Vasoactive Intestinal Peptide advances chondrogenesis and modulates pathogenic mediators in human osteoarthritis.

Vasoactive Intestinal Peptide advances chondrogenesis and modulates pathogenic mediators in human osteoarthritis. | 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 Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Vasoactive Intestinal Peptide advances chondrogenesis and modulates pathogenic mediators in human osteoarthritis. Karolina Tecza, Cristina Rodríguez-Hernández, Raúl Villanueva-Romero, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6529495/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Current therapies for osteoarthritis (OA) focus on symptom management, rather than halting disease progression. Vasoactive intestinal peptide (VIP) has shown promising effects in musculoskeletal diseases, preserving joint integrity and modulating inflammation. This study investigates the potential of VIP to promote chondrogenic differentiation of human bone marrow mesenchymal stem cells (BM-hMSC), while modulating inflammatory and cartilage extracellular matrix (ECM)-degrading mediators in human articular chondrocytes from OA patients (OA-hAC). BM-hMSC from healthy donors were cultured in pellet under chondrogenic conditions with or without VIP up to 21 days. The production of type II collagen (COL2A1), and the expression of chondrogenic ( SOX9, COL2A1 , and ACAN ) and hypertrophy ( RUNX2 , COL10A1 , and MMP13 ) genes were assessed at different time points. VIP increased the expression of the chondrogenic genes on day 12 of differentiation, compared to day 21 in untreated BM-hMSC cells, advancing chondrogenesis. Furthermore, OA-hAC were dedifferentiated in monolayer followed by redifferentiation in alginate microbeads and treated with fibronectin fragments (Fn-fs) in presence and absence of VIP. We analysed VIP effects on cell proliferation, glycosaminoglycans (GAG) production, and modulation of components of the complement system (C1R and C3) and matrix metalloproteinases (MMP1, MMP3, MMP9, and MMP13). VIP enhanced cell proliferation, increased GAG deposition, and reduced production of complement factor C1R, and metalloproteinases MMP1 and MMP13 in OA-hAC. This study highlights the potential of VIP in modulating chondrogenesis, inflammation, and cartilage degradation supporting the development of future VIP-based therapies to slow OA progression. osteoarthritis VIP chondrocytes mesenchymal stem cells MMP complement system Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Osteoarthritis (OA) is a multifactorial chronic rheumatic disease with a significant global impact, ranking among the leading causes of disability in individuals over the age of 70. Symptoms reflect a dynamic imbalance between joint tissue breakdown and repair, involving bone remodelling, synovial inflammation, and cartilage degeneration, ultimately leading to joint dysfunction and impaired quality of life[ 1 ]. Articular chondrocytes (AC) are the main resident cells in articular cartilage, playing a central role in OA pathology. In healthy cartilage, these cells maintain homeostasis between anabolic and catabolic processes in the cartilage extracellular matrix (ECM) by synthesizing essential components including glycosaminoglycans (GAG), aggrecan (ACAN), and type II collagen (COL2)[ 2 ]. During OA, this balance is disrupted, exposing these cells to inflammatory stress that alters their behaviour. This leads to the release of ECM degradation products with potential catabolic properties. Among them, fibronectin fragments (Fn-fs) stand out degrading essential ECM components, while also promoting the production of pro-inflammatory mediators, fostering further inflammation and cartilage degradation[ 3 , 4 ]. In the inflammatory microenvironment, other cells begin secreting pro-inflammatory factors, promoting chondrocyte apoptosis and hypertrophy, and reducing ECM component synthesis while increasing ECM-degrading proteases, like matrix metalloproteinases (MMP), contributing to the degradation of collagen, aggrecan, and other components of the cartilage ECM [ 1 ]. Among them, the collagenase MMP13 is critical in OA pathogenesis, being the main enzyme involved in the degradation of COL2[ 5 , 6 ]. On the other hand, a chronic low-grade inflammation is well documented, but the specific mediators involved in this pathology remain less understood. The complement system, as part of innate immunity, has been described to be involved in OA, with chondrocytes contributing to its production[ 7 , 8 ]. Increased complement protein expression has been observed in the synovial fluid from OA patients compared to healthy donors[ 9 , 10 ], being associated with pain[ 11 ]. The limited replication potential of AC reduces cartilage self-repair capacity. To address this problem, cell-based approaches and tissue engineering offer promising tools to improve cartilage repair[ 12 , 13 ]. AC cultured in monolayer tend to dedifferentiate to a fibroblastic phenotype losing their chondrogenic characteristics from early passages[ 14 ]. By contrast, three-dimensional (3D) cultures promote cell survival, functionality and morphology, bringing the in vitro conditions closer to the in vivo articular microenvironment, preventing chondrocyte dedifferentiation[ 14 , 15 ]. Further, mesenchymal stem cells (MSC) have emerged as candidates for regenerating damaged cartilage tissue due to their low immunogenicity, multipotent differentiation capability and ease of isolation from numerous sources[ 16 ]. For MSC in vitro culture, 3D pellet cultures effectively promote chondrogenic differentiation[ 17 , 18 ]. In addition, different biomaterials are currently being designed for that purpose[ 19 ]. One of the most used materials is the alginate, a biocompatible polysaccharide, ideal for culturing chondrocytes[ 20 ]. Alginate enables redifferentiation, maintaining the spherical phenotype characteristic of in vivo conditions, while promoting chondrogenic markers expression and preventing hypertrophy[ 21 ]. Among the chondrogenic markers, SRY-Box Transcription Factor 9 (SOX9) is a master transcription factor involved in the expression of target genes during chondrogenesis, including COL2A1 and ACAN [ 14 , 22 ], which levels are reduced in OA patients[ 2 , 23 ]. Conversely, Runt Related Transcription Factor 2 (RUNX2) drives chondrocyte hypertrophy, characterized by an increase in the levels of type 10 collagen (COL10)[ 21 , 24 ]. Increased levels of RUNX2 have also been described in chondrocytes from OA patients, being involved in the expression of ECM-degrading enzymes including MMP13[ 24 , 25 ]. Given the limited regenerative capacity of cartilage and the lack of effective OA treatments, it is crucial to study new therapies aimed at stopping disease progression rather than merely alleviating symptoms[ 26 ]. In this context, we have focused on studying vasoactive intestinal peptide (VIP) as a potential therapeutic tool. VIP is a neuropeptide produced by neurons, as well as endocrine and immune cells, involved in modulating the innate and adaptive immune response. Previous results point to a beneficial effect on joint integrity and functionality in musculoskeletal pathologies[ 27 , 28 ]. In OA, we have previously described the effect of VIP by inhibiting the expression of degradative enzymes and inflammatory mediators in in vitro cultures of synovial fibroblasts[ 29 , 30 ] and chondrocytes co-cultures in monolayer[ 8 ]. Recent studies also emphasize its role during osteogenic differentiation, identifying it as a potential osteoinductor[ 31 ]. Exploring the effect of VIP on human chondrogenesis, as well as its role in modulating inflammatory and ECM-degrading mediators produced by OA chondrocytes within a pro-inflammatory microenvironment, could provide valuable insights for leveraging this peptide as a potential therapeutic tool for OA. Material and Methods 3D pellet cultures of mesenchymal stem cells from healthy donors Human bone marrow mesenchymal stem cells (BM-hMSC) from four healthy donors (two men and two women, aged 30–50 years, various ethnicities) were obtained commercially from StemCell Technologies (Vancouver, Canada), characterized for MSC specific markers and tested for their ability to differentiate in vitro into the different linages. Culture flasks were previously treated with Animal Component-Free Cell Attachment Substrate (ACF-ATS; StemCell Technologies). BM-hMSC were cultured in MesenCult-ACF Plus Medium (StemCell Technologies) supplemented with 1% Glutamax (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) and 0,2% of its specific commercial supplement (StemCell Technologies). Cells were incubated at 37ºC and 5% CO 2 , changing the medium every 3 days. At confluence, 5x10 5 cells were seeded in 15-ml polypropylene conical tubes and centrifuged at 300xg for 5 minutes. Pelleted cells were incubated at 37ºC and 5% CO 2 in 500 µl of MesenCult-ACF Chondrogenic Differentiation Medium (StemCell Technologies) supplemented with 1% Glutamax, 1% penicillin/streptomycin and 5% of its specific commercial supplement (StemCell Technology) with or without VIP 10 − 8 M (Bachem, Bubendorf, Switzerland) for up to 21 days, changing the medium every 3 days, according to the supplier’s instructions. Pellets were collected for histological and immunohistochemical analysis on days 1, 3, 6, 12, 17 and 21. For gene expression, pellets were homogenised using a potter pestle. RNA from homogenised pellets and BM-hMSC was extracted using Tri Reagent (Invitrogen) according to manufacturer's instruction (Fig. 1 A). Histological analysis of cell pellets BM-hMSC cell pellets cultured in presence or absence of VIP, were recovered on days 1, 3, 6, 12, 17 and 21, fixed in paraformaldehyde, embedded in paraffin, and sectioned in 5 µm thick sections with a microtome (Leica Histocore Biocut). Sections were stained with Alcian blue and Masson's trichrome. Immunohistochemical detection of type II collagen Immunohistochemical analysis of type II collagen was performed on the previously described BM-hMSC cell pellets slides, using a primary rabbit polyclonal antibody for human collagen alpha-1(II) chain protein (1:100, Cusabio Technology, Houston, USA) and an anti-Rabbit Histofine Simple Stain (Nichirei, Tokyo, Japan). The reaction was revealed using solution of diaminobenzidine (DAB) with hydrogen peroxide as the enzyme substrate (Palex Medical S.A., Madrid, Spain), following the manufacturer’s instructions. 