Human Umbilical Cord Mesenchymal Stem Cells promote tendon functional repair in a Collagenase-Induced Tendinopathy Model

Human Umbilical Cord Mesenchymal Stem Cells promote tendon functional repair in a Collagenase-Induced Tendinopathy Model | 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 Human Umbilical Cord Mesenchymal Stem Cells promote tendon functional repair in a Collagenase-Induced Tendinopathy Model Xiangyi Sun, Zhiwei Lin, Jinyang Chen, Zhe Wang, Guangqi Zhu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4293359/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 Achilles tendon rupture is a common tendinopathy.We investigated the therapeutic effects of UC-MSCs on the inflammatory condition, and explored the preliminary mechanisms underlying their role in rabbit Achilles tendon repair.Tendon structure and functional recovery were evaluated through histological assessment, pathology, tissue hydroxyproline (Hyp) content measurement, and biomechanical testing.Then the inflammation and metabolic status of the extracellular matrix, along with potential mechanisms, were assessed through quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemical staining.This study demonstrated the satisfactory ability of UC-MSCs transplantation to promote functional repair of tendon in a rabbit model of tendon diseases. The mechanisms involved include upregulation of Collagen I and Collagen III expression, inhibition of MMP-9, and enhancement of muscle fiber contraction through increased expression of troponin C (TnC), thereby improving the structural stability at the site of inflammation. We concluded that UC-MSCs hold promising potential as an enhancement strategy for MSC-based therapy in tendon diseases. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) Achilles tendonitis Collagen type I Fluorescent tracking Mechanisms Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Achilles tendon (AT) injury is a clinically intractable tendon disorder characterized by degenerative and cumulative tissue microtrauma. The incidence rate among runners ranges from 9.1% to 10.9%, with male distance runners having a lifetime risk exceeding 50% [1-2]. This condition imposes a significant clinical burden on global healthcare systems [3]. The characteristic clinical presentations of Achilles tendon disease encompass pain, swelling, and compromised functionality, especially during ambulation and activities involving impact [1].The development of Achilles tendon disease involves various factors, including modifiable and non-modifiable risk factors [4]. Non-modifiable factors include patient demographics such as older age, male gender, compromised vascularity, hyperpronation, and other biomechanical factors, as well as systemic diseases like metabolic and rheumatic disorders. External risk factors encompass overuse, training errors (rapid increase in impact exercise volume), and certain medications known to induce tendon injury, including corticosteroids and fluoroquinolone antibiotics [5]. Treatment typically encompasses non-surgical interventions, which can be classified into passive modalities such as pharmacotherapy, injection therapies, extracorporeal shockwave therapy (ESWT), therapeutic ultrasonography, and low-level laser therapy. Active approaches may involve tendon-loading exercises, patient education, and load management [6]. However, the primary goal of treatment for most Achilles tendon disease patients is symptom relief, particularly pain, with suboptimal long-term outcomes. Naturally healed ATs often exhibit poor quality due to the formation of fibrotic scars and tendon sheath adhesion, along with an increased risk of subsequent reinjury and re-rupture during subsequent physical activities and daily tasks. Therefore, it is crucial to investigate the pathological processes of Achilles tendon disease and the mechanisms involved in tendon healing to improve current treatment strategies. In recent years, mesenchymal stem cells (MSCs) from different sources have gained popularity as a therapeutic option for tendon repair [7-8]. Takashi Oshita et al. reported on the effects of adipose-derived stem cells (ASCs) on tendon healing in a rat tendon disease model, demonstrating significantly reduced tendon degeneration at both two time points compared to the PBS group, along with a decreased ratio of type III collagen to type I collagen[9-10]. ASCs have also been shown to decrease the expression of inflammatory factors such as IL-1β, GLUT1, and CA9 in mice during the early stages of tendon injury, while inducing neovascularization [11]. The combination of human amniotic membrane (HAM) with ASCs further enhances anti-adhesion properties and accelerates tendon healing [12]. Umbilical cord-derived MSCs (UC-MSCs) exhibit greater proliferative and self-renewal potential [13] and can induce tendon regeneration at macroscopic, histological, and biomechanical levels [14]. Transplantation of IL-1β-primed UC-MSCs has demonstrated superior capacity in promoting tendon functional repair in a rat tendon disease model, improving the inflammatory response and metabolism of the extracellular matrix through the TGF-β/IL-10 pathway [15]. The activation of UC-MSCs by IL-1β may lead to alterations in their secretome, suggesting diverse mechanisms of action. For example, Fredianto et al. found that UC-MSCs under hypoxic conditions significantly improved tendon repair by upregulating TNMD and RUNX2 expression and histological scoring [8]. Additionally, combining with biomaterials such as silk fibroin and gelatin methacryloyl （GelMA） can promote the migration and proliferation of MSCs and tenocytes [16]. Furthermore, the therapeutic effect of MSCs may be attributed to paracrine effects, including the secretion of cytokines and growth factors to recruit, proliferate, and differentiate tissue-specific progenitor cells for synthesizing specific matrices [17] . Although adipose tissue and bone marrow are frequently utilized sources of MSCs, both have drawbacks such as invasive harvesting methods, low efficiencies of cell extraction, and potential donor morbidities [13]. Furthermore, MSCs derived from adult tissues demonstrate variations in their differentiation potential [18]. Additionally, the engraftment and in vivo distribution of locally injected MSCs remain unclear. This study aims to investigate the engraftment and temporal effects of UC-MSCs on AT regeneration in a rabbit model. Materials And Methods 2.1 Statement of Ethics This study received approval from the Ethics Committee of Hangzhou Hibio Pharmaceutical Research and Development Center (approval number: HB2004217 ).All animal experiments were performed in accordance with the Guidelines for Care and Use of Laboratory Animals. 2.2. Cell Isolation, Culture, and Identification UC-MSCs were isolated using a previously described protocol [19] .Human umbilical cords were obtained via cesarean section from full-term deliveries. The umbilical cords were dissected into approximately 2-3 mm tissue fragments, which were then washed with PBS. The tissue fragments were placed in a 9 cm culture dish and slowly supplemented with Dulbecco's modified eagle medium (DMEM) (Hyclone) supplemented with 10% fetal bovine serum (FBS) and 1 ％ penicillin/streptomycin. Subsequently, the culture dishes were incubated at 37℃ with 5% CO 2 . When the cells reached approximately 80% confluence, they were passaged after digestion with a trypsin-EDTA solution (0.05% trypsin, 0.53 mM EDTA). The complete culture medium was replaced every 2-3 days. The UC-MSCs were cultured until passage 5 before being used for experiments. Morphological characterization was performed, and adipogenic differentiation was confirmed through Oil Red O staining, osteogenic differentiation was verified using Alizarin Red S staining, and chondrogenic differentiation was assessed by Alcian blue and Safranin O staining. Flow cytometry analysis was conducted to detect the expression of relevant cell surface markers, including CD90, CD73, CD14, CD105, CD34, CD45, CD49d, and HLA-DR (Biolegend, USA). 2.3. Establishment of Rabbit Achilles Tendon Disease Model Healthy male New Zealand rabbits (age 17–21 weeks, weight 3000-4000g) were supplied by Hangzhou Yuhang Keliang Rabbitry Cooperative (Hangzhou, China). Anesthesia was induced in all rabbits using ketamine (30 mg/kg) and xylazine (6 mg/kg) via intramuscular injection. The left hind limb of each rabbit was used for inducing tendon disease. The models of collagenase-induced AT were constructed based on a method previously described [20]. In the low-dose group, five animals were injected with 1200 U of Type I collagenase (SOLABIO, Shanghai) using a multi-point injection technique along the left leg Achilles tendon, with 2 mL of 600 U/mL Type I collagenase injected at 1 cm intervals above the tendon. In the high-dose group, five animals were injected with 2400 U of Type I collagenase. Gait observation was performed to confirm the induction of Achilles tendonitis. On days 15 and 45 after injection, euthanasia was carried out through intravenous administration of propofol (10 mg/kg) and 19.1% potassium chloride (10 mL). Immediately after euthanasia, the gross anatomical structure of the Achilles tendon was examined. Hematoxylin-eosin (HE) staining was performed to determine the number and distribution of nucleated cells as well as the arrangement of collagen fibers. Pathological observations were made, and hydroxyproline content was measured. After determining the appropriate induction dosage, 2400 U of Type I collagenase was again used to induce tendon disease in the left hind limbs of the animals. The animals were divided into two groups: the Tendon group (injected with saline) and the UC-MSCs treatment group. P5 passage UC-MSCs or saline were injected into and around the Achilles tendon, while the corresponding right hind limb served as an untreated control. A total of four injections were administered over a duration of 70 days, with approximately 5×10 6 cells in each injection. Following euthanasia, anatomical and pathological examinations of the Achilles tendon were performed, taking into account the overall health status of all animals. Biomechanical evaluation of the tendons was conducted to determine their maximum load-bearing capacit. 