and angiogenesis. The use of exosome-hydrogel composites improved the epithelial regeneration of vaginal tissue, increased angiogenesis, and promoted smooth muscle tissue regeneration. Our findings showed that biological scaffolds loaded with exosomes can be used to persistently release exosomes and other biomolecules. The method described here is a promising cell-free regeneration strategy. Materials and Methods Identification of human umbilical cord mesenchymal stem cells (hUCMSCs) The hUCMSCs in the third passage were purchased from Qilu Cell Therapy Technology (Shandong, China) and grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, US), containing 1% penicillin/streptomycin (Gibco) and 10% exosome-depleted fetal bovine serum (Gibco) at 37°C in the presence of 5% CO 2 . The MSCs were identified following a method described in another study[ 24 ]. First, the morphological characteristics of MSCs were determined under a light microscope (Imager.D2; ZEISS, Germany). Second, osteogenic differentiation was detected via Alizarin Red staining, and adipogenic differentiation was detected via Oil Red O staining. Finally, MSC-associated surface markers (CD44, CD73, CD90, CD105, CD29, CD45, and HLA-DR) (Biolegend, US) were detected via flow cytometry analysis (FCM; FACSCanto II, BD, US). Separation and identification of hUCMSC-Exos After cell passaging was performed 4–8 times, the hUCMSC medium was collected and centrifuged for 15 min at 2,000 g and 4°C for filtering the cells. Next, the collected supernatant was centrifuged for another 30 min at 10,000 g and 4°C for filtering debris, and then, the supernatant was collected and centrifuged for 70 min at 120,000 g and 4°C. After washing with phosphate-buffered saline (PBS; Servicebio, China), the supernatant was centrifuged for 70 min at 120,000 g and 4°C. Next, pre-chilled PBS was added to suspend the precipitate, and then, 3 mg/mL (11.07 × 10 10 particles/mL) of exosomes were isolated, followed by the identification of hUCMSC-Exos[ 25 ]. First, a Flow NanoAnalyzer was used to measure the content and size of the exosomes. Then, transmission electron microscopy (TEM; Talos F200C; Thermo Scientific, US) was conducted to analyze the morphological characteristics of the exosomes. Finally, Western blotting was performed to detect hUCMSC-Exos-related surface markers, such as ALIX, CD63, and TSG101 (Servicebio, China). Synthesis of 3D printed ECM/GS/exosome bioink 1.Synthesis of decellularized extracellular matrix (dECM) hydrogel The dECM was synthesized following a method described in another study [ 13 ]. The dECM was lyophilized and pulverized to produce a powder. Then, a certain percentage of the dECM solution was prepared by digesting 0.1 g of dECM for 24 h with a 0.01 M hydrochloric acid solution (3 mL, pH 2.0), containing 60 mg of pepsin. Following total dissolution, a 10 M NaOH solution (pH 13.0) was added to adjust the pH of the dECM solution from 3.3–3.6 to 7.0–7.2. 2. Synthesis of the hydrogel Initially, 0.5% (w/v) of the photoinitiator was dissolved in tri-distilled water (3 mL) for 30 min at 50°C, and then,GelMA (purchased from Regenovo, China) was added to the solution at 8% (w/v) and mixed with 3 mL of the dECM solution before pasteurization (bioink circulation was performed thrice at 55°C and 4°C, 15 min each time) for producing the 3D bioink working solution. This procedure was conducted in the dark. Finally, the bioink was added to 0.3 g of silk fibroin and mixed thoroughly. As per the experimental requirements, we added an appropriate content of exosomes to the bioink. The original exosome content in the hydrogel was 400 µg/mL. 3 .Three-dimensional printing of the lumen scaffold of the dECM/GS/exosome bioink After the dECM/GS and dECM/GS/exosome hydrogels were prepared, the stereolithography-based 3D printer (Bio-Architect®-WS; Hangzhou Regenovo Bio-technology Ltd.) was used for fabricating the lumen scaffold (the porous grid scaffold). Following the instructions for using a 3D bioprinter, we set up and printed a scaffold that had a tubular 3D structure with a cavity. Using this technology, we produced custom brackets. During printing, dynamic projection stereolithography was used for the biofabrication of the hydrogel scaffold of the dECM/GS/exosome bioink. The bioink was exposed to visible blue light at 11 mW/cm 2 for 30 s to perform quick crosslinking. Rat vaginal reconstruction models All animal experiments were performed following the guidelines of the Ethics Committee of the Second Hospital of Hebei Medical University. First, 8–10-week-old female Sprague–Dawley rats were obtained from the Experimental Animal Center of Hebei Medical University (Shijiazhuang, China). As a part of this experiment, animals were divided randomly into dECM/GS (dEGS) scaffold group and dECM/GS scaffold-Exos (dEGS-Exos) group.We anesthetized rats using pentobarbital, and then, we removed the vaginal tissue of the experimental animals. The 3D-printed biological scaffold, which was supported by a custom-made plastic tube, was implanted. On one end of the plastic tube, we sewed the cervical tissue of the rats. The other end was fixed to the skin of the vulva. During the procedure, we were careful not to damage the urinary catheter and ureters. Then, we administered 70 mg/kg penicillin via intramuscular injection into each rat at regular intervals. After two weeks, four weeks, or eight weeks of implantation, the animals were sacrificed and their regenerated vaginal tissues (n = 6 rats at each time point) were harvested to conduct the ubsequent analyses. Histological analysis - H&E staining and Masson’s trichrome staining Following overnight fixation with 4% paraformaldehyde under ambient temperature, the sample was mixed with 70% ethanol and cultured for 48 h, followed by paraffin embedding. Then, 4-µm cross-sections were made, which were treated with Hematoxylin and Eosin (H&E) and Masson’s trichrome stain to visualize the tissue with the largest diameter. Pathological changes were visualized by performing H&E staining at different time points. A microscope and a digital camera (Zeiss, Germany) were used for taking images (at 40× and 100×). Then, the sections were stained with Masson trichrome (Solarbio, Beijing, China) following specific protocols to detect collagen deposition. Then, the slides were sealed with resin. A Zeiss Axiovert 200 light microscope was used for capturing images (magnification: 40×, 100×, and 200×). Immunohistochemistry Immunohistochemical analysis was conducted on tissue sections probed with rabbit anti-rat CD31 antibody, α-SMA antibody, and CK14 antibody to evaluate vaginal regeneration. After deparaffinization, rehydration, and antigen retrieval through heating in a microwave oven twice, the sections were treated and incubated with 3% H 2 O 2 and 5% goat serum albumin, followed by overnight incubation with primary rabbit anti-rat CD31 antibody (1:75, Abcam, USA), α-SMA antibody (1:200, Abcam, USA), and ck14 antibody (1:300, Zen-bioscience, China) at 4°C. After incubation, the sections were treated with HRP-labeled goat anti-rabbit secondary antibody for 1 h (1:200, Abcam). The DAB kit (ZSGB-BIO, China) was used for visualization. Finally, hematoxylin was used for counterstaining, and a fluorescence microscope (40FL Axioskop, Zeiss) was used for examining regenerated tissues on slides. Immunofluorescence Double-staining was performed using CD31 antibody and α-SMA antibody to analyze the angiogenesis of regenerated tissues in different groups. Immunofluorescence (IF) was then used for detecting the freshly generated endothelial surface marker CD31 along with the smooth muscle cell marker α-SMA. First, a solution containing 0.2% TritonX-100 and 3% goat serum was added to block the sections for 1.5 h. Then, the sections were incubated with anti-CD31 (1:100, Abcam, USA) and α-SMA antibodies (1:200, Abcam, USA) after dilution with the antibody diluent at 4°C overnight. Next, the sections were treated with specific fluorescent-labeled secondary antibodies (1:400, Servicebio, China) for 1.5 h at 37°C. DAPI was added to counterstain nuclei. Finally, CD31-labeled and α-SMA-labeled neovascularization was determined in every sample in five random fields of three discontinuous sections. Immunofluorescent tracking of exosomes Approximately 100 µg of exosomes were added to 50 µL of a working fluid containing 5 µL of DIR and kept for 10 min at 37°C. Next, the labeled exosomes were treated with 10 mL of PBS and extracted using the ultraionization method to remove unbound exosomes. Then, they were thoroughly mixed with “bioinks” for printing. The scaffold was embedded under the skin, and they were removed after 3, 7, and 10 days of embedding, followed by fixation using the optimal cutting temperature (OCT) compound. Finally, they were cut into 5-µm sections in a cryostat, and the distribution of exosomes in the scaffold was observed using fluorophores. Enzyme-linked immunosorbent assay (ELISA) After lysis, an ELISA Kit (Cloud-Clone Corp,China) was used to determine the concentrations of exosomes in PBS, following specific protocols. Briefly, 100 µL of the solution was added to every coated well and incubated for 60 min. After washing thrice with the wash buffer, all wells were further incubated with HRP-labeled antibodies. After washing, a color-developing solution was added to each well at 37°C and incubated for 10 min. Finally, a stop buffer was added, and then, the optical density (OD) was determined at 450 nm. The concentration of exosomes was determined using a standard curve. Western blotting assay To perform the Western blotting assay, first, RIPA lysis buffer (Servicebio, China) containing proteinase/phosphatase inhibitors (Servicebio, China) was used to extract total vaginal tissue RNA. Then, a BCA protein assay kit (Solarbio, China) was used to measure the protein content. Next, protein samples were denatured by heating at 100°C for 10 min, followed by protein separation via 10% SDS-PAGE (Biotides, China). The separated proteins were transferred onto PVDF membranes (Millipore, USA). After blocking with 5% bovine serum albumin (BSA; Sigma, USA) (contained in TBS + 0.1% Tween-20, pH = 7.4) for 1 h, the membranes were incubated using specific primary antibodies, which included CD31 (1:1200, Abcam, USA); α-antinin (1:800, Servicebio, China); CK14 (1:1200), VEGF (1:2000), total AKT (1:1000), p-AKT (1:750), total PI3K (1:1200), p-PI3K (1:1000), and GAPDH (1:1000, Zen-bioscience, China) overnight at 4°C. After washing thrice, the membranes were incubated for 1 h with M5 Goat Anti-Rabbit IgG-HRP (1:10,000, Abcam, China) under ambient temperature. Finally, the ChemiDoc MP Imaging System (Bio-Rad, USA) was used to visualize protein bands, while the Image J software was used for analysis. Statistical analysis The SPSS 21.0 software and GraphPad Prism 8.0 were used for conducting all statistical analyses. The data are presented as the mean ± SD. Two groups of data were compared using Student’s t-test. One-way ANOVA was conducted to evaluate differences among multiple groups. All differences among and between groups were considered to be statistically significant at P < 0.05. Declarations Ethical approval The animal study was reviewed and approved by Institution Animal Ethics Committee of the Second Hospital of Hebei Medical University (Approval Letter No:2023-AE083). All animal experiments were performed in accordance with the ethical guidelines approved by the Animal Care and Research Committee of Hebei Medical University. Consent for publication Not applicable. Availability of data and materials The data that support the findings of this study are available from the corresponding author upon reasonable request. Competing interests There are no conflicts to declare. Funding This study was supported by the Natural Precision Medicine Joint Fund Nurture Project of Hebei Province (H2021206463); the Medical Science Research Plan Project of Hebei Province (20210080) and the Innovative Capacity Improvement Plan Of Hebei Province (20577705D). Author contributions WXS,JHZ and XHH designed the experiments;JKZ performed synthesis and characterization of the scaffolds;XLD,ZKL and YLX created in vivo animal models. WXS and ZKL wrote the manuscript.WXS ,XHH and YFD analyzed the data.WXS ,XHH and YFD supervised experiments.XHH and WXS were responsible for the critical review of the manuscript. All authors contributed to this work, discussed the results, critically reviewed and revised the manuscript. All authors read and approved the final manuscript. Acknowledgements The authors would like to thank Dr. Yanbiao Song for his technical assistance. References Karapinar OS, Ozkan M, Okyay AG, et al. 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Int J Mol Sci. 2021;22(2):830. https://doi.org/10.3390/ijms22020830 . Parisa Khayambashi J, Iyer S, Pillai, et al. Hydrogel Encapsulation of Mesenchymal Stem Cells and Their Derived Exosomes for Tissue Engineering.I. nt J Mol Sci. 2021;22(2):684. https://doi.org/10.3390/ijms22020684 . Jiayi Yang Z, Chen D, Pan, et al. Umbilical Cord-Derived Mesenchymal Stem Cell-Derived Exosomes Combined Pluronic F127 Hydrogel Promote Chronic Diabetic Wound Healing and Complete Skin Regeneration. Int J Nanomedicine. 2020;15:5911–26. https://doi.org/10.2147/IJN.S249129 . Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Scheme.png Scheme 1. Schematic illustration of the one-step operation system for facilitating vagina reconstrusction. (A)Stereolithography-based dECM/GS/exosomes bioprinting and vagina reconstrusction implantation. (B)Migration of cells to the scaffold and controlled administration of exosomes by the 3D printed scaffolds.(C)Vaginal reconstruction. Cite Share Download PDF Status: Published Journal Publication published 27 Jun, 2024 Read the published version in Tissue Engineering and Regenerative Medicine → 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. 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(A) The morphology of hUCMSCs .Scale bar: 200 mm. (B) hUCMSCs were positive for CD73, CD90, CD44, and CD105,CD29 and were negative for CD45 and HLA-DR, as shown by flow cytometry analysis. (C) Alizarin Red staining was used for hUCMSCs osteogenic identification. (D)Oil Red O staining was conducted for adipogenic differentiation. (E) Representative images of hUCMSC-Exos under transmission electron microscopy. Scale bar: 100 nm. (F) Particle size distribution of hUCMSC-Exos was determined by Flow Nano Analyzer. (G) hUCMSC-Exos were positive for ALIX, CD63 and TSG101,which were shown by western blotting.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3203552/v1/149f620c98fb532683b57832.png"},{"id":40883695,"identity":"12b891de-1378-4909-8df7-74675b0d2af4","added_by":"auto","created_at":"2023-08-01 13:57:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1379945,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization and biocompatibility of bioink. (A)Frozen biological scanning electron microscopy.(B)The pre-print and post-print bioink morphology were shown in the figure B1 and B2; B3:Biological scaffold of pre-transplant.(C)The adhesion of the biological scaffold .(D)Rheological properties were measured to evaluate the mechanical properties of the bioink.(E)FI-RT of hydrogels.(F)Observation of exosome releasing in vitro.(D)(H)(I)\u003c/p\u003e\n\u003cp\u003eObservation of exosome releasing in vivo,respectively,3 days,7 days, and 10 days after transplantation in vovo.(DIR-stained exosomes showed red fluorescence) .Scale bar: 100 µm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3203552/v1/ac05f498f5dd7925e3f86c99.png"},{"id":40884573,"identity":"dc051d99-08a6-4c26-b1d0-5d682fbd83e3","added_by":"auto","created_at":"2023-08-01 14:05:14","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":313427,"visible":true,"origin":"","legend":"\u003cp\u003eHistological analysis of reconstructed vagina of H\u0026amp;Estaining. (A)Normal vaginal tissue.(B)Regenerated vaginal tissue in the dEGS sacffold group and (C)Regenerated vaginal tissue in the dEGS sacffold-exo group, both at 2weeks post-transplantation.