3D cultures of articular chondrocytes from osteoarthritis patients in alginate microbeads Osteoarthritis human articular chondrocytes (OA-hAC) were isolated from knee of 7 patients, 2 men and 5 women, aged between 58 and 84 years, undergoing total knee joint replacement surgery. Patients had advanced disease and were diagnosed of primary OA, excluding trauma, inflammatory disease and secondary OA. Samples were provided by the Rheumatology Service at Instituto de Investigación Biomédica (INIBIC) Complejo Hospitalario Universitario A Coruña (CHUAC, A Coruña, Spain). Informed consent was obtained from all patients before surgery. The study was approved by the Clinical Research Ethics Committee of Hospital Universitario de La Princesa and performed according to the recommendations of the Declaration of Helsinki. OA-hAC were expanded in monolayer in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5g/l glucose (Corning, NY, USA), 10% fetal bovine serum (FBS), 25mM HEPES (Lonza Ibérica SA, Barcelona, Spain), 1% Glutamax and 1% penicillin/streptomycin (Invitrogen) until passage 4. Cells were incubated at 37ºC and 5% CO 2 , changing the medium every 3 days. For gene expression analysis, total RNA was extracted from the dedifferentiated hAC using Tri Reagent (Invitrogen) according to manufacturer's instruction (Fig. 1 B, upper ). For redifferentiation studies, OA-hAC were used after passage 4, and suspended in 1% medium viscosity alginate solution (Pronova UP, MVG; NovaMatrix, Sandvika, Norway) at a density of 2x10 6 cells/ml. Alginate solution was formerly dissolved in 0.85% NaCl (Lonza Ibérica SA). Microbeads were made by dropping the resulting suspension through a 25G needle at a constant rate and height, in 15 ml of a solution composed of 100 mM CaCl 2 and 15 mM HEPES, under agitation. Then, to promote polymerization, microbeads were kept in the same container for 15 min without agitation and subsequently washed 2 times with 0.85% NaCl. Nine microbeads per well were placed in a 24-well plate and cultured in 1 ml of DMEM with 4.5g/l glucose, (Corning), 25mM HEPES (Lonza Ibérica SA), 1% ITS (composed of insulin, transferrin, bovine serum albumin, selenic acid and linoleic acid; Corning), 1% Glutamax and 1% penicillin/streptomycin (Invitrogen), ascorbic acid 50 µg/ml (Merck, Darmstadt, Germany), proline 2mM (Merck) and transformant factor beta 1 (TGF-β1; Peprotech, Thermo Fisher Scientific) 10 ng/ml to promote redifferentiation. Cells were incubated at 37ºC and 5% CO 2 for 2 weeks, changing the medium every 3 days. To verify redifferentiation of the OA-hAC, alginate microbeads were washed with 1 ml of 0.85% NaCl and dissolved with 1 ml of 0.1 M citrate buffer for 10 min. Resulting suspension was centrifuged at 1200 rpm for 10 min, obtaining 2 fractions: the upper phase containing alginate wastes and macromolecules called further-removed matrix (FRM), and the pellet with cells and their associated cellular matrix (CM). Total RNA was extracted from CM fraction using Tri Reagent (Invitrogen) according to manufacturer's instruction (Fig. 1 B, upper ). For the treatment study, after 2 weeks of redifferentiation, TGF-β1 was no longer added to the medium and OA-hAC were cultured for 7 additional days in presence or absence of VIP 10 − 8 M, 45kDa Fn-fs 10 − 8 M or the combined treatment, changing the medium every 3 days. Six replicates of each condition were set up for the experiment. Cells were incubated at 37ºC and 5% CO 2 . For protein detection, culture supernatants were collected, and CM fraction was obtained as described above (Fig. 1 B, lower ). Real-time polymerase chain reaction analysis 1,2µg of RNA were reverse transcribed using a High Capacity cDNA Reverse Transcription (RT) Kit (Applied Biosystems, Waltham, MA, USA) and semiquantitative real-time polymerase chain reaction (RT-qPCR) was performed using a TaqMan Gene Expression Master Mix (Applied Biosystems) with manufactured-predesigned primers for SOX9 (Hs00165814_m1), COL2A1 (Hs00264051_m1), ACAN (Hs00153936_m1), RUNX2 (Hs01047975_m1), COL10A1 (Hs00166657_m1), MMP13 (Hs00942584_m1). Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) (Hs99999905_m1) was used as reference gene. Results were normalized to the expression of GAPDH and plotted relative to the expression in the untreated condition, in the case of treatments, using the ΔΔ cycle threshold method for quantification. Cell proliferation assay Proliferation was determined in the redifferentiated OA-hAC cultured in microbeads after 7 days in presence of the treatments described above, using the Cell Proliferation Reagent WST-1 (Merck). This assay measures mitochondrial metabolic activity as an indicator of cell viability. Briefly, one bead per well was placed in a 96-well plate with 200 µl of DMEM in presence or absence of VIP 10 − 8 M, 45 kDa Fn-fs 10 − 8 M or the combined treatment. After 7 days, microbeads were incubated with WST-1 reagent (1:100) for 4 hours at 37ºC. 100 µl of supernatant per well were transferred to a new 96-well plate and the optical density was measured at 450 nm. The experiment was performed in duplicate. Sulfated glycosaminoglycans assay Sulfated glycosaminoglycan (GAG) content was measured in CM fractions from redifferentiated OA-hAC after 7 days of treatment, using a Blyscan Sulfated Glycosaminoglycan Assay (Biocolor Ltd, Carrickfergus, United Kingdom). Briefly, microbeads were washed with 0.85% NaCl, dissolved in 0.1M citrate buffer, and centrifuged at 1200 rpm for 10 min, obtaining the CM fraction. Papain Extraction Reagent (composed of sodium phosphate buffer pH 6.4 with sodium acetate, EDTA, cysteine-HCl and papain crystalized suspension (Sigma-Aldrich)) was added to the CM fraction. Samples were digested with the solution for 3 hours at 65ºC and the assay was done following manufacturer’s instructions. DNA content was measured using QuantiFluor dsDNA System (Promega Biotech Ibérica S.L., Madrid, Spain). Fluorescence intensity was measured using the FLUOstar Omega (BMG LABTECH) at an excitation wavelength of 504 nm and an emission wavelength of 531 nm. The assay was performed following manufacturer’s instructions. GAG content was normalized to DNA (GAG/DNA ratio). ELISA and Multiplex assays Protein expression of complement system proteins and MMP was analysed in cultured supernatants and CM fractions from redifferentiated OA-hAC after 7 days of treatment. For protein extraction, RIPA lysis buffer (Thermo Fisher Scientific) supplemented with Protease and Phosphatase Inhibitors (Thermo Fisher Scientific) was added to CM fraction. Next the samples were sonicated, and protein content was measured with a QuantiPro BCA Assay Kit (Sigma-Aldrich, San Luis, Missouri, USA). Then, a commercial ELISA kit for C1R (Merck) and a MILLIPLEX® Multiplex Assays for human C3, MMP1, MMP3, MMP9, and MMP13 (Merck) were used, following manufacturer’s instructions. Statistical analysis Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software, Inc., CA, USA). Data were subjected to normality test (Kolmogorov-Smirnov test) and equal variance test ( F -test). Statistical differences for parametric variables were assessed via t-test and analysis of variance (ANOVA) together with Sidak post hoc test, while Kruskal-Wallis test or Mann-Whitney U test were used for non-parametric variables. Results are presented as mean ± SEM (Standard Error of Mean). P -values < 0.05 were considered statistically significant (* p < 0.05; ** p < 0.01; *** p < 0.001). Results Histological analysis of pellets during BM-hMSC chondrogenesis BM-hMSC were cultured in pellet with chondrogenic medium in presence and absence of VIP for up to 21 days. Cell pellets exhibited irregular and inconsistent morphology in the early days of cultivation (days 1–6). However, as the culture progressed, they adopted a more rounded and consistent morphology (Fig. 2 A). To visualize the evolution of ECM formation, we stained cell pellets sections with Alcian blue and Masson's trichrome for monitoring GAG and collagen depositions. We noticed that ECM formation increases throughout the days in untreated (Fig. 2 B, 2 C, top ) and VIP-treated cells (Fig. 2 B, 2 C, bottom ). This deposition compacted the cell pellets giving them the appearance of cartilaginous tissue. In sections of the same pellets, we also detected the deposition of type II collagen in untreated (Fig. 2 D, top ) and VIP-treated cells (Fig. 2 D, bottom ) over the days. No staining was detected in the negative or in the isotype controls (Supplementary Fig. 1). VIP advances chondrogenesis in BM-hMSC To determine the effect of VIP during chondrogenesis, we analysed the differences in gene expression of the chondrogenic marker COL2A1 between untreated and VIP-treated BM-hMSC cultured in pellet under chondrogenic conditions for up to 21 days. While in untreated cells significant differences compared to day 1 were observed on day 21 (Fig. 3 A), in presence of VIP a significant increase was observed as early as day 12, corresponding to the highest expression of the gene (Fig. 3 B). Accordingly, when comparing both conditions at each time point, COL2A1 expression was significantly higher in VIP-treated BM-hMSC on day 6, with the greatest difference observed on day 12. This difference reversed on day 21 (Fig. 3 C). To corroborate these results, we studied the expression of additional chondrogenic ( SOX9 and ACAN ) and chondrocyte hypertrophy markers ( RUNX2 , COL10A1 and MMP13 ) in BM-hMSC cell pellets on days 1, 12 and 21. Our results show that on day 12, VIP significantly increased the expression of the chondrogenic markers SOX9 and ACAN compared to untreated cells (Fig. 3 D, E). By contrast, the expression of ACAN was higher in untreated cells on day 21 (Fig. 3 E). Regarding hypertrophy genes, initial expression levels of RUNX2 , COL10A1 and MMP13 on day 1 were higher with the treatment of VIP (Fig. 3 F, G, H). However, these differences disappeared over the days. In addition, even no significant differences were observed, the ratios COL2A1/COL10A1 and SOX9/RUNX2 were also higher in VIP-treated BM-hMSC on day 12 (Supplementary Fig. 2). Alginate microbeads allow redifferentiation of OA-hAC To corroborate that alginate microbeads promote redifferentiation of OA-hAC, we analysed mRNA expression of the chondrogenic markers SOX9 , COL2A1 and ACAN in OA-hAC previously dedifferentiated in monolayer (Fig. 4 A), as well as in the redifferentiated OA-hAC cultured in alginate microbeads for 2 weeks (Fig. 4 B, C). Additionally, we measured the expression of RUNX2 as hypertrophy marker. The results revealed that both, dedifferentiated and redifferentiated OA-hAC, expressed the studied genes, with a significant higher expression of SOX9 and COL2A1 in OA-hAC after 2 weeks of redifferentiation in microbeads (Fig. 4 D). Despite, no significant differences were observed, these cells also had a higher SOX9/RUNX2 ratio than the dedifferentiated chondrocytes (Supplementary Fig. 3). Effects of VIP and Fn-fs in the survival of OA-hAC in alginate microbeads To assess the effect of VIP in OA-hAC treated with the pro-inflammatory stimulus Fn-fs, OA-hAC redifferentiated in microbeads were subjected to 7 additional days of treatment