Mesenchymal Stem Cell Secretome Effectiveness on Healing of Chronic Tendon Injury: Analysis of Procollagen Type I N- Terminal Peptide and Procollagen Type Iii N-terminal Peptide and Histopathology in Rat’s Tendon (Rattus Norvegicus)

Mesenchymal Stem Cell Secretome Effectiveness on Healing of Chronic Tendon Injury: Analysis of Procollagen Type I N- Terminal Peptide and Procollagen Type Iii N-terminal Peptide and Histopathology in Rat’s Tendon (Rattus Norvegicus) | 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 Mesenchymal Stem Cell Secretome Effectiveness on Healing of Chronic Tendon Injury: Analysis of Procollagen Type I N- Terminal Peptide and Procollagen Type Iii N-terminal Peptide and Histopathology in Rat’s Tendon (Rattus Norvegicus) Andi Dhedie Prasatia Sam, Warsinggih Warsinggih, Muhammad Andry Usman, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5257458/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Chronic tendon injuries, such as Achilles tendinopathy, are common and challenging to treat due to the limited regenerative capacity of tendon tissue. Using mesenchymal stem cell (MSC) secretome, which contains a rich array of growth factors, holds promise for enhancing tendon healing. This study aimed to evaluate the effectiveness of MSC secretome, derived from tendon-derived stem cells (TDSCs) and adipose-derived stem cells (ASCs), on the healing of chronic Achilles tendon injuries in a rat model, focusing on the levels of Procollagen Type I N-Terminal Peptide (PINP) and Procollagen Type III N-Terminal Peptide (PIIINP), and histopathological changes. Methods A chronic tendinopathy model was induced in 16 males of Rattus norvegicus via mechanical overloading and collagenase injection. Rats were divided into four groups: TDSC secretome, ASC secretome, combined TDSC + ASC secretome, and a control group. Secretomes were administered intratendinously. Tendon healing was assessed after four weeks using enzyme-linked immunosorbent assays (ELISA) to measure PINP and PIIINP levels and histopathological analysis to evaluate collagen deposition and tissue structure. Results PINP levels were significantly higher in the TDSC + ASC group compared to the control group (p = 0.004), indicating enhanced Type I collagen synthesis. However, no significant differences were observed in PIIINP levels between the groups. The histopathological analysis did not reveal significant structural differences in tendon healing among the groups, though increased collagen alignment was observed in the TDSC + ASC group. Conclusions The combined TDSC and ASC secretome promotes Type I collagen synthesis in chronic tendon injuries, but histological improvements were insignificant. Further studies are needed to confirm the long-term benefits of secretome therapy. Mesenchymal Stem Cell Secretome Chronic Tendon Injury Tendon Healing Rat Model Figures Figure 1 INTRODUCTION Repetitive motion and age-related degeneration significantly increase the susceptibility of tendons, crucial connective tissues primarily composed of parallel collagen fibers embedded in an extracellular matrix, to injuries ( 1 ). The Achilles tendon, the largest and strongest tendon in the human body, connecting the gastrocnemius and soleus muscles, is designed to endure significant tensile forces ( 2 , 3 ). Tendon and ligament injuries are among the most frequently diagnosed musculoskeletal problems, with 30–50% of sports-related injuries involving tendons ( 4 ). Achilles tendon ruptures, particularly common in adults aged 30–50, represent the most frequent tendon ruptures in the lower extremities ( 5 ). While not life-threatening, these injuries can be severely debilitating, leading to a decline in quality of life and substantial healthcare costs ( 6 ). The healing of tendons is often poor, typically resulting in scar tissue structurally and functionally inferior to normal tendon tissue ( 7 ). Chronic tendon injuries are complicated to treat, requiring extensive intervention to restore pain-free function. Unlike acute injuries, which trigger an inflammatory response, chronic tendon injuries are characterized by collagen degeneration, a process where the collagen fibers in the tendon break down due to prolonged wear and tear and limited blood supply. This highlights the need for improved treatment strategies, especially for chronic cases.( 8 ) Recent attention has turned to cell-based therapies, mainly stem cells, which have shown significant potential in enhancing tendon repair through paracrine mechanisms. Paracrine signaling is a form of cell-to-cell communication in which a cell produces a signal to induce changes in nearby cells, altering their behavior and phenotype. The stem cell secretome, rich in growth factors like fibroblast growth factor (FGF), holds promise in improving tendon healing through these paracrine mechanisms, although its role in chronic injuries remains underexplored ( 9 , 10 ). A critical aspect of tendon healing involves collagen synthesis, particularly the production of type I and III procollagen. Procollagen type I N-terminal propeptide (PINP) and procollagen type III N-terminal propeptide (PIIINP) are markers of collagen metabolism that play a significant role in tendon repair. Elevated markers indicate active collagen synthesis, essential for tendon callus formation and overall tissue healing ( 11 , 12 ). Despite this, the specific dynamics of P1NP and PIIINP in chronic tendon injury repair, particularly in response to stem cell secretome treatment, remain inadequately studied. This study aims to investigate the effects of stem cell secretome on the repair of chronic Achilles tendon injuries. The histopathological analysis will evaluate structural changes, and healing efficacy will be assessed using immunoserological markers such as PINP and PIIINP. Our findings will provide intriguing insights into the potential of the secretome to significantly enhance tendon repair. MATERIALS & METHODS Study Design and Setting This study was conducted as an experimental laboratory to evaluate MSC-derived secretomes' effects on tendon healing in a rat model of chronic tendinopathy. The research was carried out at the Stem Cell Research and Development Center, Universitas Airlangga, and the Laboratory of Rumah Sakit Pendidikan Tinggi Negeri (RSPTN) Universitas Hasanuddin between July and December 2023. Preparation of Animal Samples A total of 16 adult male Rattus norvegicus rats, aged 8–12 weeks and weighing approximately 200–250 grams, were used in this study. The rats were housed under standard laboratory conditions with a 12-hour light/dark cycle and had free access to food and water. The Animal Ethics Committee of Universitas Airlangga approved all animal procedures. Chronic tendinopathy was induced in all rats through a combination of mechanical and enzymatic methods. The Achilles tendon of the right hindlimb was subjected to repetitive mechanical overloading by forced treadmill running at a speed of 15 m/min for 30 minutes per day over four weeks. Additionally, the tendons were injected with collagenase type I to induce collagen degradation and simulate chronic degenerative changes. After the induction of tendinopathy, the rats were randomly divided into four treatment groups (n = 4 per group): Group I, Tendon-Derived Stem Cells (TDSCs): Rats received an intratendinous injection of 50 µL of secretome derived from TDSCs. Group II, Adipose-Derived Stem Cells (ASCs): Rats received an intratendinous injection of 50 µL of secretome derived from ASCs. Group III (TDSCs + ASCs): Rats received an intratendinous injection of 25 µL of TDSC secretome and 25 µL of ASC secretome. Group IV (Control): Rats received an intratendinous injection of 50 µL of sterile saline (placebo). Preparation of MSC-Derived Secretomes and Biochemical Analysis TDSCs were isolated from the Achilles tendons of healthy donor rats, while ASCs were isolated from the inguinal adipose tissue. Both types of MSCs were cultured under standard conditions and allowed to reach confluence. The secretomes were collected from the culture media after 72 hours of incubation, filtered, and concentrated using centrifugation. The protein concentration of the secretomes was measured, and aliquots were stored at -80°C until use. Tendon healing was evaluated four weeks post-treatment using a combination of biochemical and histopathological