\u003c/p\u003e\n\u003cp\u003e(D)Regenerated vaginal tissue in the dEGS sacffold and (E)Regenerated vaginal tissue in the dEGS sacffold-exo group, both at 4 weeks post-transplantation.(G)Regenerated vaginal tissue in the dEGS sacffold and (F)Regenerated vaginal tissue in the dEGS sacffold-exo group, both at 8 weeks ost-transplantation.Scale bar: 100 µm\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3203552/v1/8d3d71994c5a032d6bff35c3.jpg"},{"id":40883694,"identity":"abb6fe88-7e01-4f87-a612-a5be4a800e6b","added_by":"auto","created_at":"2023-08-01 13:57:14","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":360577,"visible":true,"origin":"","legend":"\u003cp\u003eHistological analysis of reconstructed vagina of Masson’s trichrome taining.\u003c/p\u003e\n\u003cp\u003e(A)Normal vaginal tissue.(B) (C)Regenerated vaginal tissue in the dEGS sacffold group andin the dEGS sacffold-exo group at 2 weeks post-transplantation.(D) (E)Regenerated\u003c/p\u003e\n\u003cp\u003evaginal tissue in the dEGS sacffold group and in the dEGS sacffold-exo group at 4 weeks post-transplantatio.(G)(F)Regenerated vaginal tissue,in the dEGS sacffold-exo group at 8 weeks post-transplantation.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3203552/v1/c17a10e08ad6f1ffbaed2e29.jpg"},{"id":40886449,"identity":"12248ca7-f5f9-4bf9-9086-979101d50d36","added_by":"auto","created_at":"2023-08-01 14:13:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4811953,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of the vaginal reconstruction in each group on 2 weeks ,4 weeks and 8 weeks.(A)Regeneration of epithelial tissue.(B)Neovascularization.(C)\u003c/p\u003e\n\u003cp\u003eRegeneration of smooth muscle tissue.Respectively ,1-7 represented normal vaginal\u003c/p\u003e\n\u003cp\u003etissue,regenerate vaginal tissue of 2W scaffold group,2W scaffold-exo group,4W scaffold group, 4W scaffold-exo group,8W scaffold group and 8W scaffold-exo group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3203552/v1/6a862f18dd277a4431ae9afc.png"},{"id":40883697,"identity":"c3042826-dcee-4179-9e0b-f8d809fd4648","added_by":"auto","created_at":"2023-08-01 13:57:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1335686,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of revascularization of the vaginal reconstruction (A) blood vessels density in normal vaginal tissue.(B)(C) Angiogenesis shown in representative images of CD31-positive and α-SMA positive vessels in the scaffold group and scaffold-exo group, both at 2 weeks post-transplantation.(D) (E) Angiogenesis shown in representative images in the scaffold and scaffold-exo group,both at 4 weeks post-transplantation.(G)(F)\u003c/p\u003e\n\u003cp\u003eAngiogenesis shown in representative images,respectively in the scaffold and scaffold-exo group, both at 8 weeks post-transplantation.(red:CD31;green: α-SMA)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3203552/v1/2e732f058550808a3d8adc4c.png"},{"id":40883696,"identity":"5ca90bfb-bd1f-4aed-a36a-d446ee5f3fce","added_by":"auto","created_at":"2023-08-01 13:57:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":288981,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of different tissue components of regenerated vaginal tissue (A) Representative CD31 ,α-actinin, and CK14 bands of regenerated vaginal tissue using GAPDH as the internal reference. (B)Quantitative analysis of CD31.(C) Quantitative analysis of α-actinin.(D)Quantitative analysis of CK14.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3203552/v1/bdc0ef3dd1c4d61cb3ace7b0.png"},{"id":40888211,"identity":"31309a1e-ef68-4b74-a02a-67cf740a612c","added_by":"auto","created_at":"2023-08-01 14:21:14","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":335061,"visible":true,"origin":"","legend":"\u003cp\u003ePromoted angiogenesis by regulating PI3K/AKT pathway and secreting\u003c/p\u003e\n\u003cp\u003eproangiogenic factors.(A).Representative VEGF , p-PI3K, PI3K, p-AKT and AKT AKT bands of PI3K/AKT pathway using GAPDH as the internal reference (B)Quantitative analysis of protein expression VEGF.(C)Quantitative analysis of protein expression p-PI3K.(D).Quantitative analysis of protein expression PI3K.(E)Quantitative analysis of protein expression p-AKT.(F)Quantitative analysis of protein expression AKT.(* ,P \u0026lt; 0.05; **, P \u0026lt; 0.01; ***, P \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3203552/v1/93e5ccf143fcaca3beb35c2f.png"},{"id":61346820,"identity":"11b4c6b5-b94e-4847-aac1-d1c20490c8c0","added_by":"auto","created_at":"2024-07-29 18:13:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9268109,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3203552/v1/7103fd57-a8b5-4cc5-a393-2a7b34989ac7.pdf"},{"id":40883693,"identity":"4cd4ab5e-988d-4694-b365-e7023fe981a6","added_by":"auto","created_at":"2023-08-01 13:57:14","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":433928,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1. Schematic illustration of the one-step operation system for facilitating vagina reconstrusction. (A)Stereolithography-based dECM/GS/exosomes bioprinting and vagina reconstrusction implantation. (B)Migration of cells to the scaffold and controlled administration of exosomes by the 3D printed scaffolds.(C)Vaginal reconstruction.\u003c/p\u003e","description":"","filename":"Scheme.png","url":"https://assets-eu.researchsquare.com/files/rs-3203552/v1/e5bf1f52bd8b5dfd2fe6e731.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Desktop-stereolithography 3D printing of a decellularized extracellular matrix/mesenchymal stem cell exosome bioink for vaginal reconstruction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCongenital vaginal agenesis is a birth defect that shows a morbidity rate of 1/4000\u0026ndash;1/10000[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome accounts for the most frequently recorded form, which refers to Mullerian duct aplasia or dysplasia, and causes partial vaginal agenesis[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Vaginoplasty is a traditional way to generate a functional vagina, but it has limitations, such as contracture, necrosis, prolapse, intestinal obstruction, and malignancy. In clinical practice, traditional approaches cannot attain satisfactory results, and thus, novel management strategies need to be developed for vaginal regeneration and reconstruction for treating congenital vaginal aplasia or acquired disorders, such as male-to-female sex surgery, trauma, and gynecological tumor[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Thus, optimal vaginal reconstruction methods need to be developed.\u003c/p\u003e \u003cp\u003eIn recent years, mesenchymal stem/stromal cells (MSCs) have been used as an alternative to regenerative medicine, as they have several advantages, such as pro-angiogenic, anti-apoptotic, immunomodulatory, and lesser ethical issues. However, several studies have shown that the benefits derived from MSCs are associated with the paracrine action due to the release of extracellular vehicles (EVs), but not with cell transplantation or response at the site of injury[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Exosomes can be generated from MSCs through paracrine signaling and can be used for encapsulating proteins, liposomes, miRNAs, and mRNAs. Exosomes have various activities,including cell signaling modulation, immunity,and intercellular communication. As regenerative nano-conveyors, MSC-derived exosomes were previously suggested to exert an effect similar to the corresponding source cells on enhancing angiogenesis, proliferation, and immunomodulation[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Additionally, compared to original MSCs, exosomes are easy to store, have high biological stability, can easily perfuse in tissues, and have an immune-privileged status[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Thus, exosomes are better for tissue repair and regeneration. In this study, we determined whether MSC-exosomes might be used as a cell-free-based treatment for tissue engineering and human organ diseases.