with VIP, Fn-fs or the combined treatment. The results showed that while Fn-fs decreased cell proliferation, the presence of VIP counteracted its effects, promoting a significant increase in OA-hAC proliferation (Fig. 5 A). Modulation of glycosaminoglycans production by VIP in Fn-fs treated OA-hAC To analyse the production of GAG in the redifferentiated OA-hAC, microbeads were cultured for 7 additional days in presence of the treatments described above. The results indicated that OA-hAC treated with Fn-fs in combination with VIP, exhibited a significantly higher GAG/DNA ratio in the CM protein fraction compared to cells treated with Fn-fs alone, which supposes a greater ECM/GAG deposition in presence of the neuropeptide (Fig. 5 B). VIP decreases production of complement system proteins and MMP in Fn-fs treated OA-hAC To evaluate the effect of VIP on the production of complement system components as inflammatory mediators (C1R and C3) and its influence on the modulation of ECM-degrading enzymes (MMP1, MMP3, MMP9, and MMP13), protein levels of these mediators were analysed by ELISA and Multiplex assays. These analyses were performed on the CM fraction and culture supernatants of OA-hAC redifferentiated in microbeads treated with Fn-fs in the presence or absence of VIP. The results showed that the combined treatment with VIP tended to decrease the production of the studied mediators. Specifically, in complement proteins, a significant reduction was observed for C1R in both, CM fraction and supernatants (Fig. 6 A, B). No expression of C3 was detected in the culture supernatants. Besides, VIP significantly decreased the levels of MMP1 and MMP13 in the CM fraction (Fig. 6 C). This reduction was also significant for MMP13 in culture supernatants (Fig. 6 D). No effects were observed with the treatment of VIP alone (Supplementary Fig. 4). Discussion Human MSC have emerged as a promising therapeutic tool for OA treatment and cartilage repair[ 16 , 32 ]. Different studies have focused on optimizing hMSC differentiation through in vitro strategies[ 17 , 33 ]. Although hMSC offer promising results, further research is necessary for their application in cartilage repair. These cells can be induced to express cartilage-specific molecules like COL2 and ACAN, under the control of SOX9[ 22 ]. However, they may also develop undesirable fibrocartilage-like features and undergo hypertrophy during chondrogenesis[ 34 ], where RUNX2 and COL10 are involved[ 35 , 36 ]. In this sense, VIP and related peptides play important roles in chondrogenesis and osteogenesis. Previous studies have reported pituitary adenylate cyclase activating polypeptide (PACAP) as a positive regulator of chondrogenesis in chicken[ 37 , 38 ]. Nonetheless, the present study is the first to report the effect of VIP in this process in humans. Our findings reveal that VIP increases the expression of SOX9 , COL2A1 , and ACAN on day 12, advancing chondrogenesis in BM-hMSC, without inducing hypertrophy. These results support the use of BM-hMSC and VIP as potential therapeutic strategies for OA treatment and cartilage repair. Recent studies have reported the importance of neuro-immune interactions in OA[ 39 ]. In this context, VIP plays a complex role in the pathology. Previous findings have described reduced VIP levels in the synovial fluid and articular cartilage of OA patients, correlating with increased disease severity[ 40 ]. This downregulation may contribute to OA pathogenesis by enhancing pro-inflammatory cytokine production. Conversely, other studies suggest that VIP accumulation in joints may contribute to OA[ 41 ], promoting subchondral bone sclerosis and angiogenesis[ 42 ]. Despite these conflicting findings, several researches support that upregulation of VIP may counteract pro-inflammatory stimuli and reduce OA pain[ 29 , 30 , 41 ]. Moreover, in cell co-cultures from OA patients, VIP has demonstrated anti-inflammatory and immunomodulatory actions, potentially modulating the secretome of chondrocytes and synovial fibroblasts[ 8 ]. In the present study, to elucidate the role of VIP in OA, we have employed 3D cultures for hAC as they have shown better results in maintaining chondrocyte phenotype compared to two-dimensional monolayer cultures[ 43 ]. We corroborate that redifferentiated OA-hAC in 3D cultures in alginate microbeads show a higher expression of the chondrogenic markers SOX9 and COL2A1 compared to dedifferentiated cells in monolayer[ 43 ]. These findings highlight the importance of 3D culture systems in preserving chondrocyte phenotype for cartilage tissue engineering applications. On the other hand, since VIP requires a pro-inflammatory context to exert significant modulatory effects, we employed Fn-fs, key contributors to OA pathophysiology. These fragments contribute to the initiation and progression of the disease by inducing ECM-degrading enzymes and pro-inflammatory mediators, promoting cartilage catabolism [ 3 , 4 , 44 ]. Moreover, they are present at high concentrations in synovial fluid and cartilage from OA patients[ 45 , 46 ]. Our results reveal that in presence of Fn-fs, VIP enhances both, cell proliferation and GAG production in hAC from OA patients. These findings corroborate the beneficial effects of the neuropeptide promoting cell survival and ECM formation in a pro-inflammatory microenvironment. In relation to the catabolic process during OA progression, MMP play a crucial role, degrading essential ECM components in articular cartilage. Among these, stromelysins like MMP3, and gelatinases like MMP9, potentially contribute to cartilage destruction[ 47 , 48 ]. Notably, the collagenases, MMP1 and MMP13, whose levels are increased in OA cartilage, play key roles in the pathogenesis[ 49 ]. MMP13 is specifically expressed in OA patients and is critical in OA progression by degrading type II collagen, making it an attractive target for OA treatment. Consequently, selective MMP13 inhibitors are being developed as a promising strategy for OA therapy[ 5 , 6 ]. The up-regulation of MMP13 by Fn-fs has been previously described in bovine osteochondral explants and culture supernatants[ 50 ]. Here we demonstrated that, in Fn-fs presence, VIP decreases the production of MMP in OA-hAC, with significant reduction for the collagenases, MMP1 and MMP13, in the CM protein fraction, also with a significant decrease of MMP13 in culture supernatants from microbeads. The ability of VIP to reduce MMP13 in presence of Fn-fs has also been previously described in culture supernatants from synovial fibroblasts of OA patients[ 30 ]. Our results support the use of VIP as a potential therapeutic tool for targeting key enzymes involved in the pathology. Selectivity of VIP may be due to a combination of its mechanism of action, the differential regulation of MMP in the cellular microenvironment, and the specific pathways involved in their expression. Additional experiments could include analysing the signalling pathways involved or modulating key transcription factors to confirm these hypotheses. In addition to the catabolic process, the inflammatory microenvironment also contributes to the pathology. In this context, previous research emphasizes the significant role of the complement system in OA with an abnormally high expression and activation, associated with inflammation [ 7 , 9 ]. We previously reported that, in presence of Fn-fs, VIP reduced levels of C1R and C3 in the secretome of synovial fibroblasts and OA-hAC co-cultured in monolayer[ 8 ]. Accordingly, in this work we demonstrate that VIP decreases protein C1R expression in both CM fraction and culture supernatants from 3D OA-hAC cultures in microbeads. Although no significant differences were observed for C3, a trend toward reduction was also noted in CM fraction in presence of VIP. These results reinforce the anti-inflammatory potential of the neuropeptide. This study is the first to demonstrate the positive effects of VIP in modulating chondrogenesis of BM-hMSC, while also highlighting its potential to regulate inflammatory and catabolic mediators in 3D cultures of hAC from OA patients. Our findings suggest that targeting MMP and complement factors could serve as a potential therapeutic strategy for the treatment of OA, positioning VIP as a promising candidate for therapies aimed at slowing OA progression and improving outcomes for affected individuals. Declarations Acknowledgments and Funding Information This work was supported by grants RD21/0002/004 and RD24/0007/0014 from the Ministerio de Economía y Competitividad (Instituto de Salud Carlos III) and co‐funded by European regional development fund (ERDF) and by the UCM grants PR12/24-31572 and PR12/24-31568. We are grateful to all patients and the collaborating clinicians for their participation in this study. We also appreciate the assistance of Isabel Montero, Technician at the Department of Cell Biology and Histology at the Faculty of Biological Science, UCM, for the sectioning of paraffin embedded cell pellets. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Karolina Tecza, Cristina Rodríguez-Hernández and Selene Pérez-García. The first draft of the manuscript was written by Selene Pérez-García and Karolina Tecza, and all authors commented on previous versions of the manuscript. Funding acquisition was provided by Rosa P. Gomariz, Yasmina Juarranz and Carmen Martínez. All authors read and approved the final manuscript. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Hospital Universitario de La Princesa (06-06-24, acta CEIm 11/24). Consent to participate Informed consent was obtained from all individual participants included in the study. References Courties A, Kouki I, Soliman N, Mathieu S, Sellam J (2024) Osteoarthritis year in review 2024: Epidemiology and therapy. Osteoarthritis and cartilage / OARS. 