2.4. Labeling of UC-MSCs for fluorescent tracking UC-MSCs were labeled with red fluorescent Chloromethylbenzamido-1,1′-dioctadecyl-3,3,3′3´-tetramethylindocarb cyanine perchlorate (CM-Dil) (YEASEN, Shanghai) following a previously described protocol [21]. Briefly, UC-MSCs were trypsinized and resuspended in 1 mL of CM-Dil solution (1μmol/L). The cell suspension was incubated at 37℃ for 30 minutes, with intermittent shaking to ensure thorough mixing. After centrifugation and three washes with PBS, cells were resuspended in an appropriate amount of PBS to achieve a cell concentration of 5×10 7 /ml. The cells were then observed and photographed under a fluorescence microscope to confirm successful labeling. At day 15 post-modeling, the respective groups of rabbits underwent stem cell therapy. The treatment group received injections of CM-Dil-labeled UC-MSCs into the right Achilles tendon, while the normal control group received injections of saline. Euthanasia was performed on days 1, 3, 5, and 7 after treatment, and the heart, liver, spleen, lung, kidney, and Achilles tendon were imaged using in vivo imaging (AniView 600，Guangzhou Boruteng Biotechnology Co., Ltd.). Tissue samples were obtained from the Achilles tendon and subjected to collection, fixation, embedding, and staining with DAPI for visualization of cell nuclei. Subsequently, longitudinal sections were examined using a fluorescence microscope, and semi-quantitative analysis of fluorescence intensity was performed. 2.5.Macroscopic Evaluation After euthanizing the animals, tissue samples were collected from the Achilles tendon. For macroscopic evaluation, we employed a modified semi-quantitative system described by Stoll et al [22]. (see Additional file 2). This system consisted of 12 parameters, including tendon rupture, inflammation, tendon surface, neighboring tendon, defect level, defect size, tendon swelling/redness, connection and slidability of surrounding tissue, tendon thickness, tendon color, single muscle strain, and transition of the construct to the surrounding healthy tissue. The total macroscopic score ranged between 0 (indicating a normal tendon) and 15 (representing the most severe injury). This analysis was conducted by three blinded pathologists who graded the observed tissues according to the predetermined scoring system. 2.6. Histological Evaluation Tissue sections of the Achilles tendon were prepared as previously described [23]. In brief, the tendon was fixed in 4% paraformaldehyde, dehydrated through a standard procedure, and embedded in paraffin. Subsequently, 5 μm-thick sections were cut, stained with hematoxylin for 5 minutes and eosin for 3 to 5 minutes, and observed using a conventional light microscope. Immunofluorescence staining (IHC) was performed as previously described (Li et al., 2020). Briefly, the paraffin-embedded tissues were deparaffinized with xylene and dehydrated in a graded alcohol series. Endogenous peroxidase activity was blocked and inactivated with 0.3% hydrogen peroxide, and tissue antigens were retrieved using citrate buffer (0.01 mol/L, pH 6.0) for 10 minutes. Slides were incubated overnight at 4℃with primary antibodies including anti-Collagen I and anti-Collagen III (dilution: 1:200; Bioss). After washing the slides with PBS, they were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (dilution: 1:3000; Abcam) for 30 min at room temperature. Immunopositive expression intensity was measured using Image-Pro Plus software, with three random fields analyzed per group. The results were presented as mean ± standard deviation. 2.7. Measurement of Hydroxyproline The amount of hydroxyproline in the tendon was measured using a hydroxyproline measurement kit (Nanjing Jiancheng, Nanjing). The protocol was performed according to the manufacturer’s catalog. Briefly, approximately 80-100 mg of tissue was accurately weighed, minced, and hydrolyzed with 1 mL of 6 mol/L HCl at 95℃ for 5 hours. The hydrolysate was adjusted to pH 6.0-6.8, cooled, and 10 μl of indicator reagent was added to each tube, followed by thorough mixing. The pH-adjusted methanol and ethanol were then added to each tube until the indicator turned yellow-green, and the mixture was mixed again. After adding activated charcoal to the diluted detection solution, the mixture was thoroughly mixed and centrifuged at 3500 rpm for 10 minutes. One milliliter of the supernatant was taken for detection. The OD values of each tube were measured using a spectrophotometer(SpectraMax Plus 384®, Molecular Devices™, California, USA) at a wavelength of 540–560 nm . The hydroxyproline content (μg/mg wet weight) was calculated as follows: (OD value of sample - OD value of blank) / (OD value of standard - OD value of blank) * concentration of standard (5 μg/ml) * (total volume of hydrolysate [10 ml] / tissue wet weight [mg]). 2.8. Biomechanical Evaluation Biomechanical analysis of maximal tensile force was performed as previously described [23]. In brief, At the end of the experiment, three rabbits from each group were euthanized, and their tendons were obtained. The samples were placed between the two grips of a tensile testing machine (Instron 5900®; Instron Corporation™, MA, USA) and stretched at a rate of 10 mm/min. Three technical replicates were performed for each group, recording the maximum force at tendon rupture as the ultimate tensile strength. 2.9. Quantitative real-time polymerase chain reaction (qRT-PCR) To isolate the total RNA from tissues, RNA Rapid Extraction Kit® (Generay™, Shanghai, China) was applied according to the manufacturer’s instructions. cDNA synthesis was performed with the HiScript II Q RT SuperMix® (Vazyme™, Nanjing, China). ChamQ® SYBR Color qPCR Master Mix kit (Vazyme™, Nanjing, China) was then applied for target gene expression quantification. The gene expression fold change of each sample was referred to as relative gene expression and calculated using the 2 −ΔΔCT method. The experiment for each sample was run in triplicate. The primers used in this study were as follows, with GAPDH used as a housekeeping gene: Troponin C(TnC)：（forward 5′-AATTCTGACCACCCCCAGGA-3′，reverse 5′-ACTGTGGTTCTGGCTCTGTG-3′） MMP-9：（forward 5′-GCCCCAGCGAAAGACTCTAC-3′，reverse 5′-TTGTCCTTGTCGTAGCTGGC-3′） GAPDH：（forward 5′-TGCCGCCTGGAGAAAGC-3′，reverse 5′-CGACCTGGTCCTCGGTGTAG-3′） 2.10. Enzyme-linked immunosorbent assay After homogenizing the tissue, total proteomic content was extracted from all samples. The protein levels of TNC and MMP-9 within the tendon tissues were analyzed using the Rabbit TNC ELISA Kit（Jianglai, Shanghai, China） and the Rabbit MMP-9 ELISA Kit（Jianglai, Shanghai, China）, respectively, following the manufacturer's instructions. Subsequently, this quantification was conducted utilizing a microplate reader (SpectraMax Plus 384®, Molecular Devices™, California, USA). 2.11.Statistical analyses The statistical analysis of all data was performed using SPSS 13.0 (SPSS; Chicago, IL, USA). The quantitative values were presented as mean ± standard deviation (SD) and examined through appropriate statistical tests including the t-test, one-way analysis of variance, or one-way multivariate analysis of variance with repeated measurements. P-values of <0.05 were considered significant. Results 3.1 Identification of UC-MSCs To delineate the immunophenotype of mesenchymal stem cells derived from the umbilical cord (UC-MSCs), an inclusive array of assays targeting surface markers specific to stem cells was employed. Flow cytometry analysis revealed robust expression of CD105, CD73, CD90, and CD49d, while the presence of CD45 (leukocyte common antigen), CD34 (hematopoietic stem cell antigen), CD14 (hematopoietic stem cell antigen), and HLA-DR was conspicuously absent in UC-MSCs (Fig. 1A). These findings served as the basis for discerning cells originating from a mesenchymal lineage. Subsequently, exploration into the multidifferentiation potential of UC-MSCs commenced. Remarkably, UC-MSCs thrived as monolayers, boasting a homogeneously structured morphology characterized by gracefully elongated or fibrous shapes (Fig. 1 B). Upon exposure to chondrogenic differentiation medium, meticulous staining with Alisin Blue and Senna O facilitated the visualization of vivid blue staining spots within clustered samples (Fig. 1 C), which seamlessly transformed into resplendent red staining spots upon additional application of Senna O (Fig. 1 D). Notably, when subjected to osteogenic differentiation medium, alizarin red staining showcased the prevalence of alkaline phosphatase (ALP) activity, an early signature of osteoblasts, within the majority of cells (Fig. 1 E). Lastly, induction of adipogenesis prompted the development of lipid droplets within the cytoplasm, exquisitely stained with oil red O (Fig. 1 F). 3.2 Establishment and Preliminary Evaluation of a Tendinopathy Model In order to establish an animal model of Achilles tendonitis, various doses of type I collagenase were administered via injection into the unilateral Achilles tendon region of experimental rabbits (Fig. 2 A). After 15 days, noticeable alterations in gait emerged prominently within the 2400 U type I collagenase group, while relatively subtle changes were observed in the 1200 U group. The affected Achilles tendon exhibited erythema, swelling, and a loss of its natural luster within the 2400 U group, whereas a mild dark red hue was evident in the 1200 U group. In contrast, the control group displayed a vibrant white color with a discernible sheen (Fig. 2 B). After a duration of forty-five days, the hydroxyproline (Hyp) content was analyzed to evaluate the anabolic status of collagen at the injection site. Interestingly, a significant reduction in Hyp content was observed in the Achilles tendon (AT) group compared to the normal group, indicating a statistically significant discrepancy (Fig. 2 C). Histological examination unveiled notable disparities between the control group and the AT group. Within the control group, Achilles tendon fibrous tissues exhibited parallel and well-organized arrangements, characterized by structural integrity and distinct boundaries between adjacent fiber bundles. Eukaryotic cells were plentiful and uniformly distributed. Conversely, the AT group presented disarrayed collagen fibers, suffering from structural impairments due to substantial infiltration of inflammatory cells, and accompanied by a conspicuous decrease in the number of eukaryotic cells. Additionally, a pronounced accumulation of inflammatory cells surrounding blood vessels was observed (Fig. 2 D, E). 