analyses. Tendon samples were harvested, and the levels of PINP and PIIINP were measured using enzyme-linked immunosorbent assays (ELISA). PINP is a marker for collagen type I synthesis, while PIIINP is a marker for collagen type III synthesis. The levels of these markers provide insights into the remodeling process and the balance between collagen types I and III during tendon healing. Histopathological Analysis The harvested tendons were fixed in 10% formalin, embedded in paraffin, and sectioned for histopathological evaluation. Hematoxylin and eosin staining was performed to assess the general tissue architecture, while Masson's trichrome staining was used to evaluate collagen deposition. The sections were examined under a light microscope, and histopathological scoring was performed based on the degree of collagen alignment, angiogenesis, and cartilage formation. Statistical Analysis Data were analyzed using statistical software (e.g., SPSS, version 25.0). Continuous variables were expressed as mean ± standard deviation (SD). The differences between groups were analyzed using one-way analysis of variance (ANOVA) followed by post hoc Tukey's test for pairwise comparisons if the data is usually distributed and the Kruskal-Wallis test if the data is not normally distributed. A p-value of < 0.05 was considered statistically significant. RESULTS Procollagen I and III Levels in Tendon Samples The levels of PINP and PIIINP were measured in rat tendon samples (Rattus Norvegicus) injected with MSC secretomes derived from various sources. Four groups were included: TDSCs, ASCs, a combination of TDSCs and ASCs, and a control group. The measured levels of PINP and PIIINP in the different groups are summarized in Table 1 . The mean, standard deviation, median, minimum, and maximum values for PINP and PIIINP levels across the four groups are presented in Table 2 . A Shapiro-Wilk test was performed to assess the normality of PINP and PIIINP data. The results indicate that the data for both PINP and PIIINP are not normally distributed (p < 0.05). Therefore, non-parametric tests were used for further analysis. Table 1 Characteristics of PINP and PIIINP Levels in Rat Tendon Samples No Group PINP (ng/ml) PIIINP (ng/ml) 1 TDSCs 4 3 2 TDSCs 4 2 3 TDSCs 4 3 4 TDSCs 4 3 5 ASCs 4 3 6 ASCs 3 3 7 ASCs 4 3 8 ASCs 5 3 9 TDSCs + ASCs 5 2 10 TDSCs + ASCs 5 3 11 TDSCs + ASCs 4 3 12 Control 3 3 13 Control 2 2 14 Control 3 2 Table 2 Descriptive Statistics of PINP and PIIINP Levels Group Mean ± SD Median Min Max TDSCs 4.00 ± 0.00 4.00 4.00 4.00 ASCs 4.00 ± 0.82 4.00 3.00 5.00 TDSCs + ASCs 4.67 ± 0.58 5.00 4.00 5.00 Control 2.67 ± 0.58 3.00 2.00 3.00 Comparative Analysis of PINP and PIIINP Levels The non-parametric Kruskal-Wallis test compared the four groups' PINP and PIIINP levels. The results showed a significant difference in PINP levels (p = 0.039), while no significant difference was found among PIIINP groups (p = 0.318). Post-hoc analysis using the Mann-Whitney U test compared PINP levels between the experimental and control groups. The results indicate that combining TDSCs and ASCs significantly increased PINP levels compared to the control group (p = 0.004). The differences between the other groups and the control group were not statistically significant (p > 0.05) (Table 3 ). Table 3 Post-Hoc Analysis for PINP Levels Compared to Control Group Comparison p-value ASCs vs. Control 0.056 TDSCs vs. Control 0.056 TDSCs + ASCs vs. Control 0.004 Histopathological Findings and Analysis Histopathological evaluation of the tendon samples was conducted to assess collagen formation, angiogenesis, and cartilage formation. The scoring of histopathological findings is presented in Table 4 , and the histopathological appearance of the samples is shown in Fig. 1 . The results of the Kruskal-Wallis test for the variable Total Histopathological Scores show a p-value of 0.092, indicating no significant difference in total histopathological scores among the four groups. Table 4 Scoring and Histopathological Descriptions in Rat Tendon Samples (Rattus Norvegicus) No Sample Name Collagen Angiogenesis Cartilage Formation Total Score Remarks 1 AD 1 1 1 0 2 Hypercellular collagen 2 AD 2 1 1 0 2 Slight cut 3 AD 3 1 1 0 2 4 AD 4 1 1 1 3 Hypercellular collagen 5 AD + TD 1 2 1 0 3 Hypercellular collagen 6 AD + TD 2 2 1 0 3 Hypercellular collagen 7 AD + TD 3 1 1 0 2 Slight cut 8 KPBS 1 1 1 0 2 9 KPBS 2 2 1 0 3 Hypercellular collagen 10 KPBS 3 2 1 0 3 Hypercellular collagen 11 Healthy tendon 1 0 0 0 0 Hypocellular 12 Healthy tendon 2 1 0 0 1 Hypocellular 13 Healthy tendon 3 1 1 0 2 Hypocellular 14 Healthy tendon 4 1 1 0 2 Hypocellular DISCUSSION Our study results indicate that the group injected with the secretome of MSC, combining TDSCs and ASCs, showed a significant increase in PINP levels compared to the control group. However, the tested groups observed no significant differences between PIIINP levels. Procollagen types I and III are crucial building blocks in all connective tissues. These two markers, associated with collagen metabolism, specifically PINP and PIIINP, have been widely used in bone tissue as early predictors of the success of an intervention. PINP and PIIINP markers have recently been used to assess collagen metabolism in human Achilles tendons ( 11 , 13 ). The higher PINP levels in the combination group of TDSCs and ASCs indicate increased Type I collagen synthesis, which is essential for tendon healing. Type I collagen is the primary component of the extracellular matrix in tendons, providing structural strength and stability ( 14 ). This increase in PINP suggests that the combination of MSC secretome injection can enhance or accelerate the formation of new collagen in chronically injured tendons. In addition to the increase in PINP, it is also essential to understand the dynamics of connective tissue healing, which involves interactions between various cell types, such as fibroblasts and macrophages, and the role of growth factors released during the healing process. MSC secretome injections may modulate the microenvironment of the damaged tissue by enhancing fibroblast activity and reducing inflammation through paracrine actions, contributing to increased collagen synthesis and extracellular matrix remodeling ( 15 , 16 ). On the other hand, the lack of significant differences in PIIINP levels between groups suggests that while there is an increase in Type I collagen synthesis, there is no corresponding increase in Type III collagen synthesis. Type III collagen is typically found in the early stages of wound healing and forms granulation tissue ( 17 , 18 ). Type III collagen is later replaced by more mechanically resistant Type I collagen. The lack of significant differences in Type III collagen levels may indicate that the MSC secretome is more effective in facilitating structural repair through Type I collagen proliferation than Type III collagen. Additionally, measurements taken in the sixth-week post-operation (second-week post-injection) may correspond to the remodeling phase, where Type I collagen production begins to replace Type III collagen, leading to naturally higher PINP levels than PIIINP. This conclusion is supported by experimental studies showing that the primary increase in Type I collagen occurs during the regenerative phase, starting around 4–6 weeks post-operation ( 19 , 20 ). Furthermore, although this study did not show significant differences in PIIINP levels, previous studies have suggested that combining cellular and molecular therapies, such as the use of immunomodulated scaffolds, may yield more optimal outcomes in tendon healing, particularly in terms of long-term structural and functional repair ( 21 , 22 ). Our findings align with previous research showing that the application of MSC secretome, especially when enriched with growth factors and cytokines, can enhance tissue injury healing by increasing Type I collagen synthesis without causing excessive fibrosis ( 23 , 24 ). Histopathological analysis of the rat tendons revealed no significant differences in the total histopathological scores between the groups injected with MSC secretome and the control group. The histopathological score includes assessments of collagen quantity, angiogenesis, and cartilage formation in the tested tendons. These findings suggest that while there is an increase in Type I collagen