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Arthritis Rheum 40:2065–2074. https://doi.org/10.1002/art.1780401120 Ding L, Guo D, Homandberg GA, Buckwalter JA, Martin JA (2014) A single blunt impact on cartilage promotes fibronectin fragmentation and upregulates cartilage degrading stromelysin-1/matrix metalloproteinase-3 in a bovine ex vivo model. J Orthop Res 32:811–818. https://doi.org/10.1002/jor.22610 Supplementary Files FigS1.tif Fig. S1. BM-hMSC pellets during chondrogenic differentiation. (A) Isotype and (B) negative controls from immunohistochemistry of COL2A1 in untreated (top) and VIP-treated (bottom) cell pellets on days 1, 3, 6, 12, 17 and 21 of chondrogenic differentiation (scale bar = 200 μm). Objective 4X. Stereo microscope (Olympus SZX16) coupled to camera (Olympus SDF PLAPO 0.5XPF). FigS2.tif Fig. S2. Ratio between chondrogenic and hypertrophy markers during chondrogenesis of BM-hMSC in pellet cultures. Ratio between mRNA expression of (A) COL2A1 and COL10A1 and (B) transcription factors SOX9 and RUNX2 on days 1, 12 and 21 of chondrogenic differentiation in absence or presence of VIP, determined by RT-qPCR. Results were normalized to the expression of GADPH and plotted relative to the expression of BM-hMSC, using the ΔΔ cycle threshold method for quantification. Data are presented as mean ± SEM (n=3). FigS3.tif Fig. S3. Ratio between chondrogenic and hypertrophy markers in OA-hAC dedifferentiated in monolayer and redifferentiated in alginate microbeads. Ratio between mRNA expression of transcription factors SOX9 and RUNX2 determined by qPCR. Results were normalized to the expression of GADPH using the Δ cycle threshold method for quantification. Data are presented as mean ± SEM (n=6). FigS4.tif Fig. S4. Effect of Fn-fs and VIP on the expression of MMP and complement proteins in OA-hAC redifferentiated in alginate microbeads. Expression of complement proteins (C1R, and C3) and MMP (MMP-1, -3, -9, -13) was analysed in OA-hAC redifferentiated in alginate microbeads after 7 days of treatments. Protein levels in (A, C) CM fractions and (B, D) supernatants, were analysed by ELISA (C1R) and Multiplex (MMP1, MMP3, MMP9, MMP13, and C3). Values were normalized to total protein and plotted relative to the untreated cells (n=3). KeyMessages.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major Revisions Needed 21 Sep, 2025 Reviewers agreed at journal 13 May, 2025 Reviewers invited by journal 13 May, 2025 Editor assigned by journal 28 Apr, 2025 First submitted to journal 25 Apr, 2025 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. 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Schematic representation of the methodology used.\u003cstrong\u003e (A)\u003c/strong\u003e 3D pellet culture of BM-hMSC from healthy donors. \u003cstrong\u003e(B) \u003c/strong\u003e3D culture of hAC from OA patients in alginate microbeads. Figure created with BioRender.com\u003c/p\u003e","description":"","filename":"OnlineFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6529495/v1/5ccdc880b6eb9d5cdd61691e.png"},{"id":82890419,"identity":"ffb08c12-03a9-4a9d-97e6-4703163ba455","added_by":"auto","created_at":"2025-05-16 12:09:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":83580,"visible":true,"origin":"","legend":"\u003cp\u003eBM-hMSC pellets during chondrogenic differentiation.\u003cstrong\u003e \u003c/strong\u003eRepresentative images of\u003cstrong\u003e \u003c/strong\u003eBM-hMSC cultured in pellets for up to 21 days in chondrogenic differentiation medium. \u003cstrong\u003e(A)\u003c/strong\u003e Macroscopic morphology of untreated \u003cstrong\u003e(top)\u003c/strong\u003e and VIP 10\u003csup\u003e-8\u003c/sup\u003eM-treated \u003cstrong\u003e(bottom)\u003c/strong\u003e cell pellets on days 1, 3, 6, 12, 17 and 21 of chondrogenic differentiation. \u003cstrong\u003e(B) \u003c/strong\u003eHistological slices stained with Alcian blue, \u003cstrong\u003e(C) \u003c/strong\u003eMasson’s trichrome, and \u003cstrong\u003e(D) \u003c/strong\u003eimmunohistochemistry of COL2A1\u003cstrong\u003e \u003c/strong\u003ein \u003cstrong\u003e(top) \u003c/strong\u003euntreated and \u003cstrong\u003e(bottom) \u003c/strong\u003eVIP-treated cell pellets on days 1, 3, 6, 12, 17 and 21 of chondrogenic differentiation (scale bar = 200 μm). Objective 4X. Stereo microscope (Olympus SZX16) coupled to camera (Olympus SDF PLAPO 0.5XPF).\u003c/p\u003e","description":"","filename":"OnlineFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6529495/v1/5e633ef77f33d470594d3801.png"},{"id":82890415,"identity":"37b5e21b-9bec-43a7-9226-46bfc38be717","added_by":"auto","created_at":"2025-05-16 12:09:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":25746,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of \u003cem\u003eCOL2A1\u003c/em\u003e and comparison between untreated and VIP-treated cells during chondrogenesis of BM-hMSC in pellet cultures.\u003cstrong\u003e \u003c/strong\u003emRNA expression of \u003cem\u003eCOL2A1 \u003c/em\u003eon days 1, 3, 6, 12, 17 and 21 of chondrogenic differentiation in \u003cstrong\u003e(A)\u003c/strong\u003e absence or \u003cstrong\u003e(B)\u003c/strong\u003e presence of VIP 10\u003csup\u003e-8\u003c/sup\u003eM measured by RT-qPCR. Results were normalized to the expression of \u003cem\u003eGAPDH\u003c/em\u003e and plotted relative to the expression of BM-hMSC, using the ∆∆ cycle threshold method for quantification. Data are presented as mean ± SEM of duplicate determinations (n=4). \u003cem\u003eP\u003c/em\u003e-values indicate statistically significant differences relative to day 1.\u003cstrong\u003e (C)\u003c/strong\u003e mRNA expression of \u003cem\u003eCOL2A1\u003c/em\u003e on days 1, 3, 6, 12, 17 and 21 of chondrogenic differentiation in absence or presence of VIP 10\u003csup\u003e-8\u003c/sup\u003eM and mRNA expression of chondrogenic markers \u003cstrong\u003e(D)\u003c/strong\u003e \u003cem\u003eSOX9\u003c/em\u003e, \u003cstrong\u003e(E)\u003c/strong\u003e\u003cem\u003e ACAN\u003c/em\u003e, and hypertrophy markers \u003cstrong\u003e(F)\u003c/strong\u003e \u003cem\u003eRUNX2,\u003c/em\u003e \u003cstrong\u003e(G)\u003c/strong\u003e \u003cem\u003eCOL10A1, \u003c/em\u003e\u003cstrong\u003e(H)\u003c/strong\u003e \u003cem\u003eMMP13\u003c/em\u003e on days 1, 12 and 21 of chondrogenic differentiation in absence or presence of VIP 10\u003csup\u003e-8\u003c/sup\u003eM measured by RT-qPCR. Results were normalized to the expression of \u003cem\u003eGAPDH\u003c/em\u003e and plotted relative to the expression in the untreated condition at each time point, using the ∆∆ cycle threshold method for quantification. Data are presented as mean ± SEM of duplicate determinations (n=4) (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6529495/v1/4b9f51844f39b38fb0aa1d9c.png"},{"id":82890436,"identity":"d22efb44-c82c-4d62-84d1-fff65514a374","added_by":"auto","created_at":"2025-05-16 12:09:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":117661,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of chondrogenic and hypertrophy markers in OA-hAC dedifferentiated in monolayer and redifferentiated in alginate microbeads.\u003cstrong\u003e (A) \u003c/strong\u003eRepresentative image of chondrocytes from OA patients dedifferentiated in monolayer (scale bar = 50 μm) and \u003cstrong\u003e(B, C)\u003c/strong\u003e redifferentiated in alginate microbeads for 2 weeks. Objective 10X. Inverted microscope (Leica DM IL LED) coupled to camera (Moticam 1080), ((B) scale bar = 500 μm, (C) scale bar = 50 μm). \u003cstrong\u003e(D)\u003c/strong\u003e mRNA expression of the chondrogenic markers \u003cem\u003eSOX9\u003c/em\u003e, \u003cem\u003eCOL2A1\u003c/em\u003e, \u003cem\u003eACAN\u003c/em\u003e and the hypertrophy marker \u003cem\u003eRUNX2\u003c/em\u003e,\u003cem\u003e \u003c/em\u003ewere determined by RT-qPCR. Results were normalized to the expression of \u003cem\u003eGADPH\u003c/em\u003eand plotted relative to the expression of dedifferentiated OA chondrocytes, using the ΔΔ cycle threshold method for quantification. Data are presented as mean ± SEM of duplicate determinations (n=5) (***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"OnlineFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6529495/v1/a3572a8d1700fe1ba52e30a3.png"},{"id":82890434,"identity":"de475150-0c69-4454-b0ac-0efb96b25b3c","added_by":"auto","created_at":"2025-05-16 12:09:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":16771,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Fn-fs and VIP on cell proliferation and GAG production in OA-hAC redifferentiated in alginate microbeads. \u003cstrong\u003e(A)\u003c/strong\u003e Cell proliferation was analysed in OA-hAC redifferentiated in alginate microbeads after 7 days of treatment with VIP 10\u003csup\u003e-8\u003c/sup\u003eM, Fn-fs 10\u003csup\u003e-8\u003c/sup\u003eM or the combined treatment, using the Cell Proliferation Reagent WST-1. Values were plotted relative to the levels of untreated cells. Data are presented as mean ± SEM of duplicate determinations (n=5). \u003cstrong\u003e(B)\u003c/strong\u003e GAG concentration was analysed in OA-hAC redifferentiated in alginate microbeads after 7 days of treatment with VIP 10\u003csup\u003e-8\u003c/sup\u003eM, Fn-fs 10\u003csup\u003e-8\u003c/sup\u003eM or the combined treatment, using a Blyscan\u003csup\u003e \u003c/sup\u003eSulfated Glycosaminoglycan Assay for determination of GAG content, and a QuantiFluor dsDNA System for determination of DNA content. Values of GAG were normalized to DNA and plotted relative to the levels of untreated cells. Data are presented as mean ± SEM of duplicate determinations (n=5) (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"OnlineFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6529495/v1/794241f70eea552bfc17f633.png"},{"id":82890480,"identity":"d0a1f791-43fe-4da2-a0d8-0555e32f79ff","added_by":"auto","created_at":"2025-05-16 12:09:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":21304,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the effect of VIP on the expression of MMP and complement proteins in OA-hAC redifferentiated in alginate microbeads. Expression of complement proteins (C1R, and C3) and MMP (MMP-1, -3, -9, -13) was analysed in OA-hAC redifferentiated in alginate microbeads after 7 days of treatment with or without Fn-fs 10\u003csup\u003e-8\u003c/sup\u003eM in presence and absence of VIP 10\u003csup\u003e-8\u003c/sup\u003eM. Protein levels in \u003cstrong\u003e(A, C)\u003c/strong\u003e CM fractions and \u003cstrong\u003e(B, D) \u003c/strong\u003esupernatants,\u003cstrong\u003e \u003c/strong\u003ewere analysed by ELISA (C1R) and Multiplex (MMP1, MMP3, MMP9, MMP13, and C3). Values were normalized to total protein and plotted relative to the Fn-fs-treated cells. Data are presented as mean ± SEM of duplicate determinations (n=3) (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"OnlineFig6.png","url":"https://assets-eu.researchsquare.com/files/rs-6529495/v1/ed57588e3a851b9ae60c94b7.png"},{"id":82890546,"identity":"d6ef63a3-0dc5-42f4-9e03-c1a2ce9c2978","added_by":"auto","created_at":"2025-05-16 12:09:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1516973,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6529495/v1/e104366b-b047-4be1-b613-ccb7b079eb16.pdf"},{"id":82890427,"identity":"c32d8198-36a8-41dd-bf48-d85c89a51c04","added_by":"auto","created_at":"2025-05-16 12:09:06","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":598526,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1. \u003c/strong\u003eBM-hMSC pellets during chondrogenic differentiation. \u003cstrong\u003e(A) \u003c/strong\u003eIsotype and \u003cstrong\u003e(B) \u003c/strong\u003enegative controls from immunohistochemistry of \u003cem\u003eCOL2A1\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003ein untreated \u003cstrong\u003e(top) \u003c/strong\u003eand VIP-treated \u003cstrong\u003e(bottom) \u003c/strong\u003ecell pellets on days 1, 3, 6, 12, 17 and 21 of chondrogenic differentiation (scale bar = 200 μm). Objective 4X. Stereo microscope (Olympus SZX16) coupled to camera (Olympus SDF PLAPO 0.5XPF).