3.3 The engraftment and distribution of CM-Dil labeled UC-MSCs within the Achilles tendon To assess the effectiveness and safety of localized stem cell transplantation, we conducted pre-treatment tracing to observe the survival of UC-MSCs within the Achilles tendinopathy Model and in vivo. The CM-Dil labeled UC-MSCs emitted a vivid red fluorescence (Figure 3A). Utilizing an in vivo imaging system, we investigated the distribution of UC-MSCs within the Achilles tendon tissue before injection, as well as at 1 d, 3 d, 5 d, and 7 d post-injection. Notably, Labeled UC-MSCs treatment group exhibited fluorescent signals within the Achilles tendon tissue, while the control group showed no such fluorescence. The highest concentration of labeled UC-MSCs cells was observed at 1 d after injection, followed by a gradual decline in fluorescence intensity at 3-7 d, indicating a significant reduction in cell numbers (Supplementary Material 1). Particularly, the decrease was most prominent at 3 d. Furthermore, tissue samples were meticulously harvested from the Achilles tendons of rabbits in each group, fixed, embedded, and longitudinally sectioned for observation under a fluorescence microscope. We performed semi-quantitative analysis of the fluorescence intensity. As depicted in Figures 3B and 3C, the normal group and the control group displayed no positive signals. In contrast, positive cell counts in the treatment group followed the order of 1d > 3d > 5d > 7d, mirroring the trend of fluorescence intensity within the Achilles tendon tissues. Additionally, we examined the distribution of UC-MSCs in other organs and tissues but found no evidence of their presence through the in vivo imaging system, indicating that the injected UC-MSCs did not colonize or differentiate within the relevant organs (Supplementary Material 1). 3.4 UC-MSCs facilitate the regeneration of AT After delineating the model of Achilles tendonitis and establishing engraftment conditions, we made adjustments to the number of cell infusions and the recovery period (Fig. 4 A). Direct injection of 5×10 6 UC-MSCs was administered into the affected female rabbits (UC-MSCs group), followed by four subsequent infusions at different intervals. Molecular and histological evaluations were carried out on the 70th day after the initial cell injection, comparing the UC-MSCs group to the model group (AT group).Palpation of the Achilles tendon in the AT group revealed a lackluster appearance with slight flattening and no elasticity, while the UC-MSCs group exhibited elasticity and a glossy texture (Fig. 4 B). Macroscopic assessment incorporating parameters such as inflammation, surface characteristics, defect size, adjacent tendons, defect severity, swelling/redness, connection to surrounding tissues, and sliding ability yielded significantly higher total scores for the AT group (13.67±0.5574) compared to the UC-MSCs group (3.333±2.517) (Fig. 4 C). Furthermore, a comprehensive analysis of tissue pathology unveiled disrupted arrangement of collagen fibers and substantial infiltration of inflammatory cells, leading to structural disturbances within collagen fibers in the AT group treated with type I collagenase. Additionally, there was a notable decrease in the number of eukaryotic cells within collagen fibers and a conspicuous accumulation of inflammatory cells around blood vessels. In contrast, the UC-MSCs group exhibited orderly arranged collagen fibers, a reduced presence of inflammatory cells, and a normal number of eukaryotic cells within collagen fibers (Fig. 4 D). Comparison of HE-stained tissue sections further demonstrated that the number of eukaryotic cells within collagen fibers, both in transverse and longitudinal sections, was significantly higher in the UC-MSCs group (transverse: F= 713±16.09, P<0.001; longitudinal: F= 253±10.02, P<0.001), while the AT group showed a respective decrease of 30% and 43% (Fig. 4 E). In terms of hydroxyproline content, the UC-MSCs group displayed a significant increase compared to the AT group, and there was a significant decrease in the AT group compared to the control group (p<0.05) (Fig. 4 F). The maximum load on the Achilles tendon is closely related to the state of collagen fibers. Compared to the AT group, the UC-MSCs group exhibited a significantly higher maximum load (p<0.05) (Fig. 4 G). 3.5 Exploring Molecular Mechanisms of UC-MSCs in Therapeutic Effects on Achilles Tendon Diseases To investigate the potential molecular mechanisms underlying the therapeutic effects of UC-MSCs on Achilles tendon diseases, we conducted preliminary investigations focusing on structural stability and contraction-regulating markers at the inflammatory site. We examined the alterations of Collagen I and Collagen III in Achilles tendon tissue using immunohistochemical staining. The control group exhibited abundant brown or tan-colored Collagen I and Collagen III, while the AT group showed lower protein expression levels of Collagen I and Collagen III. In contrast, the UC-MSCs group displayed higher expression levels of Collagen I and Collagen III compared to the AT group (Figure 5A). Additionally, we employed Image-Pro Plus software to measure the optical density values of immunohistochemically positive samples. The results are presented as mean ± standard deviation. The data indicated that UC-MSCs could promote the expression of Collagen I (F=29.70, P=0.008, UC-MSCs group vs. AT group, P=0.001, Figure 5B) and Collagen II (F=8.106, P=0.0197, UC-MSCs group vs. AT group, P=0.001, Figure 5B). Furthermore, we assessed the transcriptional and protein expression levels of matrix metalloproteinase 9 (MMP-9), an enzyme involved in collagen metabolism and degradation. Compared with the AT group, UC-MSCs demonstrated the ability to inhibit MMP-9 at both the transcriptional level (mRNA level: F = 1174.69, P < 0.001, UC-MSCs group vs. AT group) and the protein level (protein level: F = 292.1, P < 0.001, UC-MSCs group vs. AT group, P < 0.001, Figure 5C). Given that the Achilles tendon is a vital structure connecting muscle and bone, we also evaluated the expression of troponin C (TnC), an important regulatory protein involved in muscle fiber contraction. Compared to the AT group, UC-MSCs significantly upregulated both the transcriptional level (mRNA level: F = 2459, P < 0.0001, UC-MSCs group vs. AT group) and the protein level (protein level: F = 56.99, P < 0.0001, UC-MSCs group vs. AT group, P = 0.0001, Figure 5D) of TnC. Discussion Achilles tendon disease, clinically diagnosed as tendinopathy, is a common degenerative overuse condition. It can be classified into insertional and mid-portion (non-insertional) tendinopathy, each with distinct characteristics and treatment strategies [24]. The repair and healing rates of Achilles tendons are relatively low due to the low cellular, vascular, and metabolic activity of tendon tissue. The process of tendon healing can be divided into three overlapping stages: inflammation, proliferation, and remodeling [25]. Conventional treatments often struggle to address all three stages adequately. Mesenchymal stem cells (MSCs), including umbilical cord-derived MSCs (UC-MSCs), have shown promising results in improving inflammation and cell death through various mechanisms, such as targeted differentiation, paracrine effects, and secretion of extracellular vesicles [26]. UC-MSCs have demonstrated satisfactory outcomes in promoting tendon repair and regeneration, not only by facilitating tissue regeneration but also by restoring the original biomechanical function of the tendon [18] . However, there is still no consensus on practical considerations regarding the source, dose, administration technique, and timing of MSC usage in clinical applications. UC-MSCs, isolated from postpartum medical waste like umbilical cords, offer advantages such as non-invasiveness and relatively lower cost compared to other adult stem cells like adipose-derived stem cells [9], dental pulp stem cells [27], and even follicle stem cells. Furthermore, UC-MSCs have a younger origin and possess higher proliferative and self-renewal differentiation potential [14]. Previous studies have also confirmed the successful repair of rotator cuff injuries using UC-MSCs without significant immunosuppression[14]. Similar positive effects on tendon function repair were observed in our rabbit model of Achilles tendon diseases. In our collagenase-induced tendonitis model, several common pathological characteristics of tendon diseases were evident, including collagen disruption, inflammatory infiltration, neovascularization, adipogenesis, and ectopic ossification, which have been extensively used to study chronic human tendon diseases [28]. Histological evaluation and semi-quantitative analysis revealed collagen fiber disarray and extensive inflammatory cell infiltration in the AT group compared to the control group, indicating structural damage to the collagen fibers (Fig 2D & Fig 4D).. To establish a reliable rabbit model of collagenase-induced tendonitis, we compared the degenerative potential of two different concentrations of type I collagenase and focused on gait analysis, macroscopic changes, and histopathology at different time points (15 days and 45 days) in rabbits. Our results align with other studies conducted in large animals, where the severity of pathology is related to the amount of injected collagenase [29-30] . The injection of 2400 U of type I collagenase induced apparent gait alterations and reddening of the Achilles tendon (Fig 2B), along with morphological changes similar to those seen in human tendon disease histology, especially at day 15. However, we observed a decrease in the number of