\u003c/p\u003e \u003cp\u003eHowever, exosomes are easily removed from the body[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Therefore, a good carrier substance is required to load exosomes and continuously release exosomes to achieve the desired tissue regeneration effect. Three-dimensional printed materials are suitable carriers of exosomes. Hence, a protective hydrogel might be used to carry exosomes as they can prevent the degradation of exosomes and act as a persistent reservoir, thus enhancing its clinical efficacy.\u003c/p\u003e \u003cp\u003eTissue engineering technologies and biomaterials are used for vaginal reconstruction, such as reconstruction with decellularized matrices[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e],small intestinal submucosa (SIS)[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and the acellular vaginal matrix (AVM)[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The AVM is a novel decellularized matrix that might be used for vaginal reconstruction. It is derived from porcine vaginal tissue and processed to remove antigenic and cellular components for decreasing immune response while retaining the natural ECM skeleton[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. There are many peptides in the decellularized ECM, including proteoglycans, fibrin, and collagen. Such peptide-exposed matricryptic sites can enhance cell migration and adhesion[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Gelatin methacryloyl (GelMA) hydrogels have properties similar to those of the native ECM, which facilitate cell proliferation and spread. They are extensively used in different biomedical applications because they have suitable bio-activities and adjustable physical features. To enhance the mechanical properties of the hydrogel, we added silk fibroin. Specifically, we added dECM to GelMA/silk fibroin hydrogels for bioprinting and tested whether this combination might be the ideal procedure for the continuous release of exosomes.\u003c/p\u003e \u003cp\u003eIn recent years, 3D bioprinting has been used in regenerative medicine for developing tissue models with high specificity based on traditional tissue engineering technologies. The use of 3D bioprinting in tissue regeneration can enhance the properties of regenerated tissue features, such as native morphological and anatomical properties, and porosity[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Three-dimensional printing technology has been widely and successfully used in organ reconstruction and plastic surgery. Typically, 3D-bioprinted tissue implants have been used in various tissues, including the heart[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], blood vessels[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], ovarian cells[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], bladder[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], ears[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], bones[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], skin[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and cornea[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Additionally, the effective role of exosomes in regenerative medicine has also been confirmed by many studies, including the regeneration of nerves, heart tissue, bones, cartilage, kidneys, liver, and muscles, and wound healing[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, studies on the effects of exosomes in vaginal regeneration are limited. Therefore, we developed a degradable biological scaffold, which was composed of dECM and gelatin methcryloyl/silk fibroin (dECM/GS) or dECM and gelatin methcryloyl/silk fibroin/exosomes (dECM/GS-exos). Our findings showed that this 3D printing system might be ideal for vaginal reconstruction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eIdentification of hUCMSCs and hUCMSC-Exos\u003c/h2\u003e\n\u003cp\u003eThe inoculated MSCs were separated from the human umbilical cord and cultured to form long fusiform cells whose morphological characteristics were similar to fibroblasts. After reaching the 4th \u0026minus;\u0026thinsp;8th passage, the cells appeared spindle-like (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Flow cytometry was conducted to characterize the immunophenotypes of the MSCs using suitable biomarkers. We found that the expression of CD73 (99.61%), CD105 (99.70%), CD44 (99.98%), CD29 (99.90%), and CD90 (99.58%) was high, while the expression of CD45 (1.02%) and HLA-DR (1.5%) was low in the hUCMSCs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Additionally, hUCMSCs were induced to develop different cell lines in the culture system. We found that the hUCMSCs stained positively with Alizarin Red and Oil Red O, which suggested that these cells might be differentiated into multiple lineages (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). The above characterizations met the standards of human MSCs. Next, hUCMSC-Exos were identified following the minimal experimental requirements for defining extracellular vesicles[\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. We found that the exosomes were cup-shaped (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE), 50\u0026ndash;100 nm in size, and had a concentration of 8.69 \u0026times; 10\u003csup\u003e10\u003c/sup\u003e particles/mL (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF). Additionally, surface markers, including the CD63, ALIX, and TSG101 proteins, were positively stained in exosomes, as determined via western blotting assays (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG). To summarize, exosomes were successfully isolated from hUCMSCs.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eCharacterization of the physicochemical structure of the hydrogel\u003c/h2\u003e\n\u003cp\u003eThe hydrogel had a typical 3D porous structure, as determined via cryo-scanning electron microscopy (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). The hydrogel was based on silk fibroin (SF) and gelatin methacrylate (GelMA) generated through sequential polymerization. The structure and biological properties of the hydrogel were tunable. We applied this prepared photocrosslinkable interpenetrating polymer network (IPN) hydrogel to an animal model. The hydrogel maintained the open 3D structure with high porosity, which allowed for nutrient transfer and facilitated continuous reconstruction. It had a low swelling ratio and degradation rate and a high compressive modulus[\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. The pre-print and post-print bioink morphology were shown in the Fig.\u0026nbsp;2B1 and 2B2; The appearance of the biostaffold of pre-transplant was shown in Fig.\u0026nbsp;2B3;The adhesion of the biological scaffold was reflected in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC.The hydrogel rheological properties are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD. The G\u0026prime; value of the hydrogel was higher than that of G\u0026Prime;, and hence, it showed solid-like properties; otherwise, it would exhibit liquid-like properties. After characterizing the hydrogel, we also performed an FT-IR analysis to assess the chemical structure of the hydrogel (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE ).\u003c/p\u003e\n\u003cp\u003eTo observe the release of exosomes in the 3D printing scaffold, we placed the scaffold loaded with exosomes in a 24-well plate, added 2 mL of PBS, and then, placed it in an incubator at 37\u0026deg;C. We collected the PBS solution after every two days and added fresh PBS solution. Then, all sample solutions collected on D1, D3, D5, D7, D9, and D11 were analyzed to determine the CD63 contents using a suitable ELISA kit to estimate the content of exosomes at different time points. The release of exosomes in vitro was a continuous process along with the degradation of the scaffold, which required a minimum of 11 days (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF). Additionally, we added the DIR-labeled exosomes to the scaffold and implanted them into the animals. The biological scaffold was removed on days 3, 7, and 10 after implantation. We found that the exosomes could persist in vivo for a long time, as determined by fluorescence imaging (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG,\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eExosomes promoted vaginal reconstruction in rats\u003c/h2\u003e\n\u003cp\u003eFor the histological analysis, H\u0026amp;E and Masson\u0026rsquo;s trichrome staining were conducted to assess tissue regeneration. We found that hUCMSC-Exos promoted epithelialization, angiogenesis, and muscle regeneration of the vagina, as determined by H\u0026amp;E staining (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e ) and Masson\u0026rsquo;s trichrome staining (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e ). Compared to normal vaginal tissue (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA), two weeks after the implantation of the scaffold in the dEGS-Exos group, the regenerated vaginal epithelium was thicker and had more papillary protrusions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). However, four weeks after implantation, the epithelium gradually became thinner and more regular, which was closest to the tissue morphology of the normal vagina. The regenerated vaginal epithelium of the dEGS group showed a slow rate of epithelial regeneration, thin epithelium, and fewer papillary epithelial protrusions.The regenerative vagina in the dEGS-Exos group had more smooth muscle cells, which indicated smooth muscle regeneration, four weeks after implantation, compared to that in the dEGS group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE). Eight weeks after implantation, the smooth muscle was thicker and more regular in the dEGS-Exos group, but muscle regeneration was irregular and discontinuous in the other group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG). The smooth muscle area was significantly greater in the dEGS-Exos group than in the dEGS group after four weeks and eight weeks of transplantation. Overall, these results indicated that hUCMSC-Exos encapsulated in hydrogel promoted vaginal reconstruction in rats.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eThe dEGS-Exos promoted angiogenesis and muscle regeneration during vaginal reconstruction in rats\u003c/h2\u003e\n\u003cp\u003eFour weeks after transplantation, the morphology and thickness of the regenerated vaginal epithelium in the dEGS-Exos group were closer to that of normal vaginal tissue, while the regenerated vagina in the dEGS group showed different thickness and an irregular morphology (Fig.\u0026nbsp;5,A2-A3). Eight weeks after transplantation, the regenerated epithelial tissue in the dEGS-Exos group was more regular, while the other presented haphazardly arranged epithelial cells (Fig.\u0026nbsp;5,A6-A7). Angiogenesis plays a key role in tissue regeneration[\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. The number of blood vessels in the vaginal regenerative tissue of the dEGS-Exos group was significantly higher than that in the vaginal regenerative tissue of the dEGS group (Fig.\u0026nbsp;5,B2-B5), two weeks and four weeks after transplantation. The number of blood vessels in the regenerated tissue was not significantly different between the groups eight weeks after transplantation(Fig.\u0026nbsp;5, B6-B7). The new smooth muscle area of the dEGS-Exos group increased considerably four weeks and eight weeks after transplantation, as determined by examining \u0026alpha;-SMA via histochemical staining, which indicated the regeneration of smooth muscles. However, muscle regeneration was not significantly different between the groups two weeks after transplantation (Fig.\u0026nbsp;5, C2-C3).However, there was a significant difference between the two groups at 4 weeks and 8 weeks post-transplantation (Fig.\u0026nbsp;5, C4-C7). Next, fluorescent double staining of CD31 and \u0026alpha;-SMA was performed to evaluate angiogenesis after treatment with dEGS-Exos. The results were similar to those of the histochemistry experiments (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). We also quantified the regenerated epithelial tissue, angiogenesis, and smooth muscle tissue of different groups at different regeneration periods by performing western blotting assays (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e), (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eActivation of the PI3K/AKT pathway for promoting angiogenesis\u003c/h2\u003e\n\u003cp\u003eTo better understand the mechanism underlying dEGS-Exos group in enhancing graft angiogenesis, pro-angiogenic proteins involved in the PI3K/AKT pathway were analyzed. The levels of p-AKT, p-PI3K, and VEGF were upregulated in the dEGS-Exos group, relative to that in the dEGS group, two weeks and four weeks after transplantation, whereas the levels of total AKT and total PI3K were not significantly different between the groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e),(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, VEGF, p-AKT, and p-PI3K (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e),(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were not significantly different eight weeks after transplantation between the dEGS-Exos and dEGS groups. Overall, we found that exosomes strongly promoted angiogenesis, probably by activating the PI3K/AKT pathway.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we fabricated a novel 3D-bioprinted vaginal scaffold, using dECM and \u0026ldquo;bioink\u0026rdquo; of dECM/Gelma/silk fibroin, and showed that exosomes can promote the reconstruction of the vagina. Our 3D scaffold extended exosome retention and promoted blood flow through neovascularization which facilitated tissue regeneration. We also showed that the scaffold with exosomes was more effective at various stages of the tissue regeneration phase compared to those without exosomes. Finally, our results suggested that exosomes might regulate the PI3K/AKT pathway to facilitate angiogenesis.\u003c/p\u003e \u003cp\u003eExosomes belong to a nano-sized subclass of EVs (40\u0026ndash;150 nm). They can be derived from paracrine effects and have been investigated by researchers in the fields of regenerative medicine, nanomedicine, and pharmacology because they are generated by cells [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. They have multiple functions and also influence tissue regeneration. Many researchers have investigated the functions of MSCs and MSC-derived exosomes for vaginal tissue repair[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].The transplantation of UC-MSC can have multiple reparative benefits, such as normal vaginal epithelial appearance, decreased fibrosis, and higher neovascularization [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In another study, exosomes derived from stem cells were shown to efficiently enhance angiogenesis and epithelization in rabbit vaginal mucosal defects[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, studies on the therapeutic efficacy of hUCMSC-derived exosomes (hUCMSC-exos) in vaginal regeneration, especially vaginal reconstruction, are limited.\u003c/p\u003e \u003cp\u003eThree-dimensional bioprinting has been successful to different degrees because of its various advantages, such as great control and reproducibility, rapid fabrication, personalized series of products, inexpensive, and easy product modifications at the designated level without any restriction on the spatial arrangement [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. By using 3D printing technology, native tissue mimics can be developed using living cells and biomaterials or active factors. Tissue models with high specificity have been prepared in regenerative medicine for improvement based on traditional tissue engineering approaches, using 3D bioprinting methods. Few studies have investigated 3D printing for vaginal tissue reconstruction.