\u003c/p\u003e","description":"","filename":"FigS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6529495/v1/4c6ef12f8c2baa081ca0a01e.tif"},{"id":82890430,"identity":"c17833c9-bd82-4e4d-abec-04f4513a4494","added_by":"auto","created_at":"2025-05-16 12:09:06","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":78928,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S2. \u003c/strong\u003eRatio between chondrogenic and hypertrophy markers during chondrogenesis of BM-hMSC in pellet cultures.\u003cstrong\u003e \u003c/strong\u003eRatio between mRNA expression of \u003cstrong\u003e(A) \u003c/strong\u003e\u003cem\u003eCOL2A1\u003c/em\u003eand \u003cem\u003eCOL10A1\u003c/em\u003e and \u003cstrong\u003e(B)\u003c/strong\u003e transcription factors \u003cem\u003eSOX9\u003c/em\u003e and \u003cem\u003eRUNX2\u003c/em\u003eon days 1, 12 and 21 of chondrogenic differentiation in absence or presence of VIP, determined by RT-qPCR. Results were normalized to the expression of \u003cem\u003eGADPH\u003c/em\u003e and plotted relative to the expression of BM-hMSC, using the ΔΔ cycle threshold method for quantification. Data are presented as mean ± SEM (n=3).\u003c/p\u003e","description":"","filename":"FigS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-6529495/v1/547788adffdcee04a6252e12.tif"},{"id":82890418,"identity":"9298fc79-2a97-4973-b27f-ff20a68cd1fb","added_by":"auto","created_at":"2025-05-16 12:09:03","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":90240,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S3. \u003c/strong\u003eRatio between chondrogenic and hypertrophy markers in OA-hAC dedifferentiated in monolayer and redifferentiated in alginate microbeads.\u003cstrong\u003e \u003c/strong\u003eRatio between mRNA expression of transcription factors \u003cem\u003eSOX9\u003c/em\u003e and \u003cem\u003eRUNX2\u003c/em\u003e determined by qPCR. Results were normalized to the expression of \u003cem\u003eGADPH\u003c/em\u003e using the Δ cycle threshold method for quantification. Data are presented as mean ± SEM (n=6).\u003c/p\u003e","description":"","filename":"FigS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-6529495/v1/6665cb8b1095f603c2009e23.tif"},{"id":82890437,"identity":"de4bd035-62bd-4a70-adfb-f728fdd1887f","added_by":"auto","created_at":"2025-05-16 12:09:08","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":154550,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S4. \u003c/strong\u003eEffect of Fn-fs and VIP on the expression of MMP and complement proteins in OA-hAC redifferentiated in alginate microbeads. Expression of complement proteins (C1R, and C3) and MMP (MMP-1, -3, -9, -13) was analysed in OA-hAC redifferentiated in alginate microbeads after 7 days of treatments. Protein levels in \u003cstrong\u003e(A, C)\u003c/strong\u003e CM fractions and \u003cstrong\u003e(B, D) \u003c/strong\u003esupernatants,\u003cstrong\u003e \u003c/strong\u003ewere analysed by ELISA (C1R) and Multiplex (MMP1, MMP3, MMP9, MMP13, and C3). Values were normalized to total protein and plotted relative to the untreated cells (n=3).\u003c/p\u003e","description":"","filename":"FigS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-6529495/v1/b5f1fc49d22ff639ea01fac1.tif"},{"id":82890431,"identity":"87c85ecb-f4c0-4886-999a-7e7272dc7c1d","added_by":"auto","created_at":"2025-05-16 12:09:06","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":16040,"visible":true,"origin":"","legend":"","description":"","filename":"KeyMessages.docx","url":"https://assets-eu.researchsquare.com/files/rs-6529495/v1/b43f8d7f70857cdc8218eb89.docx"}],"financialInterests":"","formattedTitle":"Vasoactive Intestinal Peptide advances chondrogenesis and modulates pathogenic mediators in human osteoarthritis.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteoarthritis (OA) is a multifactorial chronic rheumatic disease with a significant global impact, ranking among the leading causes of disability in individuals over the age of 70. Symptoms reflect a dynamic imbalance between joint tissue breakdown and repair, involving bone remodelling, synovial inflammation, and cartilage degeneration, ultimately leading to joint dysfunction and impaired quality of life[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eArticular chondrocytes (AC) are the main resident cells in articular cartilage, playing a central role in OA pathology. In healthy cartilage, these cells maintain homeostasis between anabolic and catabolic processes in the cartilage extracellular matrix (ECM) by synthesizing essential components including glycosaminoglycans (GAG), aggrecan (ACAN), and type II collagen (COL2)[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. During OA, this balance is disrupted, exposing these cells to inflammatory stress that alters their behaviour. This leads to the release of ECM degradation products with potential catabolic properties. Among them, fibronectin fragments (Fn-fs) stand out degrading essential ECM components, while also promoting the production of pro-inflammatory mediators, fostering further inflammation and cartilage degradation[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In the inflammatory microenvironment, other cells begin secreting pro-inflammatory factors, promoting chondrocyte apoptosis and hypertrophy, and reducing ECM component synthesis while increasing ECM-degrading proteases, like matrix metalloproteinases (MMP), contributing to the degradation of collagen, aggrecan, and other components of the cartilage ECM [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among them, the collagenase MMP13 is critical in OA pathogenesis, being the main enzyme involved in the degradation of COL2[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. On the other hand, a chronic low-grade inflammation is well documented, but the specific mediators involved in this pathology remain less understood. The complement system, as part of innate immunity, has been described to be involved in OA, with chondrocytes contributing to its production[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Increased complement protein expression has been observed in the synovial fluid from OA patients compared to healthy donors[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], being associated with pain[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe limited replication potential of AC reduces cartilage self-repair capacity. To address this problem, cell-based approaches and tissue engineering offer promising tools to improve cartilage repair[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. AC cultured in monolayer tend to dedifferentiate to a fibroblastic phenotype losing their chondrogenic characteristics from early passages[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. By contrast, three-dimensional (3D) cultures promote cell survival, functionality and morphology, bringing the \u003cem\u003ein vitro\u003c/em\u003e conditions closer to the \u003cem\u003ein vivo\u003c/em\u003e articular microenvironment, preventing chondrocyte dedifferentiation[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Further, mesenchymal stem cells (MSC) have emerged as candidates for regenerating damaged cartilage tissue due to their low immunogenicity, multipotent differentiation capability and ease of isolation from numerous sources[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. For MSC \u003cem\u003ein vitro\u003c/em\u003e culture, 3D pellet cultures effectively promote chondrogenic differentiation[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition, different biomaterials are currently being designed for that purpose[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. One of the most used materials is the alginate, a biocompatible polysaccharide, ideal for culturing chondrocytes[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Alginate enables redifferentiation, maintaining the spherical phenotype characteristic of \u003cem\u003ein vivo\u003c/em\u003e conditions, while promoting chondrogenic markers expression and preventing hypertrophy[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Among the chondrogenic markers, SRY-Box Transcription Factor 9 (SOX9) is a master transcription factor involved in the expression of target genes during chondrogenesis, including \u003cem\u003eCOL2A1\u003c/em\u003e and \u003cem\u003eACAN\u003c/em\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], which levels are reduced in OA patients[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Conversely, Runt Related Transcription Factor 2 (RUNX2) drives chondrocyte hypertrophy, characterized by an increase in the levels of type 10 collagen (COL10)[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Increased levels of \u003cem\u003eRUNX2\u003c/em\u003e have also been described in chondrocytes from OA patients, being involved in the expression of ECM-degrading enzymes including MMP13[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the limited regenerative capacity of cartilage and the lack of effective OA treatments, it is crucial to study new therapies aimed at stopping disease progression rather than merely alleviating symptoms[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In this context, we have focused on studying vasoactive intestinal peptide (VIP) as a potential therapeutic tool. VIP is a neuropeptide produced by neurons, as well as endocrine and immune cells, involved in modulating the innate and adaptive immune response. Previous results point to a beneficial effect on joint integrity and functionality in musculoskeletal pathologies[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In OA, we have previously described the effect of VIP by inhibiting the expression of degradative enzymes and inflammatory mediators in \u003cem\u003ein vitro\u003c/em\u003e cultures of synovial fibroblasts[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and chondrocytes co-cultures in monolayer[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Recent studies also emphasize its role during osteogenic differentiation, identifying it as a potential osteoinductor[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Exploring the effect of VIP on human chondrogenesis, as well as its role in modulating inflammatory and ECM-degrading mediators produced by OA chondrocytes within a pro-inflammatory microenvironment, could provide valuable insights for leveraging this peptide as a potential therapeutic tool for OA.