prokaryotic cells in the AT group compared to the control group at 45 days (Fig 2E). This contrasts with previous reports in which all treatment groups showed increased cell density [30]. We speculate that a single intratendinous injection of collagenase may cause rapid and severe damage to tendon integrity, leading to massive death of collagen cells. However, in the mouse model, an increase in the number of round resident cells was observed, indicating high cell density, which may be attributed to different animal models. Various methods have been used for MSC therapy in tendon repair, such as direct injection or systemic infusion, or combining cells with scaffolding materials [31]. Direct injection to the local site is currently the predominant administration route, but it requires clear determination of the total cell count, injection frequency, and distribution within the body [32]. Wang et al. utilized CFDA-SE-labeled UC-MSCs injected into rat tendons and detected fluorescence signals even after 1-5 weeks of transplantation. Furthermore, UC-MSCs demonstrated histological and biological improvement after 4 weeks, but most injected UC-MSCs gradually disappeared from the injured tendon over time, indicating limited residence time in vivo. In our experiment, we observed fluorescence in the AT group of CM-Dil-labeled UC-MSCs in the Achilles tendon tissue, with the highest intensity at 1 day post-injection and subsequent decreasing fluorescence from day 3 to 7 (Fig 3B), indicating a significant reduction in cell numbers (Supplementary Material 1). This could be due to the instability or metabolism of the fluorescent dye. However, studies have shown that more than half of the labeled cells are lost after 23 hours post-injection, possibly due to vascular injury during the injection process, leading to their entry into the bloodstream and migration to other injury sites [33]. To improve therapeutic effects, we employed multiple local injections as the administration technique, with an interval of 3 days for the first three injections. Furthermore, we observed that CM-Dil-labeled UC-MSCs did not settle in other visceral organs within the rabbits (Supplementary Material 1), which could be due to the relatively low number of cells injected locally and oligovascularity, as well as the non-traumatic nature of the induced tendon diseases, indicating the safety of UC-MSC treatment. In our study, we found that UC-MSCs improved the structural stability at the inflammatory site by upregulating Collagen I and Collagen III expression and inhibiting MMP-9. Mature tendons are mainly composed of Collagen I (>95%), but also include a small amount of Collagen III [34]. Wei et al. reported a significant reduction in Collagen I levels after induction of collagenase-I compared to normal tendons [35]. Similarly, our results showed a decrease in Collagen I levels of approximately 70% in the AT group compared to the control group. However, UC-MSC treatment resulted in a 1.8-fold increase in Collagen I expression (Fig 5A & Fig 5B). Additionally, Lee et al. demonstrated that MSCs transplanted into rat tendons survived for at least 12 weeks, differentiated into tendon lineage cells, and secreted type I collagen to enhance tendon repair [36]. Therefore, UC-MSCs may enhance type I collagen production. The degradation of collagen represents the opposite side, where M1 macrophage-promoted inflammatory reactions lead to the production of MMPs and a decrease in type I collagen, resulting in ECM degradation [11] . MMPs play a crucial role in tissue remodeling, and a study on rat flexor tendon healing found that MMP-9 is involved in the early stage of collagen I degradation [37]. In our study, protein and transcription levels of matrix metalloproteinase-9 (MMP-9) demonstrated an increasing trend in the AT group compared to the control group, showing approximately a 4.5-fold increase in expression. However, UC-MSC treatment significantly reduced IL-1β and inhibited MMP-9 activity (Fig 5C), consistent with the results reported by Xu et al.[38]and Wei et al.[35]. We also measured Tenascin C (TNC), a glycoprotein that interacts with collagen fibers to maintain ECM mechanical properties [39]. In the AT group, TNC levels decreased by 3-4 times compared to the control group. However, after UC-MSC treatment, the expression of TNC increased by 40%-50% (Fig 5D). Furthermore, recent studies have suggested that the healing effects of MSCs arise from their paracrine effects, such as secretion of cytokines and growth factors to recruit, proliferate, and differentiate tissue-specific progenitor cells for synthesizing specific matrices [17] . Further research using other cellular and molecular assays, such as Western blotting,, focusing on the analysis of TGF-β/SMAD2/3 pathway-related collagen protein synthesis and degradation，is warranted to improve our understanding of the mechanisms involved [15]. Although the collagenase-induced tendonitis model partially mimics natural injuries, it still differs from the natural course of the disease. Moreover, rabbit tendon diseases should be differentiated from human tendon injuries. Additionally, a comprehensive evaluation of animal behavior outcomes, including pain response and gait analysis, should be conducted. While we performed four injections of cells, the assessment of tendon healing at different time points was lacking. The timing of cell injection (acute, sub-acute, or chronic stage) influences the therapeutic effect since the local environment undergoes changes during disease progression or acute injury healing. Multiple injections may also create inconvenience for future clinical treatments. Conclusion In conclusion, this study underscores the satisfactory capacity of UC-MSCs transplantation in promoting tendon functional repair in a rabbit model of tendon disease. UC-MSC therapy improves tendon functionality at macroscopic, histological, and biomechanical levels. This process involves upregulation of Collagen I and Collagen III expression, inhibition of MMP-9 to enhance the structural stability of the inflammatory site, and an increase in muscle calcium-binding protein C (TnC) to strengthen muscle fiber contraction. Declarations Funding This work was supported by the Hangzhou Medical and Health Science and Technology Project [Grant No. B20210041]. CRediT authorship contribution statement Xiangyi Sun : experimental activity, analyzing the results, and writing the draft of the article. Zhiwei Lin and Jinyang Chen : experimental activity. Zhe Wang and Guangqi Zhu : experimental activity, conceptualization. Ruchao Long : Supervision. Zhihua Yang : conceptualization, supervision, writing, editing the final version of the article, and funding acquisition.All authors are responsible for the overall integrity of the work. Declaration of Competing Interest The authors declare that there is no conflict of interest. Data availability Data will be made available on request. References Kujala, U.M., Sarna, S. and Kaprio, J. (2005) Cumulative incidence of achilles tendon rupture and tendinopathy in male former elite athletes. Clin. J. Sport Med., 15, 133-5, http://doi:10.1097/01.jsm.0000165347.55638.23. Maffulli, N., Longo, U.G., Kadakia, A. and Spiezia, F. (2020) Achilles tendinopathy. Foot Ankle Surg, 26, 240-249, http://doi: 10.1016/j.fas.2019.03.009. Von Rickenbach, K.J., Borgstrom, H., Tenforde, A., Borg-Stein, J. and McInnis, K.C. (2021) Achilles Tendinopathy: Evaluation, Rehabilitation, and Prevention. Curr Sports Med Rep, 20, 327-334, http://doi: 10.1249/JSR.0000000000000855. Millar, N.L., Silbernagel, K.G., Thorborg, K., Kirwan, P.D., Galatz, L.M., Abrams, G.D., et al. (2021) Tendinopathy. Nat Rev Dis Primers, 7, 1, http://doi: 10.1038/s41572-020-00234-1. van Dijk, C.N., van Sterkenburg, M.N., Wiegerinck, J.I., Karlsson, J. and Maffulli, N. (2011) Terminology for Achilles tendon related disorders. Knee Surg Sports Traumatol Arthrosc, 19, 835-41, http://doi: 10.1007/s00167-010-1374-z Andia, I., Martin, J.I. and Maffulli, N. (2018) Advances with platelet rich plasma therapies for tendon regeneration. Expert Opin Biol Ther, 18, 389-398, http://doi: 10.1080/14712598.2018.1424626. Burk, J., Wittenberg-Voges, L., Schubert, S., Horstmeier, C., Brehm, W. and Geburek, F. (2023) Treatment of Naturally Occurring Tendon Disease with Allogeneic Multipotent, Cells,12(21),http://doi: 10.3390/cells12212513. Fredianto, M., Herman, H., Ismiarto, Y.D., Purba, A., Putra, A. and Hidayah, N. (2023) Combination Effect of Rotator Cuff Repair with Secretome-hypoxia MSCs Ameliorates TNMD, RUNX2, and Healing Histology Score in Rotator Cuff Tear Rats. Arch Bone Jt Surg, 11, 617-624, http://doi: 10.22038/ABJS.2023.67933.3218. Arnaud-Franco, A., Lara-Arias, J., Marino-Martinez, I.A., Cienfuegos-Jimenez, O., Barbosa-Quintana, A. and Pena-Martinez, V.M. (2022) Effect of Adipose-Derived Mesenchymal Stem Cells (ADMSCs) Application in Achilles-Tendon Injury in an Animal Model. Curr. Issues Mol. Biol., 44, 5827-5838, http://doi: 10.3390/cimb44120396. Oshita, T., Tobita, M., Tajima, S. and Mizuno, H. (2016) Adipose-Derived Stem Cells Improve Collagenase-Induced Tendinopathy in a Rat Model. Am J Sports Med, 44, 1983-9, http://doi: 10.1177/0363546516640750. Kokubu, S., Inaki, R., Hoshi, K. and Hikita, A. (2020a) Adipose-derived stem cells improve tendon repair and prevent ectopic ossification in tendinopathy by inhibiting inflammation and inducing neovascularization in the early stage of tendon healing. Regen Ther, 14, 103-110, http://doi: 10.1016/j.reth.2019.12.003. Suroto, H., Satmoko, B.A., Rarasati, T. and Prajasari, T. (2023) Long term functional outcome evaluation in post flexor digitorum profundus tendon zone I rupture repaired by palmaris longus tendon grafting augmented with human amniotic membranes and adipose derived mesenchymal stem cell: A case report. Int J Surg Case Rep, 104, 107960, http://doi: 10.1016/j.ijscr. Um, S., Ha, J., Choi, S.J., Oh, W. and Jin, H.J. (2020) Prospects for the therapeutic development of umbilical cord blood-derived mesenchymal stem cells. World J Stem Cells, 12, 1511-1528, http://doi: 10.4252/wjsc.v12.i12.1511. Yea, J.H., Bae, T.S., Kim, B.J., et al.. (2020) Regeneration of the rotator cuff tendon-to-bone interface using umbilical cord-derived mesenchymal stem cells and gradient extracellular matrix scaffolds from adipose tissue in a rat model. Acta Biomater., 114, 104-116, http://doi: 10.1016/j.actbio.2020.07.020. Wang, S., Yao, Z., Chen, L., Li, J., Chen, S. and Fan, C. (2023) Preclinical assessment of IL-1beta primed human umbilical cord mesenchymal stem cells for tendon functional repair through TGF-beta/IL-10 signaling. Heliyon, 9, e21411, http://doi: 10.1016/j.heliyon.2023.e21411. Xue, Y., Kim, H.J., Lee, J., Liu, Y., Hoffman, T., Chen, Y., et al.. (2022) Co-Electrospun Silk Fibroin and Gelatin Methacryloyl Sheet Seeded with Mesenchymal Stem Cells for Tendon Regeneration. Small, 18, e2107714, http://doi: 10.1002/smll.202107714. Zhang, B., Luo, Q., Halim, A., Ju, Y., Morita, Y. and Song, G. (2017) Directed Differentiation and Paracrine Mechanisms of Mesenchymal Stem Cells: Potential Implications for Tendon Repair and Regeneration. Curr Stem Cell Res Ther, 12, 447-454, http://doi: 10.2174/1574888X12666170502102423. Jiang, L., Lu, J., Chen, Y., Lyu, K., Long, L., Wang, X.,et al. (2023).Mesenchymal stem cells: An efficient cell therapy for tendon repair (Review). Int. J. Mol. Med., 52, http://doi: 10.3892/ijmm.2023.5273. Jiayi Yang, Zhiyi Chen, Daoyan Pan, Huaizhi Li, Jie Shen.