synthesis, as indicated by elevated PINP levels, significant structural changes at the histopathological level may not be evident in the short term or under the specific conditions of this study. This could be due to various factors, including the study's duration, the secretome dose used, or the complexity of the chronic tendon healing process ( 25 – 27 ). This study has several limitations that require further consideration. First, the results were obtained by directly injuring the previously healthy rat tendons. This differs slightly from the chronic injury process, which often includes natural degenerative aspects. Additionally, the increase in PINP with secretome injection may theoretically facilitate tendon repair and positively influence tendon injury healing success rate. However, these experimental results cannot be directly translated into clinical practice. Further research is needed to confirm these theoretical benefits of secretome use. Despite the limitations, our results may pave the way for new management approaches to improve tendon injury repair outcomes based on secretome injections. This strategy offers several advantages as MSC secretomes can be easily produced in large quantities, stored efficiently, and do not require as many cells as MSC transplantation. Additionally, this strategy avoids risks associated with cell therapy, such as immunological reactions, cells failing to survive during transplantation, cell entrapment in pulmonary capillaries, and infection ( 28 ). The significant increase in PINP levels in the combination group of TDSCs and ASCs compared to the control group suggests that this secretome combination could accelerate the healing of chronic tendon injuries. However, the histopathological results highlight that molecular-level improvements do not always correlate with observable microscopic changes in the short term. Therefore, long-term studies with continuous observation are needed to confirm the effectiveness of this therapy in repairing the structure and function of injured tendons. CONCLUSION The injection of MSC secretome, combining TDSCs and ASCs, increases Type I collagen synthesis, improves tendon microstructure, and reduces inflammation, making it effective in accelerating the healing of chronic tendon injuries in rats. Further research on larger animal models, long-term evaluations, and human clinical trials is recommended. Developing standard protocols and considering cost factors are essential for the broad application of this therapy. Declarations ETHICS APPROVAL The study protocol has been approved by the Health Research Ethics Committee of the Faculty of Medicine, Universitas Hasanuddin (No.811/UN4.6.4.5.31/PP36/2023). Each participant gave written informed consent and agreed to participate in the study. CONSENT FOR PUBLICATION Not applicable. AVAILABILITY OF DATA AND MATERIALS Not applicable. COMPETING INTEREST All authors declare that they have no competing interests. FUNDING The Indonesian Education Scholarship (BPI), Higher Education Financing Agency (BPPT), Ministry of Education, Culture, Research and Technology of the Republic of Indonesia and the Indonesia Endowment Fund for Education (LPDP), Ministry of Finance, Republic of Indonesia, fully funded this study. AUTHOR’S CONTRIBUTIONS DD performed the study concept or design, data collection, data analysis, interpretation, and paper writing. WS performed the study concept or design, data interpretation, paper writing, and validation. AN performed the study concept or design, data interpretation, paper writing, and validation. PJ performed the study concept or design, data interpretation, paper writing, and validation. HS performed the study concept or design, data interpretation, paper writing, and validation. RS performed the study concept or design, data interpretation, paper writing, and validation. SK performed the study concept or design, data interpretation, paper writing, and validation. AZ performed the study concept or design, statistical analysis, paper writing, and validation. FM performed the study concept or design, data analysis and interpretation, and paper writing. ACKNOWLEDGMENTS We want to thank the Stem Cell Research and Development Center and Airlangga University's laboratory staff for their technical support. References Lin TW, Cardenas L, Soslowsky LJ. Biomechanics of tendon injury and repair. J Biomech [Internet]. 2004;37(6):865–77. Available from: http://dx.doi.org/10.1016/j.jbiomech.2003.11.005 Maffulli N, Wong J, Almekinders LC. Types and epidemiology of tendinopathy. Clin Sports Med [Internet]. 2003;22(4):675–92. Available from: http://dx.doi.org/10.1016/s0278-5919(03)00004-8 Järvinen TAH, Kannus P, Maffulli N, Khan KM. Achilles Tendon Disorders: Etiology and Epidemiology. Foot Ankle Clin [Internet]. 2005;10(2):255–66. Available from: http://dx.doi.org/10.1016/j.fcl.2005.01.013 Bavin EP, Smith O, Baird AEG, Smith LC, Guest DJ. Equine Induced Pluripotent Stem Cells have a Reduced Tendon Differentiation Capacity Compared to Embryonic Stem Cells. Front Vet Sci [Internet]. 2015 Nov 16;2:55. Available from: https://pubmed.ncbi.nlm.nih.gov/26664982 Gossman WG, Varacallo M. Achilles Tendon Rupture. James R, Kesturu G, Balian G, Chhabra AB. Tendon: Biology, Biomechanics, Repair, Growth Factors, and Evolving Treatment Options. J Hand Surg Am [Internet]. 2008;33(1):102–12. Available from: http://dx.doi.org/10.1016/j.jhsa.2007.09.007 Gross G, Hoffmann A. Therapeutic Strategies for Tendon Healing Based on Novel Biomaterials, Factors and Cells. Pathobiology [Internet]. 2013;80(4):203–10. Available from: http://dx.doi.org/10.1159/000347059 Childress MA, Beutler A. Management of chronic tendon injuries. Am Fam Physician. 2013;87(7):486–90. Zhou X, Li J, Giannopoulos A, Kingham PJ, Backman LJ. Secretome from In Vitro Mechanically Loaded Myoblasts Induces Tenocyte Migration, Transition to a Fibroblastic Phenotype and Suppression of Collagen Production. Int J Mol Sci [Internet]. 2021 Dec 3;22(23):13089. Available from: https://pubmed.ncbi.nlm.nih.gov/34884895 Costa-Almeida R, Calejo I, Gomes ME. Mesenchymal Stem Cells Empowering Tendon Regenerative Therapies. Int J Mol Sci [Internet]. 2019 Jun 19;20(12):3002. Available from: https://pubmed.ncbi.nlm.nih.gov/31248196 Vestergaard P, Jørgensen JOL, Olesen JL, Bosnjak E, Holm L, Frystyk J, et al. Local administration of growth hormone stimulates tendon collagen synthesis in elderly men. J Appl Physiol [Internet]. 2012;113(9):1432–8. Available from: http://dx.doi.org/10.1152/japplphysiol.00816.2012 Flodin J, Juthberg R, Edman G, Flodin J. Patient-Reported Outcome and Healing Biomarkers in Patients Treated by Female versus Male Surgeons– A cohort study on Achilles tendon ruptures. Muscle Ligaments Tendons J [Internet]. 2019;09(04):531. Available from: http://dx.doi.org/10.32098/mltj.04.2019.07 Alim MA, Peterson M, Pejler G. Do Mast Cells Have a Role in Tendon Healing and Inflammation? Cells [Internet]. 2020 May 4;9(5):1134. Available from: https://pubmed.ncbi.nlm.nih.gov/32375419 Buckley MR, Evans EB, Matuszewski PE, Chen YL, Satchel LN, Elliott DM, et al. Distributions of types I, II and III collagen by region in the human supraspinatus tendon. Connect Tissue Res [Internet]. 2013;54(6):374–9. Available from: https://pubmed.ncbi.nlm.nih.gov/24088220 Gnecchi M, Zhang Z, Ni A, Dzau VJ. Paracrine mechanisms in adult stem cell signaling and therapy. Circ Res [Internet]. 2008 Nov 21;103(11):1204–19. Available from: https://pubmed.ncbi.nlm.nih.gov/19028920 Caplan AI, Dennis JE. Mesenchymal stem cells as trophic mediators. J Cell Biochem [Internet]. 2006;98(5):1076–84. Available from: http://dx.doi.org/10.1002/jcb.20886 BOYER MI, STRICKLAND JW, ENGLES DR, SACHAR K, LEVERSEDGE FJ. FLEXOR TENDON REPAIR AND REHABILITATION. J Bone Jt Surgery-American Vol [Internet]. 2002;84(9):1684–706. Available from: http://dx.doi.org/10.2106/00004623-200209000-00025 Müller SA, Todorov A, Heisterbach PE, Martin I, Majewski M. Tendon healing: an overview of physiology, biology, and pathology of tendon healing and systematic review of state of the art in tendon bioengineering. Knee Surgery, Sport Traumatol Arthrosc [Internet]. 2013;23(7):2097–105. Available from: http://dx.doi.org/10.1007/s00167-013-2680-z Sandrey MA. Acute and Chronic Tendon Injuries: Factors Affecting the Healing Response and Treatment. J Sport Rehabil [Internet]. 