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e3D pellet cultures of mesenchymal stem cells from healthy donors\u003c/h2\u003e \u003cp\u003eHuman bone marrow mesenchymal stem cells (BM-hMSC) from four healthy donors (two men and two women, aged 30\u0026ndash;50 years, various ethnicities) were obtained commercially from StemCell Technologies (Vancouver, Canada), characterized for MSC specific markers and tested for their ability to differentiate \u003cem\u003ein vitro\u003c/em\u003e into the different linages.\u003c/p\u003e \u003cp\u003eCulture flasks were previously treated with Animal Component-Free Cell Attachment Substrate (ACF-ATS; StemCell Technologies). BM-hMSC were cultured in MesenCult-ACF Plus Medium (StemCell Technologies) supplemented with 1% Glutamax (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) and 0,2% of its specific commercial supplement (StemCell Technologies). Cells were incubated at 37\u0026ordm;C and 5% CO\u003csub\u003e2\u003c/sub\u003e, changing the medium every 3 days.\u003c/p\u003e \u003cp\u003eAt confluence, 5x10\u003csup\u003e5\u003c/sup\u003e cells were seeded in 15-ml polypropylene conical tubes and centrifuged at 300xg for 5 minutes. Pelleted cells were incubated at 37\u0026ordm;C and 5% CO\u003csub\u003e2\u003c/sub\u003e in 500 \u0026micro;l of MesenCult-ACF Chondrogenic Differentiation Medium (StemCell Technologies) supplemented with 1% Glutamax, 1% penicillin/streptomycin and 5% of its specific commercial supplement (StemCell Technology) with or without VIP 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003eM (Bachem, Bubendorf, Switzerland) for up to 21 days, changing the medium every 3 days, according to the supplier\u0026rsquo;s instructions. Pellets were collected for histological and immunohistochemical analysis on days 1, 3, 6, 12, 17 and 21. For gene expression, pellets were homogenised using a potter pestle. RNA from homogenised pellets and BM-hMSC was extracted using Tri Reagent (Invitrogen) according to manufacturer's instruction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistological analysis of cell pellets\u003c/h3\u003e\n\u003cp\u003eBM-hMSC cell pellets cultured in presence or absence of VIP, were recovered on days 1, 3, 6, 12, 17 and 21, fixed in paraformaldehyde, embedded in paraffin, and sectioned in 5 \u0026micro;m thick sections with a microtome (Leica Histocore Biocut). Sections were stained with Alcian blue and Masson's trichrome.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemical detection of type II collagen\u003c/h3\u003e\n\u003cp\u003eImmunohistochemical analysis of type II collagen was performed on the previously described BM-hMSC cell pellets slides, using a primary rabbit polyclonal antibody for human collagen alpha-1(II) chain protein (1:100, Cusabio Technology, Houston, USA) and an anti-Rabbit Histofine Simple Stain (Nichirei, Tokyo, Japan). The reaction was revealed using solution of diaminobenzidine (DAB) with hydrogen peroxide as the enzyme substrate (Palex Medical S.A., Madrid, Spain), following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003ch3\u003e3D cultures of articular chondrocytes from osteoarthritis patients in alginate microbeads\u003c/h3\u003e\n\u003cp\u003eOsteoarthritis human articular chondrocytes (OA-hAC) were isolated from knee of 7 patients, 2 men and 5 women, aged between 58 and 84 years, undergoing total knee joint replacement surgery. Patients had advanced disease and were diagnosed of primary OA, excluding trauma, inflammatory disease and secondary OA. Samples were provided by the Rheumatology Service at Instituto de Investigaci\u0026oacute;n Biom\u0026eacute;dica (INIBIC) Complejo Hospitalario Universitario A Coru\u0026ntilde;a (CHUAC, A Coru\u0026ntilde;a, Spain). Informed consent was obtained from all patients before surgery. The study was approved by the Clinical Research Ethics Committee of Hospital Universitario de La Princesa and performed according to the recommendations of the Declaration of Helsinki.\u003c/p\u003e \u003cp\u003eOA-hAC were expanded in monolayer in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) with 4.5g/l glucose (Corning, NY, USA), 10% fetal bovine serum (FBS), 25mM HEPES (Lonza Ib\u0026eacute;rica SA, Barcelona, Spain), 1% Glutamax and 1% penicillin/streptomycin (Invitrogen) until passage 4. Cells were incubated at 37\u0026ordm;C and 5% CO\u003csub\u003e2\u003c/sub\u003e, changing the medium every 3 days. For gene expression analysis, total RNA was extracted from the dedifferentiated hAC using Tri Reagent (Invitrogen) according to manufacturer's instruction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cem\u003eupper\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eFor redifferentiation studies, OA-hAC were used after passage 4, and suspended in 1% medium viscosity alginate solution (Pronova UP, MVG; NovaMatrix, Sandvika, Norway) at a density of 2x10\u003csup\u003e6\u003c/sup\u003e cells/ml. Alginate solution was formerly dissolved in 0.85% NaCl (Lonza Ib\u0026eacute;rica SA). Microbeads were made by dropping the resulting suspension through a 25G needle at a constant rate and height, in 15 ml of a solution composed of 100 mM CaCl\u003csub\u003e2\u003c/sub\u003e and 15 mM HEPES, under agitation. Then, to promote polymerization, microbeads were kept in the same container for 15 min without agitation and subsequently washed 2 times with 0.85% NaCl.\u003c/p\u003e \u003cp\u003eNine microbeads per well were placed in a 24-well plate and cultured in 1 ml of DMEM with 4.5g/l glucose, (Corning), 25mM HEPES (Lonza Ib\u0026eacute;rica SA), 1% ITS (composed of insulin, transferrin, bovine serum albumin, selenic acid and linoleic acid; Corning), 1% Glutamax and 1% penicillin/streptomycin (Invitrogen), ascorbic acid 50 \u0026micro;g/ml (Merck, Darmstadt, Germany), proline 2mM (Merck) and transformant factor beta 1 (TGF-β1; Peprotech, Thermo Fisher Scientific) 10 ng/ml to promote redifferentiation. Cells were incubated at 37\u0026ordm;C and 5% CO\u003csub\u003e2\u003c/sub\u003e for 2 weeks, changing the medium every 3 days.\u003c/p\u003e \u003cp\u003eTo verify redifferentiation of the OA-hAC, alginate microbeads were washed with 1 ml of 0.85% NaCl and dissolved with 1 ml of 0.1 M citrate buffer for 10 min. Resulting suspension was centrifuged at 1200 rpm for 10 min, obtaining 2 fractions: the upper phase containing alginate wastes and macromolecules called further-removed matrix (FRM), and the pellet with cells and their associated cellular matrix (CM). Total RNA was extracted from CM fraction using Tri Reagent (Invitrogen) according to manufacturer's instruction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cem\u003eupper\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eFor the treatment study, after 2 weeks of redifferentiation, TGF-β1 was no longer added to the medium and OA-hAC were cultured for 7 additional days in presence or absence of VIP 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003eM, 45kDa Fn-fs 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003eM or the combined treatment, changing the medium every 3 days. Six replicates of each condition were set up for the experiment. Cells were incubated at 37\u0026ordm;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. For protein detection, culture supernatants were collected, and CM fraction was obtained as described above (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cem\u003elower\u003c/em\u003e).\u003c/p\u003e\n\u003ch3\u003eReal-time polymerase chain reaction analysis\u003c/h3\u003e\n\u003cp\u003e1,2\u0026micro;g of RNA were reverse transcribed using a High Capacity cDNA Reverse Transcription (RT) Kit (Applied Biosystems, Waltham, MA, USA) and semiquantitative real-time polymerase chain reaction (RT-qPCR) was performed using a TaqMan Gene Expression Master Mix (Applied Biosystems) with manufactured-predesigned primers for \u003cem\u003eSOX9\u003c/em\u003e (Hs00165814_m1), \u003cem\u003eCOL2A1\u003c/em\u003e (Hs00264051_m1), \u003cem\u003eACAN\u003c/em\u003e (Hs00153936_m1), \u003cem\u003eRUNX2\u003c/em\u003e (Hs01047975_m1), \u003cem\u003eCOL10A1\u003c/em\u003e (Hs00166657_m1), \u003cem\u003eMMP13\u003c/em\u003e (Hs00942584_m1). Glyceraldehyde 3-phosphate dehydrogenase (\u003cem\u003eGAPDH\u003c/em\u003e) (Hs99999905_m1) was used as reference gene. Results were normalized to the expression of \u003cem\u003eGAPDH\u003c/em\u003e and plotted relative to the expression in the untreated condition, in the case of treatments, using the ΔΔ cycle threshold method for quantification.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation assay\u003c/h2\u003e \u003cp\u003eProliferation was determined in the redifferentiated OA-hAC cultured in microbeads after 7 days in presence of the treatments described above, using the Cell Proliferation Reagent WST-1 (Merck). This assay measures mitochondrial metabolic activity as an indicator of cell viability. Briefly, one bead per well was placed in a 96-well plate with 200 \u0026micro;l of DMEM in presence or absence of VIP 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003eM, 45 kDa Fn-fs 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003eM or the combined treatment. After 7 days, microbeads were incubated with WST-1 reagent (1:100) for 4 hours at 37\u0026ordm;C. 100 \u0026micro;l of supernatant per well were transferred to a new 96-well plate and the optical density was measured at 450 nm. The experiment was performed in duplicate.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSulfated glycosaminoglycans assay\u003c/h3\u003e\n\u003cp\u003eSulfated glycosaminoglycan (GAG) content was measured in CM fractions from redifferentiated OA-hAC after 7 days of treatment, using a Blyscan Sulfated Glycosaminoglycan Assay (Biocolor Ltd, Carrickfergus, United Kingdom). Briefly, microbeads were washed with 0.85% NaCl, dissolved in 0.1M citrate buffer, and centrifuged at 1200 rpm for 10 min, obtaining the CM fraction. Papain Extraction Reagent (composed of sodium phosphate buffer pH 6.4 with sodium acetate, EDTA, cysteine-HCl and papain crystalized suspension (Sigma-Aldrich)) was added to the CM fraction. Samples were digested with the solution for 3 hours at 65\u0026ordm;C and the assay was done following manufacturer\u0026rsquo;s instructions. DNA content was measured using QuantiFluor dsDNA System (Promega Biotech Ib\u0026eacute;rica S.L., Madrid, Spain). Fluorescence intensity was measured using the FLUOstar Omega (BMG LABTECH) at an excitation wavelength of 504 nm and an emission wavelength of 531 nm. The assay was performed following manufacturer\u0026rsquo;s instructions. GAG content was normalized to DNA (GAG/DNA ratio).