(2020) Umbilical Cord-Derived Mesenchymal Stem Cell-Derived Exosomes Combined Pluronic F127 Hydrogel Promote Chronic Diabetic Wound Healing and Complete Skin Regeneration. Int J Nanomedicine, 15, 5911-5926.http://doi: 10.2147/IJN.S249129. Choi, H.J., Choi, S., Kim, J.G., Song, M.H., Shim, K.S., et al. (2020) Enhanced tendon restoration effects of anti-inflammatory lactoferrin-immobilized, heparin-polymeric nanoparticles in an Achilles tendinitis rat model. Carbohydr Poly, 241:116284. , http://doi: 10.1016/j.carbpol.2020.116284. Yuan, K., Lai, C., Wei, L., Feng, T., Yang, Q., Zhang, T., et al. (2019) The Effect of Vascular Endothelial Growth Factor on Bone Marrow Mesenchymal Stem Cell Engraftment in Rat Fibrotic Liver upon Transplantation. Stem Cells Int., 2019, 5310202, http://doi: 10.1155/2019/5310202. Stoll, C., John, T., Conrad, C., Lohan, A., Hondke, S., Ertel, W., et al. (2011) Healing parameters in a rabbit partial tendon defect following tenocyte/biomaterial implantation. Biomaterials,32,4806-15 ,http://doi: 10.1016/j.biomaterials.2011.03.026 Li, J., Yao, Z., Xiong, H., Cui, H., Wang, X., Zheng, W., et al. (2020) Extracellular vesicles from hydroxycamptothecin primed umbilical cord stem cells enhance anti-adhesion potential for treatment of tendon injury. Stem Cell Res. Ther., 11, 500, http://doi: 10.1186/s13287-020-02016-8. Chimenti, R.L., Cychosz, C.C., Hall, M.M. and Phisitkul, P. (2017) Current Concepts Review Update: Insertional Achilles Tendinopathy. Foot Ankle Int., 38, 1160-1169, http:// doi: 10.1177/1071100717723127. Andarawis-Puri, N., Flatow, E.L. and Soslowsky, L.J. (2015) Tendon basic science: Development, repair, regeneration, and healing. J. Orthop. Res., 33, 780-4, http://doi: 10.1002/jor.22869. Kokubu, S., Inaki, R., Hoshi, K. and Hikita, A. (2020b) Adipose-derived stem cells improve tendon repair and prevent ectopic ossification in tendinopathy by inhibiting inflammation and inducing neovascularization in the early stage of tendon healing. Regen Ther, 14, 103-110, http://doi: 10.1016/j.reth.2019.12.003 Li, F., Wang, X., Shi, J., Wu, S., Xing, W. and He, Y. (2023) Anti-inflammatory effect of dental pulp stem cells. Front Immunol, 14, 1284868, http://doi: 10.3389/fimmu.2023.1284868. Liu, A., Wang, Q., Zhao, Z., Wu, R., Wang, M., Li, J., et al. (2021) Nitric Oxide Nanomotor Driving Exosomes-Loaded Microneedles for Achilles Tendinopathy Healing. ACS Nano, 15, 13339-13350, http://doi: 10.1021/acsnano.1c03177. Ghelfi, J., Bacle, M., Stephanov, O., de Forges, H., Soulairol, I., Roger, P., et al. (2021) Collagenase-Induced Patellar Tendinopathy with Neovascularization: First Results towards a Piglet Model of Musculoskeletal Embolization. Biomedicines, 10, http:// doi: 10.3390/biomedicines10010002. Perucca, O.C., Lovati, A.B., Vigano, M., Stanco, D., Bottagisio, M., Di Giancamillo, A., et al. (2016) Dose-Related and Time-Dependent Development of Collagenase-Induced Tendinopathy in Rats. PLoS One, 11, e0161590, http://doi: 10.1371/journal.pone.0161590. Shojaee, A. and Parham, A. (2019) Strategies of tenogenic differentiation of equine stem cells for tendon repair: current status and challenges. Stem Cell Res. Ther., 10, 181, http://doi: 10.1186/s13287-019-1291-0. Bowers, K., Amelse, L., Bow, A., Newby, S., MacDonald, A., Sun, X., et al. (2022) Mesenchymal Stem Cell Use in Acute Tendon Injury: In Vitro Tenogenic Potential vs. In Vivo Dose Response. Bioengineering (Basel), 9, http://doi: 10.3390/bioengineering9080407. Becerra, P., Valdes, V.M., Dudhia, J., Fiske-Jackson, A.R., Neves, F., Hartman, N.G, et al.. (2013) Distribution of injected technetium(99m)-labeled mesenchymal stem cells in horses with naturally occurring tendinopathy. J. Orthop. Res., 31, 1096-102, http://doi: 10.1002/jor.22338. Cho, Y., Kim, H.S., Kang, D., Kim, H., Lee, N., Yun, J., et al.(2021) CTRP3 exacerbates tendinopathy by dysregulating tendon stem cell differentiation and altering extracellular matrix composition. Sci Adv, 7, eabg6069, http://doi: 10.1126/sciadv.abg6069. Wei, B., Ji, M., Lin, Y., Wang, S., Liu, Y., Geng, R.,et al. (2023) Mitochondrial transfer from bone mesenchymal stem cells protects against tendinopathy both in vitro and in vivo. Stem Cell Res. Ther., 14, 104, http://doi: 10.1186/s13287-023-03329-0. Lee, S.Y., Kwon, B., Lee, K., Son, Y.H. and Chung, S.G. (2017) Therapeutic Mechanisms of Human Adipose-Derived Mesenchymal Stem Cells in a Rat Tendon Injury Model. Am J Sports Med, 45, 1429-1439, http://doi: 10.1177/0363546517689874. Bramono, D.S., Richmond, J.C., Weitzel, P.P., Kaplan, D.L. and Altman, G.H. (2004) Matrix metalloproteinases and their clinical applications in orthopaedics. Clin Orthop Relat Res, 272-85, http://doi: 10.1097/01.blo.0000144166.66737.3a. Xu, B., Wang, Y., He, G., Tang, K.L., Guo, L. and Chen, W. (2024) A novel and efficient murine model for investigating tendon-to-bone healing. J. Orthop. Surg. Res., 19, 90, http://doi: 10.1186/s13018-023-04496-9. Jarvinen, T.A., Jozsa, L., Kannus, P., Jarvinen, T.L., Hurme, T., Kvist, M., et al. (2003) Mechanical loading regulates the expression of tenascin-C in the myotendinous junction and tendon but does not induce de novo synthesis in the skeletal muscle. J. Cell Sci., 116, 857-66, http://doi: 10.1242/jcs.00303. Additional Declarations No competing interests reported. Supplementary Files AppendixA.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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injury is a clinically intractable tendon disorder characterized by degenerative and cumulative tissue microtrauma. The incidence rate among runners ranges from 9.1% to 10.9%, with male distance runners having a lifetime risk exceeding 50%\u0026nbsp;[1-2].\u0026nbsp;This condition imposes a significant clinical burden on global healthcare systems\u0026nbsp;[3].\u003c/p\u003e\n\u003cp\u003eThe characteristic clinical presentations of Achilles tendon disease encompass pain, swelling, and compromised functionality, especially during ambulation and activities involving impact\u0026nbsp;[1].The development of Achilles tendon disease involves various factors, including modifiable and non-modifiable risk factors\u0026nbsp;[4]. Non-modifiable factors include patient demographics such as older age, male gender, compromised vascularity, hyperpronation, and other biomechanical factors, as well as systemic diseases like metabolic and rheumatic disorders. External risk factors encompass overuse, training errors (rapid increase in impact exercise volume), and certain medications known to induce tendon injury, including corticosteroids and fluoroquinolone antibiotics\u0026nbsp;[5]. Treatment typically encompasses non-surgical interventions, which can be classified into passive modalities such as pharmacotherapy, injection therapies, extracorporeal shockwave therapy (ESWT), therapeutic ultrasonography, and low-level laser therapy. Active approaches may involve tendon-loading exercises, patient education, and load management\u0026nbsp;[6]. However, the primary goal of treatment for most Achilles tendon disease patients is symptom relief, particularly pain, with suboptimal long-term outcomes. Naturally healed ATs often exhibit poor quality due to the formation of fibrotic scars and tendon sheath adhesion, along with an increased risk of subsequent reinjury and re-rupture during subsequent physical activities and daily tasks. Therefore, it is crucial to investigate the pathological processes of Achilles tendon disease and the mechanisms involved in tendon healing to improve current treatment strategies.\u003c/p\u003e\n\u003cp\u003eIn recent years, mesenchymal stem cells (MSCs) from different sources have gained popularity as a therapeutic option for tendon repair\u0026nbsp;[7-8]. Takashi Oshita et al. reported on the effects of adipose-derived stem cells (ASCs) on tendon healing in a rat tendon disease model, demonstrating significantly reduced tendon degeneration at both two time points compared to the PBS group, along with a decreased ratio of type III collagen to type I collagen[9-10]. ASCs have also been shown to decrease the expression of inflammatory factors such as IL-1\u0026beta;, GLUT1, and CA9 in mice during the early stages of tendon injury, while inducing neovascularization\u0026nbsp;[11]. The combination of human amniotic membrane (HAM) with ASCs further enhances anti-adhesion properties and accelerates tendon healing\u0026nbsp;[12]. Umbilical cord-derived MSCs (UC-MSCs) exhibit greater proliferative and self-renewal potential\u0026nbsp;[13]\u0026nbsp;and can induce tendon regeneration at macroscopic, histological, and biomechanical levels\u0026nbsp;[14]. Transplantation of IL-1\u0026beta;-primed UC-MSCs has demonstrated superior capacity in promoting tendon functional repair in a rat tendon disease model, improving the inflammatory response and metabolism of the extracellular matrix through the TGF-\u0026beta;/IL-10 pathway\u0026nbsp;[15]. The activation of UC-MSCs by IL-1\u0026beta; may lead to alterations in their secretome, suggesting diverse mechanisms of action. For example, Fredianto et al. found that UC-MSCs under hypoxic conditions significantly improved tendon repair by upregulating TNMD and RUNX2 expression and histological scoring\u0026nbsp;[8]. Additionally, combining with biomaterials such as silk fibroin and gelatin methacryloyl\u0026nbsp;（GelMA）\u0026nbsp;can promote the migration and proliferation of MSCs and tenocytes\u0026nbsp;[16]. Furthermore, the therapeutic effect of MSCs may be attributed to paracrine effects, including the secretion of cytokines and growth factors to recruit, proliferate, and differentiate tissue-specific progenitor cells for synthesizing specific matrices\u0026nbsp;[17]\u0026nbsp;.