\u003c/p\u003e \u003cp\u003eBased on the above-mentioned results, we used exosomes along with 3D bioprinting technology for vaginal tissue regeneration and found that the exosomes improved vaginal reconstruction in rats and promoted the revascularization of grafts, which in turn facilitated tissue reconstruction. Our findings confirmed the therapeutic effect of exosomes in vaginal reconstruction and expanded their application in regenerative medicine.\u003c/p\u003e \u003cp\u003eGood biomaterials are a key element of 3D printing technology. Biomaterials are natural or synthesized substances or their combinations, which can autonomously substitute and restore tissues in the body if used as a part of the functional system[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The application of biomaterials for vaginal regeneration, such as SIS and hydrogels, was the candidate method for delivering and maintaining the effective acting factor in the target organ[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, such biomaterials might not be able to completely mimic the complicated extracellular ecological environment for cell survival[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The ECM can enhance cell differentiation, infiltration, and recruitment with no need for additional growth factors[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Decellularized ECM (dECM), which is found in native tissues and has special and complicated tissue-specific biochemical characteristics, has been extensively investigated in the field of tissue engineering[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The dECM-based bioinks have drawn the attention of researchers for 3D bioprinting [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], tissue remodeling, and recovery of functions [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In the dECM synthesized in our study, most cells were removed, and the native tissue composition and structure were preserved [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The 3D printing scaffold was successfully prepared using the dECM \u0026ldquo;bioink\u0026rdquo; that encapsulated bone marrow mesenchymal stem cells (BMSCs). The scaffold prepared in this study was used for vaginal reconstruction [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Additionally, dECM hydrogels promoted cell migration, which matched the results of another study [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough the bioink showed good biocompatibility, this natural material-derived bioink had poorer mechanical properties than synthesized materials and limited the use of such hydrogels. Adding silk fibroin (SF) improved the composite hydrogel regarding its mechanical performance and thermostability. Additionally, SF combined with gelatin balanced the degradation rate and the mechanical performance for matching the new vaginal tissue. Such a dually-optimized scaffold was suitable for vaginal reconstruction not only for its suitable degradation rate and continuous release of exosomes but also for its desirable mechanical properties for efficient cell recruitment and proliferation.\u003c/p\u003e \u003cp\u003eIn this study, we applied the dECM-bioink-encapsulating exosomes and printed lumen bioscaffolds, which provided structural support and promoted the recruitment of cells in situ. The 3D scaffold fabricated had a similar structure to that of the body and allowed recreating the complexity of the \u003cem\u003ein-vivo\u003c/em\u003e native tissue milieu. Thus, it overcame the limitations related to the 2D culture system and provided structural support between the tissue environment and the cell culture environment [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral combinations of different biomaterials and exosomes have shown good regenerative effects in the fields of cardiac regeneration, skin regeneration, bone regeneration, cartilage regeneration, and muscle regeneration [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In this study, we analyzed the therapeutic efficacy of hUCMSC-derived exosomes (hUCMSC-exos) combined with biomaterials, such as hydrogels, for vaginal reconstruction. We found that the exosomes promoted vaginal reconstruction, which involved the generation of new vessels and muscles at different periods after scaffold transplantation. Hence, hydrogels with encapsulated MSC-derived exosomes might be used for cell-free treatment and vaginal reconstruction. However, the metabolic pathway of these exosomes is not known, and the mechanisms underlying the role of exosomes in vaginal reconstruction need to be further determined.\u003c/p\u003e \u003cp\u003eVaginal reconstruction is a complicated process that involves various cells, such as epithelial cells, vascular endothelial cells, smooth muscle cells, macrophages, and nerve cells, along with various biochemical factors generated by them. Angiogenesis also promotes regeneration. We found that exosomes promoted the formation of blood vessels in regenerative tissues. This result was similar to that reported by Yang, who found that biomaterial-based exosome therapy can enhance early angiogenesis of diabetic wounds while promoting skin remodeling and wound healing [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The PI3K/AKT pathway promotes angiogenesis. Our results showed that the expression of p-PI3K, p-AKT, and VEGF in the dEGS-Exos group was upregulated, which suggested that the activation of the PI3K/AKT pathway was probably related to the regulation of the effect of exosomes on angiogenesis. We analyzed angiogenesis and its signaling pathways, and we aim to further study the mechanism by which exosomes promote vascularization. The smooth muscle regeneration in the dEGS-Exos group also needs to be further investigated. More studies on effective muscle regeneration and the anatomical structure of vaginal regeneration tissue need to be conducted to fully understand the regeneration of vaginal tissue.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTo summarize, we synthesized hUCMSC-exos and dECM/GS hydrogels via stereolithography for vaginal reconstruction. The dECM/GS hydrogels persistently released exosomes, thus promoting angiogenesis and smooth muscle regeneration. In vivo, these complexes promoted the biological activities of exosomes and, thus, enhanced vaginal tissue regeneration and angiogenesis. The use of exosome-hydrogel composites improved the epithelial regeneration of vaginal tissue, increased angiogenesis, and promoted smooth muscle tissue regeneration. Our findings showed that biological scaffolds loaded with exosomes can be used to persistently release exosomes and other biomolecules. The method described here is a promising cell-free regeneration strategy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eIdentification of human umbilical cord mesenchymal stem cells (hUCMSCs)\u003c/p\u003e \u003cp\u003eThe hUCMSCs in the third passage were purchased from Qilu Cell Therapy Technology (Shandong, China) and grown in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM; Gibco, US), containing 1% penicillin/streptomycin (Gibco) and 10% exosome-depleted fetal bovine serum (Gibco) at 37\u0026deg;C in the presence of 5% CO\u003csub\u003e2\u003c/sub\u003e. The MSCs were identified following a method described in another study[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. First, the morphological characteristics of MSCs were determined under a light microscope (Imager.D2; ZEISS, Germany). Second, osteogenic differentiation was detected via Alizarin Red staining, and adipogenic differentiation was detected via Oil Red O staining. Finally, MSC-associated surface markers (CD44, CD73, CD90, CD105, CD29, CD45, and HLA-DR) (Biolegend, US) were detected via flow cytometry analysis (FCM; FACSCanto II, BD, US).