\u003c/p\u003e\n\u003ch3\u003eELISA and Multiplex assays\u003c/h3\u003e\n\u003cp\u003eProtein expression of complement system proteins and MMP was analysed in cultured supernatants and CM fractions from redifferentiated OA-hAC after 7 days of treatment. For protein extraction, RIPA lysis buffer (Thermo Fisher Scientific) supplemented with Protease and Phosphatase Inhibitors (Thermo Fisher Scientific) was added to CM fraction. Next the samples were sonicated, and protein content was measured with a QuantiPro BCA Assay Kit (Sigma-Aldrich, San Luis, Missouri, USA). Then, a commercial ELISA kit for C1R (Merck) and a MILLIPLEX\u0026reg; Multiplex Assays for human C3, MMP1, MMP3, MMP9, and MMP13 (Merck) were used, following manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software, Inc., CA, USA). Data were subjected to normality test (Kolmogorov-Smirnov test) and equal variance test (\u003cem\u003eF\u003c/em\u003e-test). Statistical differences for parametric variables were assessed via t-test and analysis of variance (ANOVA) together with Sidak \u003cem\u003epost hoc\u003c/em\u003e test, while Kruskal-Wallis test or Mann-Whitney U test were used for non-parametric variables. Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (Standard Error of Mean). \u003cem\u003eP\u003c/em\u003e-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant (*\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis of pellets during BM-hMSC chondrogenesis\u003c/h2\u003e \u003cp\u003eBM-hMSC were cultured in pellet with chondrogenic medium in presence and absence of VIP for up to 21 days. Cell pellets exhibited irregular and inconsistent morphology in the early days of cultivation (days 1\u0026ndash;6). However, as the culture progressed, they adopted a more rounded and consistent morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo visualize the evolution of ECM formation, we stained cell pellets sections with Alcian blue and Masson's trichrome for monitoring GAG and collagen depositions. We noticed that ECM formation increases throughout the days in untreated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cem\u003etop\u003c/em\u003e) and VIP-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cem\u003ebottom\u003c/em\u003e). This deposition compacted the cell pellets giving them the appearance of cartilaginous tissue.\u003c/p\u003e \u003cp\u003eIn sections of the same pellets, we also detected the deposition of type II collagen in untreated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cem\u003etop\u003c/em\u003e) and VIP-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cem\u003ebottom\u003c/em\u003e) over the days. No staining was detected in the negative or in the isotype controls (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eVIP advances chondrogenesis in BM-hMSC\u003c/h2\u003e \u003cp\u003eTo determine the effect of VIP during chondrogenesis, we analysed the differences in gene expression of the chondrogenic marker \u003cem\u003eCOL2A1\u003c/em\u003e between untreated and VIP-treated BM-hMSC cultured in pellet under chondrogenic conditions for up to 21 days. While in untreated cells significant differences compared to day 1 were observed on day 21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), in presence of VIP a significant increase was observed as early as day 12, corresponding to the highest expression of the gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Accordingly, when comparing both conditions at each time point, \u003cem\u003eCOL2A1\u003c/em\u003e expression was significantly higher in VIP-treated BM-hMSC on day 6, with the greatest difference observed on day 12. This difference reversed on day 21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo corroborate these results, we studied the expression of additional chondrogenic (\u003cem\u003eSOX9\u003c/em\u003e and \u003cem\u003eACAN\u003c/em\u003e) and chondrocyte hypertrophy markers (\u003cem\u003eRUNX2\u003c/em\u003e, \u003cem\u003eCOL10A1\u003c/em\u003e and \u003cem\u003eMMP13\u003c/em\u003e) in BM-hMSC cell pellets on days 1, 12 and 21. Our results show that on day 12, VIP significantly increased the expression of the chondrogenic markers \u003cem\u003eSOX9\u003c/em\u003e and \u003cem\u003eACAN\u003c/em\u003e compared to untreated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). By contrast, the expression of \u003cem\u003eACAN\u003c/em\u003e was higher in untreated cells on day 21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Regarding hypertrophy genes, initial expression levels of \u003cem\u003eRUNX2\u003c/em\u003e, \u003cem\u003eCOL10A1\u003c/em\u003e and \u003cem\u003eMMP13\u003c/em\u003e on day 1 were higher with the treatment of VIP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G, H). However, these differences disappeared over the days. In addition, even no significant differences were observed, the ratios \u003cem\u003eCOL2A1/COL10A1\u003c/em\u003e and \u003cem\u003eSOX9/RUNX2\u003c/em\u003e were also higher in VIP-treated BM-hMSC on day 12 (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAlginate microbeads allow redifferentiation of OA-hAC\u003c/h2\u003e \u003cp\u003eTo corroborate that alginate microbeads promote redifferentiation of OA-hAC, we analysed mRNA expression of the chondrogenic markers \u003cem\u003eSOX9\u003c/em\u003e, \u003cem\u003eCOL2A1\u003c/em\u003e and \u003cem\u003eACAN\u003c/em\u003e in OA-hAC previously dedifferentiated in monolayer (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), as well as in the redifferentiated OA-hAC cultured in alginate microbeads for 2 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). Additionally, we measured the expression of \u003cem\u003eRUNX2\u003c/em\u003e as hypertrophy marker. The results revealed that both, dedifferentiated and redifferentiated OA-hAC, expressed the studied genes, with a significant higher expression of \u003cem\u003eSOX9\u003c/em\u003e and \u003cem\u003eCOL2A1\u003c/em\u003e in OA-hAC after 2 weeks of redifferentiation in microbeads (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Despite, no significant differences were observed, these cells also had a higher \u003cem\u003eSOX9/RUNX2\u003c/em\u003e ratio than the dedifferentiated chondrocytes (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffects of VIP and Fn-fs in the survival of OA-hAC in alginate microbeads\u003c/h2\u003e \u003cp\u003eTo assess the effect of VIP in OA-hAC treated with the pro-inflammatory stimulus Fn-fs, OA-hAC redifferentiated in microbeads were subjected to 7 additional days of treatment with VIP, Fn-fs or the combined treatment. The results showed that while Fn-fs decreased cell proliferation, the presence of VIP counteracted its effects, promoting a significant increase in OA-hAC proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eModulation of glycosaminoglycans production by VIP in Fn-fs treated OA-hAC\u003c/h2\u003e \u003cp\u003eTo analyse the production of GAG in the redifferentiated OA-hAC, microbeads were cultured for 7 additional days in presence of the treatments described above. The results indicated that OA-hAC treated with Fn-fs in combination with VIP, exhibited a significantly higher GAG/DNA ratio in the CM protein fraction compared to cells treated with Fn-fs alone, which supposes a greater ECM/GAG deposition in presence of the neuropeptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eVIP decreases production of complement system proteins and MMP in Fn-fs treated OA-hAC\u003c/h2\u003e \u003cp\u003eTo evaluate the effect of VIP on the production of complement system components as inflammatory mediators (C1R and C3) and its influence on the modulation of ECM-degrading enzymes (MMP1, MMP3, MMP9, and MMP13), protein levels of these mediators were analysed by ELISA and Multiplex assays. These analyses were performed on the CM fraction and culture supernatants of OA-hAC redifferentiated in microbeads treated with Fn-fs in the presence or absence of VIP. The results showed that the combined treatment with VIP tended to decrease the production of the studied mediators. Specifically, in complement proteins, a significant reduction was observed for C1R in both, CM fraction and supernatants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). No expression of C3 was detected in the culture supernatants. Besides, VIP significantly decreased the levels of MMP1 and MMP13 in the CM fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). This reduction was also significant for MMP13 in culture supernatants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). No effects were observed with the treatment of VIP alone (Supplementary Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHuman MSC have emerged as a promising therapeutic tool for OA treatment and cartilage repair[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Different studies have focused on optimizing hMSC differentiation through \u003cem\u003ein vitro\u003c/em\u003e strategies[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Although hMSC offer promising results, further research is necessary for their application in cartilage repair. These cells can be induced to express cartilage-specific molecules like COL2 and ACAN, under the control of SOX9[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, they may also develop undesirable fibrocartilage-like features and undergo hypertrophy during chondrogenesis[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], where RUNX2 and COL10 are involved[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this sense, VIP and related peptides play important roles in chondrogenesis and osteogenesis. Previous studies have reported pituitary adenylate cyclase activating polypeptide (PACAP) as a positive regulator of chondrogenesis in chicken[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Nonetheless, the present study is the first to report the effect of VIP in this process in humans. Our findings reveal that VIP increases the expression of \u003cem\u003eSOX9\u003c/em\u003e, \u003cem\u003eCOL2A1\u003c/em\u003e, and \u003cem\u003eACAN\u003c/em\u003e on day 12, advancing chondrogenesis in BM-hMSC, without inducing hypertrophy. These results support the use of BM-hMSC and VIP as potential therapeutic strategies for OA treatment and cartilage repair.