\u003c/p\u003e\n\u003cp\u003eAlthough adipose tissue and bone marrow are frequently utilized sources of MSCs, both have drawbacks such as invasive harvesting methods, low efficiencies of cell extraction, and potential donor morbidities [13]. Furthermore, MSCs derived from adult tissues demonstrate variations in their differentiation potential [18]. \u0026nbsp;Additionally, the engraftment and in vivo distribution of locally injected MSCs remain unclear. This study aims to investigate the engraftment and temporal effects of UC-MSCs on AT regeneration in a rabbit model.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Statement of Ethics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study received approval from the Ethics Committee of Hangzhou Hibio Pharmaceutical Research and Development Center\u0026nbsp;(approval number: HB2004217\u0026nbsp;).All animal experiments were performed in accordance with the Guidelines for Care and Use of Laboratory Animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Cell Isolation, Culture, and Identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUC-MSCs were isolated using a previously described protocol [19] .Human umbilical cords were obtained via cesarean section from full-term deliveries. The umbilical cords were dissected into approximately 2-3 mm tissue fragments, which were then washed with PBS. The tissue fragments were placed in a 9 cm culture dish and slowly supplemented with \u0026nbsp;Dulbecco\u0026apos;s modified eagle medium (DMEM) (Hyclone) \u0026nbsp;supplemented with 10% fetal bovine serum (FBS) and \u0026nbsp;1 ％ penicillin/streptomycin. Subsequently, the culture dishes were incubated at 37℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e. When the cells reached approximately\u0026nbsp;80% confluence, they were passaged after digestion with a trypsin-EDTA solution (0.05% trypsin, 0.53 mM EDTA). The complete culture medium was replaced every 2-3 days. The UC-MSCs were cultured until passage 5 before being used for experiments. Morphological characterization was performed, and adipogenic differentiation was confirmed through Oil Red O staining, osteogenic differentiation was verified using Alizarin Red S staining, and chondrogenic differentiation was assessed by Alcian blue and Safranin O staining. Flow cytometry analysis was conducted to detect the expression of relevant cell surface markers, including CD90, CD73, CD14, CD105, CD34, CD45, CD49d, and HLA-DR\u0026nbsp;(Biolegend, USA).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Establishment of Rabbit Achilles Tendon Disease Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHealthy male New Zealand rabbits\u0026nbsp;(age 17\u0026ndash;21 weeks, weight 3000-4000g) were supplied by Hangzhou Yuhang Keliang Rabbitry Cooperative (Hangzhou, China). Anesthesia was induced in all rabbits using ketamine (30 mg/kg) and xylazine (6 mg/kg) via intramuscular injection. The left hind limb of each rabbit was used for inducing tendon disease. The models of collagenase-induced AT were constructed based on a method previously described\u0026nbsp;[20]. In the low-dose group, five animals were injected with 1200 U of Type I collagenase (SOLABIO, Shanghai) using a multi-point injection technique along the left leg Achilles tendon, with 2 mL of 600 U/mL Type I collagenase injected at 1 cm intervals above the tendon. In the high-dose group, five animals were injected with 2400 U of Type I collagenase.\u003c/p\u003e\n\u003cp\u003eGait observation was performed to confirm the induction of Achilles tendonitis. On days 15 and 45 after injection, euthanasia was carried out through intravenous administration of propofol (10 mg/kg) and 19.1% potassium chloride (10 mL). Immediately after euthanasia, the gross anatomical structure of the Achilles tendon was examined. Hematoxylin-eosin (HE) staining was performed to determine the number and distribution of nucleated cells as well as the arrangement of collagen fibers. Pathological observations were made, and hydroxyproline content was measured.\u003c/p\u003e\n\u003cp\u003eAfter determining the appropriate induction dosage, 2400 U of Type I collagenase was again used to induce tendon disease in the left hind limbs of the animals. The animals were divided into two groups: the Tendon group (injected with saline) and the UC-MSCs treatment group. P5 passage UC-MSCs or saline were injected into and around the Achilles tendon, while the corresponding right hind limb served as an untreated control. A total of four injections were administered over a duration of 70 days, with approximately 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells in each injection. Following euthanasia, anatomical and pathological examinations of the Achilles tendon were performed, taking into account the overall health status of all animals. Biomechanical evaluation of the tendons was conducted to determine their maximum load-bearing capacit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2.4. Labeling of UC-MSCs for fluorescent tracking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUC-MSCs were labeled with red fluorescent Chloromethylbenzamido-1,1\u0026prime;-dioctadecyl-3,3,3\u0026prime;3\u0026acute;-tetramethylindocarb cyanine perchlorate (CM-Dil) (YEASEN, Shanghai) following a previously described protocol\u0026nbsp;[21]. Briefly, UC-MSCs were trypsinized and resuspended in 1 mL of CM-Dil solution (1\u0026mu;mol/L). The cell suspension was incubated at 37℃\u0026nbsp;for 30 minutes, with intermittent shaking to ensure thorough mixing. After centrifugation and three washes with PBS, cells were resuspended in an appropriate amount of PBS to achieve a cell concentration of 5\u0026times;10\u003csup\u003e7\u003c/sup\u003e/ml. The cells were then observed and photographed under a fluorescence microscope to confirm successful labeling. At day 15 post-modeling, the respective groups of rabbits underwent stem cell therapy. The treatment group received injections of CM-Dil-labeled UC-MSCs into the right Achilles tendon, while the normal control group received injections of saline. Euthanasia was performed on days 1, 3, 5, and 7 after treatment, and the heart, liver, spleen, lung, kidney, and Achilles tendon were imaged using in vivo imaging (AniView 600，Guangzhou Boruteng Biotechnology Co., Ltd.). Tissue samples were obtained from the Achilles tendon and subjected to collection, fixation, embedding, and staining with DAPI for visualization of cell nuclei. Subsequently, longitudinal sections were examined using a fluorescence microscope, and semi-quantitative analysis of fluorescence intensity was performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2.5.Macroscopic Evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter euthanizing the animals, tissue samples were collected from the Achilles tendon. For macroscopic evaluation, we employed a modified semi-quantitative system described by Stoll et al\u0026nbsp;[22]. (see Additional file 2). This system consisted of 12 parameters, including tendon rupture, inflammation, tendon surface, neighboring tendon, defect level, defect size, tendon swelling/redness, connection and slidability of surrounding tissue, tendon thickness, tendon color, single muscle strain, and transition of the construct to the surrounding healthy tissue. The total macroscopic score ranged between 0 (indicating a normal tendon) and 15 (representing the most severe injury). This analysis was conducted by three blinded pathologists who graded the observed tissues according to the predetermined scoring system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6. Histological Evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue sections of the Achilles tendon were prepared as previously described\u0026nbsp;[23]. In brief, the tendon was fixed in 4% paraformaldehyde, dehydrated through a standard procedure, and embedded in paraffin. Subsequently, 5 \u0026mu;m-thick sections were cut, stained with hematoxylin for 5 minutes and eosin for 3 to 5 minutes, and observed using a conventional light microscope.