2003;12(1):70–91. Available from: http://dx.doi.org/10.1123/jsr.12.1.70 Valkering KP, Aufwerber S, Ranuccio F, Lunini E, Edman G, Ackermann PW. Functional weight-bearing mobilization after Achilles tendon rupture enhances early healing response: a single-blinded randomized controlled trial. Knee Surg Sports Traumatol Arthrosc [Internet]. 2016/08/18. 2017 Jun;25(6):1807–16. Available from: https://pubmed.ncbi.nlm.nih.gov/27539402 Oryan A, Moshiri A, Meimandi-Parizi A. Role of Embedded Pure Xenogenous Bovine Platelet Gel on Experimental Tendon Healing, Modelling and Remodelling. BioDrugs [Internet]. 2014;28(6):537–56. Available from: http://dx.doi.org/10.1007/s40259-014-0107-0 Lohan P, Murphy N, Treacy O, Lynch K, Morcos M, Chen B, et al. Third-Party Allogeneic Mesenchymal Stromal Cells Prevent Rejection in a Pre-sensitized High-Risk Model of Corneal Transplantation. Front Immunol [Internet]. 2018 Nov 20;9:2666. Available from: https://pubmed.ncbi.nlm.nih.gov/30515159 Chen T, You Y, Jiang H, Wang ZZ. Epithelial-mesenchymal transition (EMT): A biological process in the development, stem cell differentiation, and tumorigenesis. J Cell Physiol [Internet]. 2017/04/10. 2017 Dec;232(12):3261–72. Available from: https://pubmed.ncbi.nlm.nih.gov/28079253 Tamama K, Kerpedjieva SS. Acceleration of Wound Healing by Multiple Growth Factors and Cytokines Secreted from Multipotential Stromal Cells/Mesenchymal Stem Cells. Adv wound care [Internet]. 2012 Aug;1(4):177–82. Available from: https://pubmed.ncbi.nlm.nih.gov/24527301 Pajala A, Melkko J, Leppilahti J, Ohtonen P, Soini Y, Risteli J. Tenascin-C and type I and III collagen expression in total Achilles tendon rupture. An immunohistochemical study. Histol Histopathol. 2009;24(10):1207. Kjær M, Langberg H, Heinemeier K, Bayer ML, Hansen M, Holm L, et al. From mechanical loading to collagen synthesis, structural changes and function in human tendon. Scand J Med Sci Sports. 2009;19(4):500–10. Christensen B, Dyrberg E, Aagaard P, Kjaer M, Langberg H. Short-term immobilization and recovery affect skeletal muscle but not collagen tissue turnover in humans. J Appl Physiol. 2008;105(6):1845–51. Lange-Consiglio A, Rossi D, Tassan S, Perego R, Cremonesi F, Parolini O. Conditioned Medium from Horse Amniotic Membrane-Derived Multipotent Progenitor Cells: Immunomodulatory Activity In Vitro and First Clinical Application in Tendon and Ligament Injuries In Vivo. Stem Cells Dev [Internet]. 2013;22(22):3015–24. Available from: http://dx.doi.org/10.1089/scd.2013.0214 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5257458","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":366249788,"identity":"8b2f73fb-2233-439f-a335-a50b17e3267a","order_by":0,"name":"Andi Dhedie Prasatia Sam","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIie3QvQqCUBTA8SOCkx/rbdFXuBJEj6METg5B0BKUENQzRPUORuB84kAuUauQQy1NBU7NqUQ0XRsb7n+6HPjBORdAJvvPOIKGAATKBYDVI9R/JCr/mUBNSsg+MxGxltMNwjC3zVTdj4ywC9YcFbwLCMv3fYTjrd0iLTgbCQN28GC3Eq2VhRyVGfkx6Z2aQFbeJVrMeZNJTNZzUBGnifA38TjpmloR3kTcLOijdyR3QVq7tU6Y7h78SHiLnfW2RTEkxzzRtXgkY9tOiQrRj9V5X+9yJSVqAjKZTCZr6AWPTFOkgnoL7AAAAABJRU5ErkJggg==","orcid":"","institution":"Muslim University of Indonesia","correspondingAuthor":true,"prefix":"","firstName":"Andi","middleName":"Dhedie Prasatia","lastName":"Sam","suffix":""},{"id":366249789,"identity":"8e688283-ab12-4282-85d6-99a4c01dbf9c","order_by":1,"name":"Warsinggih Warsinggih","email":"","orcid":"","institution":"Hasanuddin University","correspondingAuthor":false,"prefix":"","firstName":"Warsinggih","middleName":"","lastName":"Warsinggih","suffix":""},{"id":366249790,"identity":"bdcfeab3-7c24-4de4-889e-d6d6ee5f0ad3","order_by":2,"name":"Muhammad Andry Usman","email":"","orcid":"","institution":"Hasanuddin University","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Andry","lastName":"Usman","suffix":""},{"id":366249791,"identity":"054b48dc-9a4f-4049-8d7e-0672de6c96cd","order_by":3,"name":"Muhammad Phetrus Johan","email":"","orcid":"","institution":"Hasanuddin University","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Phetrus","lastName":"Johan","suffix":""},{"id":366249792,"identity":"67432bd9-6021-4212-803a-aef7b9684c89","order_by":4,"name":"Heri Suroto","email":"","orcid":"","institution":"Airlangga University","correspondingAuthor":false,"prefix":"","firstName":"Heri","middleName":"","lastName":"Suroto","suffix":""},{"id":366249793,"identity":"0965e4d8-e67e-4868-8511-6e7f4770083b","order_by":5,"name":"M. Ruksal Saleh","email":"","orcid":"","institution":"Hasanuddin University","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"Ruksal","lastName":"Saleh","suffix":""},{"id":366249794,"identity":"7da1a4d9-fca4-4968-8a33-5c14856daa0d","order_by":6,"name":"Muhammad Sakti","email":"","orcid":"","institution":"Hasanuddin University","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Sakti","suffix":""},{"id":366249797,"identity":"412721f0-609a-466a-ae7c-551d6f92d53d","order_by":7,"name":"Andi Alfian Zainuddin","email":"","orcid":"","institution":"Hasanuddin University","correspondingAuthor":false,"prefix":"","firstName":"Andi","middleName":"Alfian","lastName":"Zainuddin","suffix":""},{"id":366249799,"identity":"55f81ca5-be85-4dfc-b700-fa09e1479ae2","order_by":8,"name":"Andi Firman Mubarak","email":"","orcid":"","institution":"Muslim University of Indonesia","correspondingAuthor":false,"prefix":"","firstName":"Andi","middleName":"Firman","lastName":"Mubarak","suffix":""}],"badges":[],"createdAt":"2024-10-14 02:38:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5257458/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5257458/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":66807333,"identity":"c17ea65a-111c-496e-8cd2-b3fa6d63c15f","added_by":"auto","created_at":"2024-10-16 16:06:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":308640,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological appearance of the samples at 20x magnification. (A) Healthy tendon, hypocellular appearance with mild changes of less than 25% irregular collagen fibers and moderate tissue infiltration with arterioles. (B) AD, hypercellular collagen with mild changes of less than 25% irregular collagen fibers, presence of capillaries, and isolated hyaline cartilage nodules. (C) AD + DT, hypercellular collagen appearance with moderate changes with less than 50% irregular collagen fibers, and presence of capillaries. (D) KPBS, hypercellular collagen appearance with moderate changes with irregular collagen fibers between 25 - 50% and presence of capillaries.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5257458/v1/fc82f980c9a528ff3f715011.png"},{"id":66909881,"identity":"dcb1e4f9-40b5-4622-8e03-2ec231bc39b9","added_by":"auto","created_at":"2024-10-17 20:46:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1055217,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5257458/v1/c31746ab-0f25-41e9-ad86-d538ec8355d0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eMesenchymal Stem Cell Secretome Effectiveness on Healing of Chronic Tendon Injury: Analysis of Procollagen Type I N- Terminal Peptide and Procollagen Type Iii N-terminal Peptide and Histopathology in Rat’s Tendon (Rattus Norvegicus)\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eRepetitive motion and age-related degeneration significantly increase the susceptibility of tendons, crucial connective tissues primarily composed of parallel collagen fibers embedded in an extracellular matrix, to injuries (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The Achilles tendon, the largest and strongest tendon in the human body, connecting the gastrocnemius and soleus muscles, is designed to endure significant tensile forces (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTendon and ligament injuries are among the most frequently diagnosed musculoskeletal problems, with 30\u0026ndash;50% of sports-related injuries involving tendons (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Achilles tendon ruptures, particularly common in adults aged 30\u0026ndash;50, represent the most frequent tendon ruptures in the lower extremities (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). While not life-threatening, these injuries can be severely debilitating, leading to a decline in quality of life and substantial healthcare costs (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe healing of tendons is often poor, typically resulting in scar tissue structurally and functionally inferior to normal tendon tissue (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Chronic tendon injuries are complicated to treat, requiring extensive intervention to restore pain-free function. Unlike acute injuries, which trigger an inflammatory response, chronic tendon injuries are characterized by collagen degeneration, a process where the collagen fibers in the tendon break down due to prolonged wear and tear and limited blood supply. This highlights the need for improved treatment strategies, especially for chronic cases.