\u003c/p\u003e \u003cp\u003eSeparation and identification of hUCMSC-Exos\u003c/p\u003e \u003cp\u003eAfter cell passaging was performed 4\u0026ndash;8 times, the hUCMSC medium was collected and centrifuged for 15 min at 2,000 \u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C for filtering the cells. Next, the collected supernatant was centrifuged for another 30 min at 10,000 \u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C for filtering debris, and then, the supernatant was collected and centrifuged for 70 min at 120,000 \u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C. After washing with phosphate-buffered saline (PBS; Servicebio, China), the supernatant was centrifuged for 70 min at 120,000 \u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C. Next, pre-chilled PBS was added to suspend the precipitate, and then, 3 mg/mL (11.07 \u0026times; 10\u003csup\u003e10\u003c/sup\u003e particles/mL) of exosomes were isolated, followed by the identification of hUCMSC-Exos[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. First, a Flow NanoAnalyzer was used to measure the content and size of the exosomes. Then, transmission electron microscopy (TEM; Talos F200C; Thermo Scientific, US) was conducted to analyze the morphological characteristics of the exosomes. Finally, Western blotting was performed to detect hUCMSC-Exos-related surface markers, such as ALIX, CD63, and TSG101 (Servicebio, China).\u003c/p\u003e \u003cp\u003eSynthesis of 3D printed ECM/GS/exosome bioink\u003c/p\u003e \u003cp\u003e1.Synthesis of decellularized extracellular matrix (dECM) hydrogel\u003c/p\u003e \u003cp\u003eThe dECM was synthesized following a method described in another study [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The dECM was lyophilized and pulverized to produce a powder. Then, a certain percentage of the dECM solution was prepared by digesting 0.1 g of dECM for 24 h with a 0.01 M hydrochloric acid solution (3 mL, pH 2.0), containing 60 mg of pepsin. Following total dissolution, a 10 M NaOH solution (pH 13.0) was added to adjust the pH of the dECM solution from 3.3\u0026ndash;3.6 to 7.0\u0026ndash;7.2.\u003c/p\u003e \u003cp\u003e2. Synthesis of the hydrogel\u003c/p\u003e \u003cp\u003eInitially, 0.5% (w/v) of the photoinitiator was dissolved in tri-distilled water (3 mL) for 30 min at 50\u0026deg;C, and then,GelMA (purchased from Regenovo, China) was added to the solution at 8% (w/v) and mixed with 3 mL of the dECM solution before pasteurization (bioink circulation was performed thrice at 55\u0026deg;C and 4\u0026deg;C, 15 min each time) for producing the 3D bioink working solution. This procedure was conducted in the dark. Finally, the bioink was added to 0.3 g of silk fibroin and mixed thoroughly. As per the experimental requirements, we added an appropriate content of exosomes to the bioink. The original exosome content in the hydrogel was 400 \u0026micro;g/mL.\u003c/p\u003e \u003cp\u003e3 .Three-dimensional printing of the lumen scaffold of the dECM/GS/exosome bioink\u003c/p\u003e \u003cp\u003eAfter the dECM/GS and dECM/GS/exosome hydrogels were prepared, the stereolithography-based 3D printer (Bio-Architect\u0026reg;-WS; Hangzhou Regenovo Bio-technology Ltd.) was used for fabricating the lumen scaffold (the porous grid scaffold). Following the instructions for using a 3D bioprinter, we set up and printed a scaffold that had a tubular 3D structure with a cavity. Using this technology, we produced custom brackets. During printing, dynamic projection stereolithography was used for the biofabrication of the hydrogel scaffold of the dECM/GS/exosome bioink. The bioink was exposed to visible blue light at 11 mW/cm\u003csup\u003e2\u003c/sup\u003e for 30 s to perform quick crosslinking.\u003c/p\u003e \u003cp\u003eRat vaginal reconstruction models\u003c/p\u003e \u003cp\u003e All animal experiments were performed following the guidelines of the Ethics Committee of the Second Hospital of Hebei Medical University. First, 8\u0026ndash;10-week-old female Sprague\u0026ndash;Dawley rats were obtained from the Experimental Animal Center of Hebei Medical University (Shijiazhuang, China). As a part of this experiment,\u003c/p\u003e \u003cp\u003eanimals were divided randomly into dECM/GS (dEGS) scaffold group and dECM/GS scaffold-Exos (dEGS-Exos) group.We anesthetized rats using pentobarbital, and then, we removed the vaginal tissue of the experimental animals. The 3D-printed biological scaffold, which was supported by a custom-made plastic tube, was implanted. On one end of the plastic tube, we sewed the cervical tissue of the rats. The other end was fixed to the skin of the vulva. During the procedure, we were careful not to damage the urinary catheter and ureters. Then, we administered 70 mg/kg penicillin via intramuscular injection into each rat at regular intervals. After two weeks, four weeks, or eight weeks of implantation, the animals were sacrificed and their regenerated vaginal tissues (n\u0026thinsp;=\u0026thinsp;6 rats at each time point) were harvested to conduct the ubsequent analyses.\u003c/p\u003e \u003cp\u003eHistological analysis - H\u0026amp;E staining and Masson\u0026rsquo;s trichrome staining\u003c/p\u003e \u003cp\u003eFollowing overnight fixation with 4% paraformaldehyde under ambient temperature, the sample was mixed with 70% ethanol and cultured for 48 h, followed by paraffin embedding. Then, 4-\u0026micro;m cross-sections were made, which were treated with Hematoxylin and Eosin (H\u0026amp;E) and Masson\u0026rsquo;s trichrome stain to visualize the tissue with the largest diameter. Pathological changes were visualized by performing H\u0026amp;E staining at different time points. A microscope and a digital camera (Zeiss, Germany) were used for taking images (at 40\u0026times; and 100\u0026times;). Then, the sections were stained with Masson trichrome (Solarbio, Beijing, China) following specific protocols to detect collagen deposition. Then, the slides were sealed with resin. A Zeiss Axiovert 200 light microscope was used for capturing images (magnification: 40\u0026times;, 100\u0026times;, and 200\u0026times;).\u003c/p\u003e \u003cp\u003eImmunohistochemistry\u003c/p\u003e \u003cp\u003eImmunohistochemical analysis was conducted on tissue sections probed with rabbit anti-rat CD31 antibody, α-SMA antibody, and CK14 antibody to evaluate vaginal regeneration. After deparaffinization, rehydration, and antigen retrieval through heating in a microwave oven twice, the sections were treated and incubated with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 5% goat serum albumin, followed by overnight incubation with primary rabbit anti-rat CD31 antibody (1:75, Abcam, USA), α-SMA antibody (1:200, Abcam, USA), and ck14 antibody (1:300, Zen-bioscience, China) at 4\u0026deg;C. After incubation, the sections were treated with HRP-labeled goat anti-rabbit secondary antibody for 1 h (1:200, Abcam). The DAB kit (ZSGB-BIO, China) was used for visualization. Finally, hematoxylin was used for counterstaining, and a fluorescence microscope (40FL Axioskop, Zeiss) was used for examining regenerated tissues on slides.\u003c/p\u003e \u003cp\u003eImmunofluorescence\u003c/p\u003e \u003cp\u003eDouble-staining was performed using CD31 antibody and α-SMA antibody to analyze the angiogenesis of regenerated tissues in different groups. Immunofluorescence (IF) was then used for detecting the freshly generated endothelial surface marker CD31 along with the smooth muscle cell marker α-SMA. First, a solution containing 0.2% TritonX-100 and 3% goat serum was added to block the sections for 1.5 h. Then, the sections were incubated with anti-CD31 (1:100, Abcam, USA) and α-SMA antibodies (1:200, Abcam, USA) after dilution with the antibody diluent at 4\u0026deg;C overnight. Next, the sections were treated with specific fluorescent-labeled secondary antibodies (1:400, Servicebio, China) for 1.5 h at 37\u0026deg;C. DAPI was added to counterstain nuclei. Finally, CD31-labeled and α-SMA-labeled neovascularization was determined in every sample in five random fields of three discontinuous sections.