\u003c/p\u003e\n\u003cp\u003eImmunofluorescence staining (IHC) was performed as previously described\u0026nbsp;(Li et al., 2020). Briefly, the paraffin-embedded tissues were deparaffinized with xylene and dehydrated in a graded alcohol series. Endogenous peroxidase activity was blocked and inactivated with 0.3% hydrogen peroxide, and tissue antigens were retrieved using citrate buffer (0.01 mol/L, pH 6.0) for 10 minutes. Slides were incubated overnight at 4℃with primary antibodies including anti-Collagen I and anti-Collagen III (dilution: 1:200; Bioss). After washing the slides with PBS, they were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (dilution: 1:3000; Abcam) for 30 min at room temperature. Immunopositive expression intensity was measured using Image-Pro Plus software, with three random fields analyzed per group. The results were presented as mean\u0026nbsp;\u0026plusmn;\u0026nbsp;standard deviation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7. Measurement of Hydroxyproline\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe amount of hydroxyproline in the tendon was measured using a hydroxyproline measurement kit (Nanjing Jiancheng, Nanjing). \u0026nbsp;The protocol was performed according to the manufacturer\u0026rsquo;s catalog. Briefly, approximately 80-100 mg of tissue was accurately weighed, minced, and hydrolyzed with 1 mL of 6 mol/L HCl at 95℃ for 5 hours. The hydrolysate was adjusted to pH 6.0-6.8, cooled, and 10 \u0026mu;l of indicator reagent was added to each tube, followed by thorough mixing. The pH-adjusted methanol and ethanol were then added to each tube until the indicator turned yellow-green, and the mixture was mixed again. After adding activated charcoal to the diluted detection solution, the mixture was thoroughly mixed and centrifuged at 3500 rpm for 10 minutes. One milliliter of the supernatant was taken for detection. The OD values of each tube were measured using a spectrophotometer(SpectraMax Plus 384\u0026reg;, Molecular Devices\u0026trade;, California, USA) at a wavelength of 540\u0026ndash;560 nm . The hydroxyproline content (\u0026mu;g/mg wet weight) was calculated as follows: (OD value of sample - OD value of blank) / (OD value of standard - OD value of blank) * concentration of standard (5 \u0026mu;g/ml) * (total volume of hydrolysate [10 ml] / tissue wet weight [mg]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8. Biomechanical Evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiomechanical analysis of maximal tensile force was performed as previously described\u0026nbsp;[23]. In brief, At the end of the experiment, three rabbits from each group were euthanized, and their tendons were obtained. The samples were placed between the two grips of a tensile testing machine (Instron 5900\u0026reg;; Instron Corporation\u0026trade;, MA, USA) and stretched at a rate of 10 mm/min. Three technical replicates were performed for each group, recording the maximum force at tendon rupture as the ultimate tensile strength.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9. Quantitative real-time polymerase chain reaction (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo isolate the total RNA from tissues, RNA Rapid Extraction Kit\u0026reg; (Generay\u0026trade;, Shanghai, China)\u0026nbsp;was applied according to the manufacturer\u0026rsquo;s instructions.\u0026nbsp;cDNA synthesis was performed with the HiScript II Q RT SuperMix\u0026reg; (Vazyme\u0026trade;, Nanjing, China). ChamQ\u0026reg; SYBR Color qPCR Master Mix kit (Vazyme\u0026trade;, Nanjing, China)\u0026nbsp;was then applied for target gene expression quantification. The gene expression fold change of each sample was referred to as relative gene expression and calculated using the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;CT\u003c/sup\u003e method. The experiment for each sample was run in triplicate. The primers used in this study were as follows, with GAPDH used as a housekeeping gene:\u003c/p\u003e\n\u003cp\u003eTroponin C(TnC)：（forward 5\u0026prime;-AATTCTGACCACCCCCAGGA-3\u0026prime;，reverse 5\u0026prime;-ACTGTGGTTCTGGCTCTGTG-3\u0026prime;）\u003c/p\u003e\n\u003cp\u003eMMP-9：（forward 5\u0026prime;-GCCCCAGCGAAAGACTCTAC-3\u0026prime;，reverse 5\u0026prime;-TTGTCCTTGTCGTAGCTGGC-3\u0026prime;）\u003c/p\u003e\n\u003cp\u003eGAPDH：（forward 5\u0026prime;-TGCCGCCTGGAGAAAGC-3\u0026prime;，reverse 5\u0026prime;-CGACCTGGTCCTCGGTGTAG-3\u0026prime;）\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10. Enzyme-linked immunosorbent assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter homogenizing the tissue, total proteomic content was extracted from all samples. The protein levels of TNC and MMP-9 within the tendon tissues were analyzed using the Rabbit TNC ELISA Kit（Jianglai, Shanghai, China）\u0026nbsp;and the \u0026nbsp;Rabbit MMP-9 ELISA Kit（Jianglai, Shanghai, China）, respectively, following the manufacturer\u0026apos;s instructions. Subsequently, this quantification was conducted utilizing a microplate reader (SpectraMax Plus 384\u0026reg;, Molecular Devices\u0026trade;, California, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11.Statistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe statistical analysis of all data was performed using SPSS 13.0 (SPSS; Chicago, IL, USA). The quantitative values were presented as mean \u0026plusmn; standard deviation (SD) and examined through appropriate statistical tests including the t-test, one-way analysis of variance, or one-way multivariate analysis of variance with repeated measurements. P-values of \u0026lt;0.05 were considered significant.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Identification of UC-MSCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo delineate the immunophenotype of mesenchymal stem cells derived from the umbilical cord (UC-MSCs), an inclusive array of assays targeting surface markers specific to stem cells was employed. Flow cytometry analysis revealed robust expression of CD105, CD73, CD90, and CD49d, while the presence of CD45 (leukocyte common antigen), CD34 (hematopoietic stem cell antigen), CD14 (hematopoietic stem cell antigen), and HLA-DR was conspicuously absent in UC-MSCs (Fig. 1A). These findings served as the basis for discerning cells originating from a mesenchymal lineage. Subsequently, exploration into the multidifferentiation potential of UC-MSCs commenced. Remarkably, UC-MSCs thrived as monolayers, boasting a homogeneously structured morphology characterized by gracefully elongated or fibrous shapes (Fig. 1 B). Upon exposure to chondrogenic differentiation medium, meticulous staining with Alisin Blue and Senna O facilitated the visualization of vivid blue staining spots within clustered samples (Fig. 1 C), which seamlessly transformed into resplendent red staining spots upon additional application of Senna O (Fig. 1 D). Notably, when subjected to osteogenic differentiation medium, alizarin red staining showcased the prevalence of alkaline phosphatase (ALP) activity, an early signature of osteoblasts, within the majority of cells (Fig. 1 E). Lastly, induction of adipogenesis prompted the development of lipid droplets within the cytoplasm, exquisitely stained with oil red O (Fig. 1 F).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Establishment and Preliminary Evaluation of a Tendinopathy Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to establish an animal model of Achilles tendonitis, various doses of type I collagenase were administered via injection into the unilateral Achilles tendon region of experimental rabbits (Fig. 2 A). After 15 days, noticeable alterations in gait emerged prominently within the 2400 U type I collagenase group, while relatively subtle changes were observed in the 1200 U group. The affected Achilles tendon exhibited erythema, swelling, and a loss of its natural luster within the 2400 U group, whereas a mild dark red hue was evident in the 1200 U group. In contrast, the control group displayed a vibrant white color with a discernible sheen (Fig. 2 B).\u003c/p\u003e\n\u003cp\u003eAfter a duration of forty-five days, the hydroxyproline (Hyp) content was analyzed to evaluate the anabolic status of collagen at the injection site. Interestingly, a significant reduction in Hyp content was observed in the Achilles tendon (AT) group compared to the normal group, indicating a statistically significant discrepancy (Fig. 2 C).\u003c/p\u003e\n\u003cp\u003eHistological examination unveiled notable disparities between the control group and the AT group. Within the control group, Achilles tendon fibrous tissues exhibited parallel and well-organized arrangements, characterized by structural integrity and distinct boundaries between adjacent fiber bundles. Eukaryotic cells were plentiful and uniformly distributed. Conversely, the AT group presented disarrayed collagen fibers, suffering from structural impairments due to substantial infiltration of inflammatory cells, and accompanied by a conspicuous decrease in the number of eukaryotic cells. Additionally, a pronounced accumulation of inflammatory cells surrounding blood vessels was observed (Fig. 2 D, E).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e3.3 The engraftment and distribution of CM-Dil labeled UC-MSCs within the Achilles tendon\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the effectiveness and safety of localized stem cell transplantation, we conducted pre-treatment tracing to observe the survival of UC-MSCs within the Achilles tendinopathy Model and in vivo. The CM-Dil labeled UC-MSCs emitted a vivid red fluorescence (Figure 3A). Utilizing an in vivo imaging system, we investigated the distribution of UC-MSCs within the Achilles tendon tissue before injection, as well as at 1 d, 3 d, 5 d, and 7 d post-injection. Notably, Labeled UC-MSCs treatment group exhibited fluorescent signals within the Achilles tendon tissue, while the control group showed no such fluorescence. The highest concentration of labeled UC-MSCs cells was observed at 1 d after injection, followed by a gradual decline in fluorescence intensity at 3-7 d, indicating a significant reduction in cell numbers (Supplementary Material 1). Particularly, the decrease was most prominent at 3 d. Furthermore, tissue samples were meticulously harvested from the Achilles tendons of rabbits in each group, fixed, embedded, and longitudinally sectioned for observation under a fluorescence microscope. We performed semi-quantitative analysis of the fluorescence intensity. As depicted in Figures 3B and 3C, the normal group and the control group displayed no positive signals. In contrast, positive cell counts in the treatment group followed the order of 1d \u0026gt; 3d \u0026gt; 5d \u0026gt; 7d, mirroring the trend of fluorescence intensity within the Achilles tendon tissues. Additionally, we examined the distribution of UC-MSCs in other organs and tissues but found no evidence of their presence through the in vivo imaging system, indicating that the injected UC-MSCs did not colonize or differentiate within the relevant organs (Supplementary Material 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 UC-MSCs facilitate the regeneration of AT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter delineating the model of Achilles tendonitis and establishing engraftment conditions, we made adjustments to the number of cell infusions and the recovery period (Fig. 4 A). Direct injection of 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e UC-MSCs was administered into the affected female rabbits (UC-MSCs group), followed by four subsequent infusions at different intervals. Molecular and histological evaluations were carried out on the 70th day after the initial cell injection, comparing the UC-MSCs group to the model group (AT group).Palpation of the Achilles tendon in the AT group revealed a lackluster appearance with slight flattening and no elasticity, while the UC-MSCs group exhibited elasticity and a glossy texture (Fig. 4 B). Macroscopic assessment incorporating parameters such as inflammation, surface characteristics, defect size, adjacent tendons, defect severity, swelling/redness, connection to surrounding tissues, and sliding ability yielded significantly higher total scores for the AT group (13.67\u0026plusmn;0.5574) compared to the UC-MSCs group (3.333\u0026plusmn;2.517) (Fig. 4 C).