(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eRecent attention has turned to cell-based therapies, mainly stem cells, which have shown significant potential in enhancing tendon repair through paracrine mechanisms. Paracrine signaling is a form of cell-to-cell communication in which a cell produces a signal to induce changes in nearby cells, altering their behavior and phenotype. The stem cell secretome, rich in growth factors like fibroblast growth factor (FGF), holds promise in improving tendon healing through these paracrine mechanisms, although its role in chronic injuries remains underexplored (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA critical aspect of tendon healing involves collagen synthesis, particularly the production of type I and III procollagen. Procollagen type I N-terminal propeptide (PINP) and procollagen type III N-terminal propeptide (PIIINP) are markers of collagen metabolism that play a significant role in tendon repair. Elevated markers indicate active collagen synthesis, essential for tendon callus formation and overall tissue healing (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Despite this, the specific dynamics of P1NP and PIIINP in chronic tendon injury repair, particularly in response to stem cell secretome treatment, remain inadequately studied.\u003c/p\u003e \u003cp\u003eThis study aims to investigate the effects of stem cell secretome on the repair of chronic Achilles tendon injuries. The histopathological analysis will evaluate structural changes, and healing efficacy will be assessed using immunoserological markers such as PINP and PIIINP. Our findings will provide intriguing insights into the potential of the secretome to significantly enhance tendon repair.\u003c/p\u003e"},{"header":"MATERIALS \u0026 METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design and Setting\u003c/h2\u003e \u003cp\u003eThis study was conducted as an experimental laboratory to evaluate MSC-derived secretomes' effects on tendon healing in a rat model of chronic tendinopathy. The research was carried out at the Stem Cell Research and Development Center, Universitas Airlangga, and the Laboratory of Rumah Sakit Pendidikan Tinggi Negeri (RSPTN) Universitas Hasanuddin between July and December 2023.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of Animal Samples\u003c/h3\u003e\n\u003cp\u003eA total of 16 adult male Rattus norvegicus rats, aged 8\u0026ndash;12 weeks and weighing approximately 200\u0026ndash;250 grams, were used in this study. The rats were housed under standard laboratory conditions with a 12-hour light/dark cycle and had free access to food and water. The Animal Ethics Committee of Universitas Airlangga approved all animal procedures.\u003c/p\u003e \u003cp\u003eChronic tendinopathy was induced in all rats through a combination of mechanical and enzymatic methods. The Achilles tendon of the right hindlimb was subjected to repetitive mechanical overloading by forced treadmill running at a speed of 15 m/min for 30 minutes per day over four weeks. Additionally, the tendons were injected with collagenase type I to induce collagen degradation and simulate chronic degenerative changes.\u003c/p\u003e \u003cp\u003eAfter the induction of tendinopathy, the rats were randomly divided into four treatment groups (n\u0026thinsp;=\u0026thinsp;4 per group):\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eGroup I, Tendon-Derived Stem Cells (TDSCs): Rats received an intratendinous injection of 50 \u0026micro;L of secretome derived from TDSCs.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eGroup II, Adipose-Derived Stem Cells (ASCs): Rats received an intratendinous injection of 50 \u0026micro;L of secretome derived from ASCs.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eGroup III (TDSCs\u0026thinsp;+\u0026thinsp;ASCs): Rats received an intratendinous injection of 25 \u0026micro;L of TDSC secretome and 25 \u0026micro;L of ASC secretome.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eGroup IV (Control): Rats received an intratendinous injection of 50 \u0026micro;L of sterile saline (placebo).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e\n\u003ch3\u003ePreparation of MSC-Derived Secretomes and Biochemical Analysis\u003c/h3\u003e\n\u003cp\u003eTDSCs were isolated from the Achilles tendons of healthy donor rats, while ASCs were isolated from the inguinal adipose tissue. Both types of MSCs were cultured under standard conditions and allowed to reach confluence. The secretomes were collected from the culture media after 72 hours of incubation, filtered, and concentrated using centrifugation. The protein concentration of the secretomes was measured, and aliquots were stored at -80\u0026deg;C until use. Tendon healing was evaluated four weeks post-treatment using a combination of biochemical and histopathological analyses.\u003c/p\u003e \u003cp\u003eTendon samples were harvested, and the levels of PINP and PIIINP were measured using enzyme-linked immunosorbent assays (ELISA). PINP is a marker for collagen type I synthesis, while PIIINP is a marker for collagen type III synthesis. The levels of these markers provide insights into the remodeling process and the balance between collagen types I and III during tendon healing.\u003c/p\u003e\n\u003ch3\u003eHistopathological Analysis\u003c/h3\u003e\n\u003cp\u003eThe harvested tendons were fixed in 10% formalin, embedded in paraffin, and sectioned for histopathological evaluation. Hematoxylin and eosin staining was performed to assess the general tissue architecture, while Masson's trichrome staining was used to evaluate collagen deposition. The sections were examined under a light microscope, and histopathological scoring was performed based on the degree of collagen alignment, angiogenesis, and cartilage formation.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using statistical software (e.g., SPSS, version 25.0). Continuous variables were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The differences between groups were analyzed using one-way analysis of variance (ANOVA) followed by post hoc Tukey's test for pairwise comparisons if the data is usually distributed and the Kruskal-Wallis test if the data is not normally distributed. A p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eProcollagen I and III Levels in Tendon Samples\u003c/h2\u003e \u003cp\u003eThe levels of PINP and PIIINP were measured in rat tendon samples (Rattus Norvegicus) injected with MSC secretomes derived from various sources. Four groups were included: TDSCs, ASCs, a combination of TDSCs and ASCs, and a control group. The measured levels of PINP and PIIINP in the different groups are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The mean, standard deviation, median, minimum, and maximum values for PINP and PIIINP levels across the four groups are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. A Shapiro-Wilk test was performed to assess the normality of PINP and PIIINP data. The results indicate that the data for both PINP and PIIINP are not normally distributed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Therefore, non-parametric tests were used for further analysis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of PINP and PIIINP Levels in Rat Tendon Samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePINP (ng/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePIIINP (ng/ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTDSCs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTDSCs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTDSCs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTDSCs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eASCs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eASCs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eASCs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eASCs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTDSCs\u0026thinsp;+\u0026thinsp;ASCs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTDSCs\u0026thinsp;+\u0026thinsp;ASCs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTDSCs\u0026thinsp;+\u0026thinsp;ASCs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDescriptive Statistics of PINP and PIIINP Levels\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMax\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTDSCs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASCs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTDSCs\u0026thinsp;+\u0026thinsp;ASCs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eComparative Analysis of PINP and PIIINP Levels\u003c/h3\u003e\n\u003cp\u003eThe non-parametric Kruskal-Wallis test compared the four groups' PINP and PIIINP levels. The results showed a significant difference in PINP levels (p\u0026thinsp;=\u0026thinsp;0.039), while no significant difference was found among PIIINP groups (p\u0026thinsp;=\u0026thinsp;0.318). Post-hoc analysis using the Mann-Whitney U test compared PINP levels between the experimental and control groups. The results indicate that combining TDSCs and ASCs significantly increased PINP levels compared to the control group (p\u0026thinsp;=\u0026thinsp;0.004). The differences between the other groups and the control group were not statistically significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePost-Hoc Analysis for PINP Levels Compared to Control\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup Comparison\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASCs vs. Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.056\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTDSCs vs. Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.056\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTDSCs\u0026thinsp;+\u0026thinsp;ASCs vs. Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological Findings and Analysis\u003c/h2\u003e \u003cp\u003eHistopathological evaluation of the tendon samples was conducted to assess collagen formation, angiogenesis, and cartilage formation. The scoring of histopathological findings is presented in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, and the histopathological appearance of the samples is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The results of the Kruskal-Wallis test for the variable Total Histopathological Scores show a p-value of 0.092, indicating no significant difference in total histopathological scores among the four groups.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eScoring and Histopathological Descriptions in Rat Tendon Samples (Rattus Norvegicus)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCollagen\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAngiogenesis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCartilage Formation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTotal Score\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRemarks\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAD 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHypercellular collagen\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAD 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSlight cut\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAD 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAD 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHypercellular collagen\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAD\u0026thinsp;+\u0026thinsp;TD 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHypercellular collagen\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAD\u0026thinsp;+\u0026thinsp;TD 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHypercellular collagen\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAD\u0026thinsp;+\u0026thinsp;TD 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSlight cut\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKPBS 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKPBS 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHypercellular collagen\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKPBS 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHypercellular collagen\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHealthy tendon 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHypocellular\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHealthy tendon 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHypocellular\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHealthy tendon 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHypocellular\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHealthy tendon 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHypocellular\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur study results indicate that the group injected with the secretome of MSC, combining TDSCs and ASCs, showed a significant increase in PINP levels compared to the control group. However, the tested groups observed no significant differences between PIIINP levels.\u003c/p\u003e \u003cp\u003eProcollagen types I and III are crucial building blocks in all connective tissues. These two markers, associated with collagen metabolism, specifically PINP and PIIINP, have been widely used in bone tissue as early predictors of the success of an intervention. PINP and PIIINP markers have recently been used to assess collagen metabolism in human Achilles tendons (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe higher PINP levels in the combination group of TDSCs and ASCs indicate increased Type I collagen synthesis, which is essential for tendon healing. Type I collagen is the primary component of the extracellular matrix in tendons, providing structural strength and stability (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). This increase in PINP suggests that the combination of MSC secretome injection can enhance or accelerate the formation of new collagen in chronically injured tendons.\u003c/p\u003e \u003cp\u003eIn addition to the increase in PINP, it is also essential to understand the dynamics of connective tissue healing, which involves interactions between various cell types, such as fibroblasts and macrophages, and the role of growth factors released during the healing process. MSC secretome injections may modulate the microenvironment of the damaged tissue by enhancing fibroblast activity and reducing inflammation through paracrine actions, contributing to increased collagen synthesis and extracellular matrix remodeling (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOn the other hand, the lack of significant differences in PIIINP levels between groups suggests that while there is an increase in Type I collagen synthesis, there is no corresponding increase in Type III collagen synthesis. Type III collagen is typically found in the early stages of wound healing and forms granulation tissue (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Type III collagen is later replaced by more mechanically resistant Type I collagen. The lack of significant differences in Type III collagen levels may indicate that the MSC secretome is more effective in facilitating structural repair through Type I collagen proliferation than Type III collagen. Additionally, measurements taken in the sixth-week post-operation (second-week post-injection) may correspond to the remodeling phase, where Type I collagen production begins to replace Type III collagen, leading to naturally higher PINP levels than PIIINP. This conclusion is supported by experimental studies showing that the primary increase in Type I collagen occurs during the regenerative phase, starting around 4\u0026ndash;6 weeks post-operation (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, although this study did not show significant differences in PIIINP levels, previous studies have suggested that combining cellular and molecular therapies, such as the use of immunomodulated scaffolds, may yield more optimal outcomes in tendon healing, particularly in terms of long-term structural and functional repair (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Our findings align with previous research showing that