\u003c/p\u003e \u003cp\u003eImmunofluorescent tracking of exosomes\u003c/p\u003e \u003cp\u003eApproximately 100 \u0026micro;g of exosomes were added to 50 \u0026micro;L of a working fluid containing 5 \u0026micro;L of DIR and kept for 10 min at 37\u0026deg;C. Next, the labeled exosomes were treated with 10 mL of PBS and extracted using the ultraionization method to remove unbound exosomes. Then, they were thoroughly mixed with \u0026ldquo;bioinks\u0026rdquo; for printing. The scaffold was embedded under the skin, and they were removed after 3, 7, and 10 days of embedding, followed by fixation using the optimal cutting temperature (OCT) compound. Finally, they were cut into 5-\u0026micro;m sections in a cryostat, and the distribution of exosomes in the scaffold was observed using fluorophores.\u003c/p\u003e \u003cp\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/p\u003e \u003cp\u003eAfter lysis, an ELISA Kit (Cloud-Clone Corp,China) was used to determine the concentrations of exosomes in PBS, following specific protocols. Briefly, 100 \u0026micro;L of the solution was added to every coated well and incubated for 60 min. After washing thrice with the wash buffer, all wells were further incubated with HRP-labeled antibodies. After washing, a color-developing solution was added to each well at 37\u0026deg;C and incubated for 10 min. Finally, a stop buffer was added, and then, the optical density (OD) was determined at 450 nm. The concentration of exosomes was determined using a standard curve.\u003c/p\u003e \u003cp\u003eWestern blotting assay\u003c/p\u003e \u003cp\u003eTo perform the Western blotting assay, first, RIPA lysis buffer (Servicebio, China) containing proteinase/phosphatase inhibitors (Servicebio, China) was used to extract total vaginal tissue RNA. Then, a BCA protein assay kit (Solarbio, China) was used to measure the protein content. Next, protein samples were denatured by heating at 100\u0026deg;C for 10 min, followed by protein separation via 10% SDS-PAGE (Biotides, China). The separated proteins were transferred onto PVDF membranes (Millipore, USA). After blocking with 5% bovine serum albumin (BSA; Sigma, USA) (contained in TBS\u0026thinsp;+\u0026thinsp;0.1% Tween-20, pH\u0026thinsp;=\u0026thinsp;7.4) for 1 h, the membranes were incubated using specific primary antibodies, which included CD31 (1:1200, Abcam, USA); α-antinin\u003c/p\u003e \u003cp\u003e(1:800, Servicebio, China); CK14 (1:1200), VEGF (1:2000), total AKT (1:1000), p-AKT (1:750), total PI3K (1:1200), p-PI3K (1:1000), and GAPDH (1:1000, Zen-bioscience, China) overnight at 4\u0026deg;C. After washing thrice, the membranes were incubated for 1 h with M5 Goat Anti-Rabbit IgG-HRP (1:10,000, Abcam, China) under ambient temperature. Finally, the ChemiDoc MP Imaging System (Bio-Rad, USA) was used to visualize protein bands, while the Image J software was used for analysis.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe SPSS 21.0 software and GraphPad Prism 8.0 were used for conducting all statistical analyses. The data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Two groups of data were compared using Student\u0026rsquo;s t-test. One-way ANOVA was conducted to evaluate differences among multiple groups. All differences among and between groups were considered to be statistically significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study was reviewed and approved by Institution Animal Ethics Committee of the Second Hospital of Hebei Medical University (Approval Letter No:2023-AE083). All animal experiments were performed in accordance with the ethical guidelines approved by the Animal Care and Research Committee of Hebei Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no conflicts to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Natural Precision Medicine Joint Fund Nurture Project of Hebei Province (H2021206463); the Medical Science Research Plan Project of Hebei Province (20210080) and the Innovative Capacity Improvement Plan Of Hebei Province (20577705D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWXS,JHZ and XHH designed the\u0026nbsp;experiments;JKZ performed synthesis and characterization of the scaffolds;XLD,ZKL and YLX\u0026nbsp;created in vivo animal models.\u0026nbsp;WXS and ZKL\u0026nbsp;wrote the manuscript.WXS ,XHH and YFD\u0026nbsp;analyzed the data.WXS ,XHH and YFD\u0026nbsp;supervised experiments.XHH and WXS were responsible for the critical review of the manuscript. All authors contributed to this work, discussed the results, critically reviewed and revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Dr. Yanbiao Song for his technical assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKarapinar OS, Ozkan M, Okyay AG, et al. Evaluation of vaginal agenesis treated with the modified McIndoe technique: a retrospective study. J Turk Ger Gynecol Assoc. 2016;17:101\u0026ndash;5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5152/jtgga.2016.16013\u003c/span\u003e\u003cspan address=\"10.5152/jtgga.2016.16013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMotta GL, Tavares PM, Burttet LM, et al. 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Int J Nanomedicine. 2020;15:5911\u0026ndash;26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/IJN.S249129\u003c/span\u003e\u003cspan address=\"10.2147/IJN.S249129\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"Vaginal reconstruction, 3D printing, Vascularization, Decellularized extracellular matrix, PI3K/AKT signaling pathway","lastPublishedDoi":"10.21203/rs.3.rs-3203552/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3203552/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Abstarct\nBackground\n3D-printing is widely used in regenerative medicine and is expected to achieve vaginal morphological restoration and true functional reconstruction.Mesenchymal stem cells-derived exosomes (MSCs-Exos) were applyed in the regeneration of various tissues.The current study aimed to explore the effctive of MSCs-Exos in vaginal reconstruction.\nResults\nIn this work, hydrogel was designed using decellularized extracellular matrix (dECM) and gelatin methacrylate (GelMA) and silk fibroin (SF).The biological scaffolds was constructed using desktop-stereolithography.The physicochemical properties of the hydrogels were evaluated.It was observed that the sustained release property of exosomes in the hydrogel both in vitro and in vitro.The results revealed that 3D scaffold encapsulating exosomes expressed significant effects on the vascularization and musule regeneration of the regenerative vagina tissue.Also, MSCs-Exos strongly promoted vascularization in the vaginal reconstruction of rats,which may through the PI3K/AKT signaling pathway.\nConclusions\nOur results indicated that the 3D-printed, lumenal scaffold encapsulating exosomes might be used as a cell-free alternative treatment strategy for vaginal reconstruction.","manuscriptTitle":"Desktop-stereolithography 3D printing of a decellularized extracellular matrix/mesenchymal stem cell exosome bioink for vaginal reconstruction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-01 13:57:09","doi":"10.21203/rs.3.rs-3203552/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":"e185ad93-54ee-405e-88fd-946ddb2f6c1a","owner":[],"postedDate":"August 1st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-07-29T18:12:52+00:00","versionOfRecord":{"articleIdentity":"rs-3203552","link":"https://doi.org/10.1007/s13770-024-00649-x","journal":{"identity":"tissue-engineering-and-regenerative-medicine","isVorOnly":false,"title":"Tissue Engineering and Regenerative Medicine"},"publishedOn":"2024-06-27 18:12:52","publishedOnDateReadable":"June 27th, 2024"},"versionCreatedAt":"2023-08-01 13:57:09","video":"","vorDoi":"10.1007/s13770-024-00649-x","vorDoiUrl":"https://doi.org/10.1007/s13770-024-00649-x","workflowStages":[]},"version":"v1","identity":"rs-3203552","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3203552","identity":"rs-3203552","version":["v1"]},"buildId":"zQwnuV7TCBrMSSSToR1PI","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}