\u003c/p\u003e \u003cp\u003eRecent studies have reported the importance of neuro-immune interactions in OA[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this context, VIP plays a complex role in the pathology. Previous findings have described reduced VIP levels in the synovial fluid and articular cartilage of OA patients, correlating with increased disease severity[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This downregulation may contribute to OA pathogenesis by enhancing pro-inflammatory cytokine production. Conversely, other studies suggest that VIP accumulation in joints may contribute to OA[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], promoting subchondral bone sclerosis and angiogenesis[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Despite these conflicting findings, several researches support that upregulation of VIP may counteract pro-inflammatory stimuli and reduce OA pain[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Moreover, in cell co-cultures from OA patients, VIP has demonstrated anti-inflammatory and immunomodulatory actions, potentially modulating the secretome of chondrocytes and synovial fibroblasts[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In the present study, to elucidate the role of VIP in OA, we have employed 3D cultures for hAC as they have shown better results in maintaining chondrocyte phenotype compared to two-dimensional monolayer cultures[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. We corroborate that redifferentiated OA-hAC in 3D cultures in alginate microbeads show a higher expression of the chondrogenic markers \u003cem\u003eSOX9\u003c/em\u003e and \u003cem\u003eCOL2A1\u003c/em\u003e compared to dedifferentiated cells in monolayer[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. These findings highlight the importance of 3D culture systems in preserving chondrocyte phenotype for cartilage tissue engineering applications. On the other hand, since VIP requires a pro-inflammatory context to exert significant modulatory effects, we employed Fn-fs, key contributors to OA pathophysiology. These fragments contribute to the initiation and progression of the disease by inducing ECM-degrading enzymes and pro-inflammatory mediators, promoting cartilage catabolism [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Moreover, they are present at high concentrations in synovial fluid and cartilage from OA patients[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Our results reveal that in presence of Fn-fs, VIP enhances both, cell proliferation and GAG production in hAC from OA patients. These findings corroborate the beneficial effects of the neuropeptide promoting cell survival and ECM formation in a pro-inflammatory microenvironment.\u003c/p\u003e \u003cp\u003eIn relation to the catabolic process during OA progression, MMP play a crucial role, degrading essential ECM components in articular cartilage. Among these, stromelysins like MMP3, and gelatinases like MMP9, potentially contribute to cartilage destruction[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Notably, the collagenases, MMP1 and MMP13, whose levels are increased in OA cartilage, play key roles in the pathogenesis[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. MMP13 is specifically expressed in OA patients and is critical in OA progression by degrading type II collagen, making it an attractive target for OA treatment. Consequently, selective MMP13 inhibitors are being developed as a promising strategy for OA therapy[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The up-regulation of MMP13 by Fn-fs has been previously described in bovine osteochondral explants and culture supernatants[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Here we demonstrated that, in Fn-fs presence, VIP decreases the production of MMP in OA-hAC, with significant reduction for the collagenases, MMP1 and MMP13, in the CM protein fraction, also with a significant decrease of MMP13 in culture supernatants from microbeads. The ability of VIP to reduce MMP13 in presence of Fn-fs has also been previously described in culture supernatants from synovial fibroblasts of OA patients[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Our results support the use of VIP as a potential therapeutic tool for targeting key enzymes involved in the pathology. Selectivity of VIP may be due to a combination of its mechanism of action, the differential regulation of MMP in the cellular microenvironment, and the specific pathways involved in their expression. Additional experiments could include analysing the signalling pathways involved or modulating key transcription factors to confirm these hypotheses.\u003c/p\u003e \u003cp\u003eIn addition to the catabolic process, the inflammatory microenvironment also contributes to the pathology. In this context, previous research emphasizes the significant role of the complement system in OA with an abnormally high expression and activation, associated with inflammation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. We previously reported that, in presence of Fn-fs, VIP reduced levels of C1R and C3 in the secretome of synovial fibroblasts and OA-hAC co-cultured in monolayer[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Accordingly, in this work we demonstrate that VIP decreases protein C1R expression in both CM fraction and culture supernatants from 3D OA-hAC cultures in microbeads. Although no significant differences were observed for C3, a trend toward reduction was also noted in CM fraction in presence of VIP. These results reinforce the anti-inflammatory potential of the neuropeptide.\u003c/p\u003e \u003cp\u003eThis study is the first to demonstrate the positive effects of VIP in modulating chondrogenesis of BM-hMSC, while also highlighting its potential to regulate inflammatory and catabolic mediators in 3D cultures of hAC from OA patients. Our findings suggest that targeting MMP and complement factors could serve as a potential therapeutic strategy for the treatment of OA, positioning VIP as a promising candidate for therapies aimed at slowing OA progression and improving outcomes for affected individuals.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments and Funding Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants RD21/0002/004 and RD24/0007/0014 from the Ministerio de Econom\u0026iacute;a y Competitividad (Instituto de Salud Carlos III) and co‐funded by European regional development fund (ERDF) and by the UCM grants PR12/24-31572 and PR12/24-31568. We are grateful to all patients and the collaborating clinicians for their participation in this study. We also appreciate the assistance of Isabel Montero, Technician at the Department of Cell Biology and Histology at the Faculty of Biological Science, UCM, for the sectioning of paraffin embedded cell pellets.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Karolina Tecza, Cristina Rodr\u0026iacute;guez-Hern\u0026aacute;ndez and Selene P\u0026eacute;rez-Garc\u0026iacute;a. The first draft of the manuscript was written by Selene P\u0026eacute;rez-Garc\u0026iacute;a and Karolina Tecza, and all authors commented on previous versions of the manuscript. Funding acquisition was provided by Rosa P. Gomariz, Yasmina Juarranz and Carmen Mart\u0026iacute;nez. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Hospital Universitario de La Princesa (06-06-24, acta CEIm 11/24).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCourties A, Kouki I, Soliman N, Mathieu S, Sellam J (2024) Osteoarthritis year in review 2024: Epidemiology and therapy. Osteoarthritis and cartilage / OARS. 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J Orthop Res 32:811\u0026ndash;818. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jor.22610\u003c/span\u003e\u003cspan address=\"10.1002/jor.22610\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmme","sideBox":"Learn more about [Journal of Molecular Medicine](https://www.springer.com/journal/109)","snPcode":"109","submissionUrl":"https://submission.nature.com/new-submission/109/3","title":"Journal of Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"osteoarthritis, VIP, chondrocytes, mesenchymal stem cells, MMP, complement system","lastPublishedDoi":"10.21203/rs.3.rs-6529495/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6529495/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCurrent therapies for osteoarthritis (OA) focus on symptom management, rather than halting disease progression. Vasoactive intestinal peptide (VIP) has shown promising effects in musculoskeletal diseases, preserving joint integrity and modulating inflammation. This study investigates the potential of VIP to promote chondrogenic differentiation of human bone marrow mesenchymal stem cells (BM-hMSC), while modulating inflammatory and cartilage extracellular matrix (ECM)-degrading mediators in human articular chondrocytes from OA patients (OA-hAC). BM-hMSC from healthy donors were cultured in pellet under chondrogenic conditions with or without VIP up to 21 days. The production of type II collagen (COL2A1), and the expression of chondrogenic (\u003cem\u003eSOX9, COL2A1\u003c/em\u003e, and \u003cem\u003eACAN\u003c/em\u003e) and hypertrophy (\u003cem\u003eRUNX2\u003c/em\u003e, \u003cem\u003eCOL10A1\u003c/em\u003e, and \u003cem\u003eMMP13\u003c/em\u003e) genes were assessed at different time points. VIP increased the expression of the chondrogenic genes on day 12 of differentiation, compared to day 21 in untreated BM-hMSC cells, advancing chondrogenesis. Furthermore, OA-hAC were dedifferentiated in monolayer followed by redifferentiation in alginate microbeads and treated with fibronectin fragments (Fn-fs) in presence and absence of VIP. We analysed VIP effects on cell proliferation, glycosaminoglycans (GAG) production, and modulation of components of the complement system (C1R and C3) and matrix metalloproteinases (MMP1, MMP3, MMP9, and MMP13). VIP enhanced cell proliferation, increased GAG deposition, and reduced production of complement factor C1R, and metalloproteinases MMP1 and MMP13 in OA-hAC. This study highlights the potential of VIP in modulating chondrogenesis, inflammation, and cartilage degradation supporting the development of future VIP-based therapies to slow OA progression.\u003c/p\u003e","manuscriptTitle":"Vasoactive Intestinal Peptide advances chondrogenesis and modulates pathogenic mediators in human osteoarthritis.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-16 12:08:39","doi":"10.21203/rs.3.rs-6529495/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2025-09-21T09:39:53+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-05-13T21:17:05+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-13T15:47:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-28T06:16:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Molecular Medicine","date":"2025-04-25T09:42:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmme","sideBox":"Learn more about [Journal of Molecular Medicine](https://www.springer.com/journal/109)","snPcode":"109","submissionUrl":"https://submission.nature.com/new-submission/109/3","title":"Journal of Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c5e65fb2-b9be-4879-bbba-504060303b0d","owner":[],"postedDate":"May 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-08T09:16:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-16 12:08:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6529495","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6529495","identity":"rs-6529495","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}