the application of MSC secretome, especially when enriched with growth factors and cytokines, can enhance tissue injury healing by increasing Type I collagen synthesis without causing excessive fibrosis (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHistopathological analysis of the rat tendons revealed no significant differences in the total histopathological scores between the groups injected with MSC secretome and the control group. The histopathological score includes assessments of collagen quantity, angiogenesis, and cartilage formation in the tested tendons. These findings suggest that while there is an increase in Type I collagen synthesis, as indicated by elevated PINP levels, significant structural changes at the histopathological level may not be evident in the short term or under the specific conditions of this study. This could be due to various factors, including the study's duration, the secretome dose used, or the complexity of the chronic tendon healing process (\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study has several limitations that require further consideration. First, the results were obtained by directly injuring the previously healthy rat tendons. This differs slightly from the chronic injury process, which often includes natural degenerative aspects. Additionally, the increase in PINP with secretome injection may theoretically facilitate tendon repair and positively influence tendon injury healing success rate. However, these experimental results cannot be directly translated into clinical practice. Further research is needed to confirm these theoretical benefits of secretome use.\u003c/p\u003e \u003cp\u003eDespite the limitations, our results may pave the way for new management approaches to improve tendon injury repair outcomes based on secretome injections. This strategy offers several advantages as MSC secretomes can be easily produced in large quantities, stored efficiently, and do not require as many cells as MSC transplantation. Additionally, this strategy avoids risks associated with cell therapy, such as immunological reactions, cells failing to survive during transplantation, cell entrapment in pulmonary capillaries, and infection (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe significant increase in PINP levels in the combination group of TDSCs and ASCs compared to the control group suggests that this secretome combination could accelerate the healing of chronic tendon injuries. However, the histopathological results highlight that molecular-level improvements do not always correlate with observable microscopic changes in the short term. Therefore, long-term studies with continuous observation are needed to confirm the effectiveness of this therapy in repairing the structure and function of injured tendons.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe injection of MSC secretome, combining TDSCs and ASCs, increases Type I collagen synthesis, improves tendon microstructure, and reduces inflammation, making it effective in accelerating the healing of chronic tendon injuries in rats. Further research on larger animal models, long-term evaluations, and human clinical trials is recommended. Developing standard protocols and considering cost factors are essential for the broad application of this therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eETHICS APPROVAL\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe study protocol has been approved by the Health Research Ethics Committee of the Faculty of Medicine, Universitas Hasanuddin (No.811/UN4.6.4.5.31/PP36/2023). Each participant gave written informed consent and agreed to participate in the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAVAILABILITY OF DATA AND MATERIALS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eCOMPETING INTEREST\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eFUNDING\u003c/p\u003e\n\u003cp\u003eThe Indonesian Education Scholarship (BPI), Higher Education Financing Agency (BPPT), Ministry of Education, Culture, Research and Technology of the\u0026nbsp;Republic of Indonesia and the Indonesia Endowment Fund for Education (LPDP), Ministry of Finance, Republic of Indonesia, fully funded this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR\u0026rsquo;S CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDD performed the study concept or design, data collection, data analysis, interpretation, and paper writing.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWS performed the study concept or design, data interpretation, paper writing, and validation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAN performed the study concept or design, data interpretation, paper writing, and validation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePJ performed the study concept or design, data interpretation, paper writing, and validation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHS performed the study concept or design, data interpretation, paper writing, and validation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRS performed the study concept or design, data interpretation, paper writing, and validation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSK performed the study concept or design, data interpretation, paper writing, and validation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAZ performed the study concept or design, statistical analysis, paper writing, and validation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFM performed the study concept or design, data analysis and interpretation, and paper writing.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eACKNOWLEDGMENTS\u003c/p\u003e\n\u003cp\u003eWe want to thank the Stem Cell Research and Development Center and Airlangga University\u0026apos;s laboratory staff for their technical support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLin TW, Cardenas L, Soslowsky LJ. Biomechanics of tendon injury and repair. J Biomech [Internet]. 2004;37(6):865\u0026ndash;77. Available from: http://dx.doi.org/10.1016/j.jbiomech.2003.11.005\u003c/li\u003e\n\u003cli\u003eMaffulli N, Wong J, Almekinders LC. Types and epidemiology of tendinopathy. Clin Sports Med [Internet]. 2003;22(4):675\u0026ndash;92. Available from: http://dx.doi.org/10.1016/s0278-5919(03)00004-8\u003c/li\u003e\n\u003cli\u003eJ\u0026auml;rvinen TAH, Kannus P, Maffulli N, Khan KM. Achilles Tendon Disorders: Etiology and Epidemiology. Foot Ankle Clin [Internet]. 2005;10(2):255\u0026ndash;66. Available from: http://dx.doi.org/10.1016/j.fcl.2005.01.013\u003c/li\u003e\n\u003cli\u003eBavin EP, Smith O, Baird AEG, Smith LC, Guest DJ. Equine Induced Pluripotent Stem Cells have a Reduced Tendon Differentiation Capacity Compared to Embryonic Stem Cells. Front Vet Sci [Internet]. 2015 Nov 16;2:55. Available from: https://pubmed.ncbi.nlm.nih.gov/26664982\u003c/li\u003e\n\u003cli\u003eGossman WG, Varacallo M. Achilles Tendon Rupture. \u003c/li\u003e\n\u003cli\u003eJames R, Kesturu G, Balian G, Chhabra AB. Tendon: Biology, Biomechanics, Repair, Growth Factors, and Evolving Treatment Options. J Hand Surg Am [Internet]. 2008;33(1):102\u0026ndash;12. Available from: http://dx.doi.org/10.1016/j.jhsa.2007.09.007\u003c/li\u003e\n\u003cli\u003eGross G, Hoffmann A. Therapeutic Strategies for Tendon Healing Based on Novel Biomaterials, Factors and Cells. Pathobiology [Internet]. 2013;80(4):203\u0026ndash;10. Available from: http://dx.doi.org/10.1159/000347059\u003c/li\u003e\n\u003cli\u003eChildress MA, Beutler A. Management of chronic tendon injuries. Am Fam Physician. 2013;87(7):486\u0026ndash;90. \u003c/li\u003e\n\u003cli\u003eZhou X, Li J, Giannopoulos A, Kingham PJ, Backman LJ. Secretome from In Vitro Mechanically Loaded Myoblasts Induces Tenocyte Migration, Transition to a Fibroblastic Phenotype and Suppression of Collagen Production. Int J Mol Sci [Internet]. 2021 Dec 3;22(23):13089. Available from: https://pubmed.ncbi.nlm.nih.gov/34884895\u003c/li\u003e\n\u003cli\u003eCosta-Almeida R, Calejo I, Gomes ME. Mesenchymal Stem Cells Empowering Tendon Regenerative Therapies. Int J Mol Sci [Internet]. 2019 Jun 19;20(12):3002. Available from: https://pubmed.ncbi.nlm.nih.gov/31248196\u003c/li\u003e\n\u003cli\u003eVestergaard P, J\u0026oslash;rgensen JOL, Olesen JL, Bosnjak E, Holm L, Frystyk J, et al. Local administration of growth hormone stimulates tendon collagen synthesis in elderly men. J Appl Physiol [Internet]. 2012;113(9):1432\u0026ndash;8. Available from: http://dx.doi.org/10.1152/japplphysiol.00816.2012\u003c/li\u003e\n\u003cli\u003eFlodin J, Juthberg R, Edman G, Flodin J. Patient-Reported Outcome and Healing Biomarkers in Patients Treated by Female versus Male Surgeons\u0026ndash; A cohort study on Achilles tendon ruptures. Muscle Ligaments Tendons J [Internet]. 2019;09(04):531. Available from: http://dx.doi.org/10.32098/mltj.04.2019.07\u003c/li\u003e\n\u003cli\u003eAlim MA, Peterson M, Pejler G. Do Mast Cells Have a Role in Tendon Healing and Inflammation? Cells [Internet]. 2020 May 4;9(5):1134. Available from: https://pubmed.ncbi.nlm.nih.gov/32375419\u003c/li\u003e\n\u003cli\u003eBuckley MR, Evans EB, Matuszewski PE, Chen YL, Satchel LN, Elliott DM, et al. Distributions of types I, II and III collagen by region in the human supraspinatus tendon. Connect Tissue Res [Internet]. 2013;54(6):374\u0026ndash;9. Available from: https://pubmed.ncbi.nlm.nih.gov/24088220\u003c/li\u003e\n\u003cli\u003eGnecchi M, Zhang Z, Ni A, Dzau VJ. Paracrine mechanisms in adult stem cell signaling and therapy. Circ Res [Internet]. 2008 Nov 21;103(11):1204\u0026ndash;19. Available from: https://pubmed.ncbi.nlm.nih.gov/19028920\u003c/li\u003e\n\u003cli\u003eCaplan AI, Dennis JE. Mesenchymal stem cells as trophic mediators. J Cell Biochem [Internet]. 2006;98(5):1076\u0026ndash;84. Available from: http://dx.doi.org/10.1002/jcb.20886\u003c/li\u003e\n\u003cli\u003eBOYER MI, STRICKLAND JW, ENGLES DR, SACHAR K, LEVERSEDGE FJ. FLEXOR TENDON REPAIR AND REHABILITATION. J Bone Jt Surgery-American Vol [Internet]. 2002;84(9):1684\u0026ndash;706. Available from: http://dx.doi.org/10.2106/00004623-200209000-00025\u003c/li\u003e\n\u003cli\u003eM\u0026uuml;ller SA, Todorov A, Heisterbach PE, Martin I, Majewski M. Tendon healing: an overview of physiology, biology, and pathology of tendon healing and systematic review of state of the art in tendon bioengineering. Knee Surgery, Sport Traumatol Arthrosc [Internet]. 2013;23(7):2097\u0026ndash;105. Available from: http://dx.doi.org/10.1007/s00167-013-2680-z\u003c/li\u003e\n\u003cli\u003eSandrey MA. Acute and Chronic Tendon Injuries: Factors Affecting the Healing Response and Treatment. J Sport Rehabil [Internet]. 2003;12(1):70\u0026ndash;91. Available from: http://dx.doi.org/10.1123/jsr.12.1.70\u003c/li\u003e\n\u003cli\u003eValkering KP, Aufwerber S, Ranuccio F, Lunini E, Edman G, Ackermann PW. Functional weight-bearing mobilization after Achilles tendon rupture enhances early healing response: a single-blinded randomized controlled trial. Knee Surg Sports Traumatol Arthrosc [Internet]. 2016/08/18. 2017 Jun;25(6):1807\u0026ndash;16. Available from: https://pubmed.ncbi.nlm.nih.gov/27539402\u003c/li\u003e\n\u003cli\u003eOryan A, Moshiri A, Meimandi-Parizi A. Role of Embedded Pure Xenogenous Bovine Platelet Gel on Experimental Tendon Healing, Modelling and Remodelling. BioDrugs [Internet]. 2014;28(6):537\u0026ndash;56. Available from: http://dx.doi.org/10.1007/s40259-014-0107-0\u003c/li\u003e\n\u003cli\u003eLohan P, Murphy N, Treacy O, Lynch K, Morcos M, Chen B, et al. Third-Party Allogeneic Mesenchymal Stromal Cells Prevent Rejection in a Pre-sensitized High-Risk Model of Corneal Transplantation. Front Immunol [Internet]. 2018 Nov 20;9:2666. Available from: https://pubmed.ncbi.nlm.nih.gov/30515159\u003c/li\u003e\n\u003cli\u003eChen T, You Y, Jiang H, Wang ZZ. Epithelial-mesenchymal transition (EMT): A biological process in the development, stem cell differentiation, and tumorigenesis. J Cell Physiol [Internet]. 2017/04/10. 2017 Dec;232(12):3261\u0026ndash;72. Available from: https://pubmed.ncbi.nlm.nih.gov/28079253\u003c/li\u003e\n\u003cli\u003eTamama K, Kerpedjieva SS. Acceleration of Wound Healing by Multiple Growth Factors and Cytokines Secreted from Multipotential Stromal Cells/Mesenchymal Stem Cells. Adv wound care [Internet]. 2012 Aug;1(4):177\u0026ndash;82. Available from: https://pubmed.ncbi.nlm.nih.gov/24527301\u003c/li\u003e\n\u003cli\u003ePajala A, Melkko J, Leppilahti J, Ohtonen P, Soini Y, Risteli J. Tenascin-C and type I and III collagen expression in total Achilles tendon rupture. An immunohistochemical study. Histol Histopathol. 2009;24(10):1207. \u003c/li\u003e\n\u003cli\u003eKj\u0026aelig;r M, Langberg H, Heinemeier K, Bayer ML, Hansen M, Holm L, et al. From mechanical loading to collagen synthesis, structural changes and function in human tendon. Scand J Med Sci Sports. 2009;19(4):500\u0026ndash;10. \u003c/li\u003e\n\u003cli\u003eChristensen B, Dyrberg E, Aagaard P, Kjaer M, Langberg H. Short-term immobilization and recovery affect skeletal muscle but not collagen tissue turnover in humans. J Appl Physiol. 2008;105(6):1845\u0026ndash;51. \u003c/li\u003e\n\u003cli\u003eLange-Consiglio A, Rossi D, Tassan S, Perego R, Cremonesi F, Parolini O. Conditioned Medium from Horse Amniotic Membrane-Derived Multipotent Progenitor Cells: Immunomodulatory Activity In Vitro and First Clinical Application in Tendon and Ligament Injuries In Vivo. Stem Cells Dev [Internet]. 2013;22(22):3015\u0026ndash;24. Available from: http://dx.doi.org/10.1089/scd.2013.0214\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Mesenchymal Stem Cell Secretome, Chronic Tendon Injury, Tendon Healing, Rat Model","lastPublishedDoi":"10.21203/rs.3.rs-5257458/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5257458/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eChronic tendon injuries, such as Achilles tendinopathy, are common and challenging to treat due to the limited regenerative capacity of tendon tissue. Using mesenchymal stem cell (MSC) secretome, which contains a rich array of growth factors, holds promise for enhancing tendon healing. This study aimed to evaluate the effectiveness of MSC secretome, derived from tendon-derived stem cells (TDSCs) and adipose-derived stem cells (ASCs), on the healing of chronic Achilles tendon injuries in a rat model, focusing on the levels of Procollagen Type I N-Terminal Peptide (PINP) and Procollagen Type III N-Terminal Peptide (PIIINP), and histopathological changes.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA chronic tendinopathy model was induced in 16 males of Rattus norvegicus via mechanical overloading and collagenase injection. Rats were divided into four groups: TDSC secretome, ASC secretome, combined TDSC\u0026thinsp;+\u0026thinsp;ASC secretome, and a control group. Secretomes were administered intratendinously. Tendon healing was assessed after four weeks using enzyme-linked immunosorbent assays (ELISA) to measure PINP and PIIINP levels and histopathological analysis to evaluate collagen deposition and tissue structure.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ePINP levels were significantly higher in the TDSC\u0026thinsp;+\u0026thinsp;ASC group compared to the control group (p\u0026thinsp;=\u0026thinsp;0.004), indicating enhanced Type I collagen synthesis. However, no significant differences were observed in PIIINP levels between the groups. The histopathological analysis did not reveal significant structural differences in tendon healing among the groups, though increased collagen alignment was observed in the TDSC\u0026thinsp;+\u0026thinsp;ASC group.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe combined TDSC and ASC secretome promotes Type I collagen synthesis in chronic tendon injuries, but histological improvements were insignificant. Further studies are needed to confirm the long-term benefits of secretome therapy.\u003c/p\u003e","manuscriptTitle":"Mesenchymal Stem Cell Secretome Effectiveness on Healing of Chronic Tendon Injury: Analysis of Procollagen Type I N- Terminal Peptide and Procollagen Type Iii N-terminal Peptide and Histopathology in Rat’s Tendon (Rattus Norvegicus)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-16 16:06:30","doi":"10.21203/rs.3.rs-5257458/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"910aacac-0078-4d66-a92d-4f551665dd38","owner":[],"postedDate":"October 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-17T20:38:22+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-16 16:06:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5257458","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5257458","identity":"rs-5257458","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}