\u003c/p\u003e\n\u003cp\u003eFurthermore, a comprehensive analysis of tissue pathology unveiled disrupted arrangement of collagen fibers and substantial infiltration of inflammatory cells, leading to structural disturbances within collagen fibers in the AT group treated with type I collagenase. Additionally, there was a notable decrease in the number of eukaryotic cells within collagen fibers and a conspicuous accumulation of inflammatory cells around blood vessels. In contrast, the UC-MSCs group exhibited orderly arranged collagen fibers, a reduced presence of inflammatory cells, and a normal number of eukaryotic cells within collagen fibers (Fig. 4 D). Comparison of HE-stained tissue sections further demonstrated that the number of eukaryotic cells within collagen fibers, both in transverse and longitudinal sections, was significantly higher in the UC-MSCs group (transverse: F= 713\u0026plusmn;16.09, P\u0026lt;0.001; longitudinal: F= 253\u0026plusmn;10.02, P\u0026lt;0.001), while the AT group showed a respective decrease of 30% and 43% (Fig. 4 E).\u003c/p\u003e\n\u003cp\u003eIn terms of hydroxyproline content, the UC-MSCs group displayed a significant increase compared to the AT group, and there was a significant decrease in the AT group compared to the control group (p\u0026lt;0.05) (Fig. 4 F). The maximum load on the Achilles tendon is closely related to the state of collagen fibers. Compared to the AT group, the UC-MSCs group exhibited a significantly higher maximum load (p\u0026lt;0.05) (Fig. 4 G).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e3.5 Exploring Molecular Mechanisms of UC-MSCs in Therapeutic Effects on Achilles Tendon Diseases\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the potential molecular mechanisms underlying the therapeutic effects of UC-MSCs on Achilles tendon diseases, we conducted preliminary investigations focusing on structural stability and contraction-regulating markers at the inflammatory site. We examined the alterations of Collagen I and Collagen III in Achilles tendon tissue using immunohistochemical staining. The control group exhibited abundant brown or tan-colored Collagen I and Collagen III, while the AT group showed lower protein expression levels of Collagen I and Collagen III. In contrast, the UC-MSCs group displayed higher expression levels of Collagen I and Collagen III compared to the AT group (Figure 5A). Additionally, we employed Image-Pro Plus software to measure the optical density values of immunohistochemically positive samples. The results are presented as mean \u0026plusmn; standard deviation. The data indicated that UC-MSCs could promote the expression of Collagen I (F=29.70, P=0.008, UC-MSCs group vs. AT group, P=0.001, Figure 5B) and Collagen II (F=8.106, P=0.0197, UC-MSCs group vs. AT group, P=0.001, Figure 5B).\u003c/p\u003e\n\u003cp\u003eFurthermore, we assessed the transcriptional and protein expression levels of matrix metalloproteinase 9 (MMP-9), an enzyme involved in collagen metabolism and degradation. Compared with the AT group, UC-MSCs demonstrated the ability to inhibit MMP-9 at both the transcriptional level (mRNA level: F = 1174.69, P \u0026lt; 0.001, UC-MSCs group vs. AT group) and the protein level (protein level: F = 292.1, P \u0026lt; 0.001, UC-MSCs group vs. AT group, P \u0026lt; 0.001, Figure 5C).\u003c/p\u003e\n\u003cp\u003eGiven that the Achilles tendon is a vital structure connecting muscle and bone, we also evaluated the expression of troponin C (TnC), an important regulatory protein involved in muscle fiber contraction. Compared to the AT group, UC-MSCs significantly upregulated both the transcriptional level (mRNA level: F = 2459, P \u0026lt; 0.0001, UC-MSCs group vs. AT group) and the protein level (protein level: F = 56.99, P \u0026lt; 0.0001, UC-MSCs group vs. AT group, P = 0.0001, Figure 5D) of TnC.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAchilles tendon disease, clinically diagnosed as tendinopathy, is a common degenerative overuse condition. It can be classified into insertional and mid-portion (non-insertional) tendinopathy, each with distinct characteristics and treatment strategies\u0026nbsp;[24]. The repair and healing rates of Achilles tendons are relatively low due to the low cellular, vascular, and metabolic activity of tendon tissue. The process of tendon healing can be divided into three overlapping stages: inflammation, proliferation, and remodeling\u0026nbsp;[25]. Conventional treatments often struggle to address all three stages adequately.\u003c/p\u003e\n\u003cp\u003eMesenchymal stem cells (MSCs), including umbilical cord-derived MSCs (UC-MSCs), have shown promising results in improving inflammation and cell death through various mechanisms, such as targeted differentiation, paracrine effects, and secretion of extracellular vesicles\u0026nbsp;[26]. UC-MSCs have demonstrated satisfactory outcomes in promoting tendon repair and regeneration, not only by facilitating tissue regeneration but also by restoring the original biomechanical function of the tendon\u0026nbsp;[18]\u0026nbsp;. However, there is still no consensus on practical considerations regarding the source, dose, administration technique, and timing of MSC usage in clinical applications.\u003c/p\u003e\n\u003cp\u003eUC-MSCs, isolated from postpartum medical waste like umbilical cords, offer advantages such as non-invasiveness and relatively lower cost compared to other adult stem cells like adipose-derived stem cells\u0026nbsp;[9], dental pulp stem cells\u0026nbsp;[27], and even follicle stem cells. Furthermore, UC-MSCs have a younger origin and possess higher proliferative and self-renewal differentiation potential\u0026nbsp;[14]. Previous studies have also confirmed the successful repair of rotator cuff injuries using UC-MSCs without significant immunosuppression[14]. Similar positive effects on tendon function repair were observed in our rabbit model of Achilles tendon diseases.\u003c/p\u003e\n\u003cp\u003eIn our collagenase-induced tendonitis model, several common pathological characteristics of tendon diseases were evident, including collagen disruption, inflammatory infiltration, neovascularization, adipogenesis, and ectopic ossification, which have been extensively used to study chronic human tendon diseases\u0026nbsp;[28]. Histological evaluation and semi-quantitative analysis revealed collagen fiber disarray and extensive inflammatory cell infiltration in the AT group compared to the control group, indicating structural damage to the collagen fibers (Fig 2D \u0026amp; Fig 4D).. To establish a reliable rabbit model of collagenase-induced tendonitis, we compared the degenerative potential of two different concentrations of type I collagenase and focused on gait analysis, macroscopic changes, and histopathology at different time points (15 days and 45 days) in rabbits. Our results align with other studies conducted in large animals, where the severity of pathology is related to the amount of injected collagenase\u0026nbsp;[29-30]\u0026nbsp;. The injection of 2400 U of type I collagenase induced apparent gait alterations and reddening of the Achilles tendon (Fig 2B), along with morphological changes similar to those seen in human tendon disease histology, especially at day 15. However, we observed a decrease in the number of prokaryotic cells in the AT group compared to the control group at 45 days (Fig 2E). This contrasts with previous reports in which all treatment groups showed increased cell density\u0026nbsp;[30]. We speculate that a single intratendinous injection of collagenase may cause rapid and severe damage to tendon integrity, leading to massive death of collagen cells. However, in the mouse model, an increase in the number of round resident cells was observed, indicating high cell density, which may be attributed to different animal models.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVarious methods have been used for MSC therapy in tendon repair, such as direct injection or systemic infusion, or combining cells with scaffolding materials\u0026nbsp;[31]. Direct injection to the local site is currently the predominant administration route, but it requires clear determination of the total cell count, injection frequency, and distribution within the body\u0026nbsp;[32]. Wang et al. utilized CFDA-SE-labeled UC-MSCs injected into rat tendons and detected fluorescence signals even after 1-5 weeks of transplantation. Furthermore, UC-MSCs demonstrated histological and biological improvement after 4 weeks, but most injected UC-MSCs gradually disappeared from the injured tendon over time, indicating limited residence time in vivo. In our experiment, we observed fluorescence in the AT group of CM-Dil-labeled UC-MSCs in the Achilles tendon tissue, with the highest intensity at 1 day post-injection and subsequent decreasing fluorescence from day 3 to 7 (Fig 3B), indicating a significant reduction in cell numbers (Supplementary Material 1). This could be due to the instability or metabolism of the fluorescent dye. However, studies have shown that more than half of the labeled cells are lost after 23 hours post-injection, possibly due to vascular injury during the injection process, leading to their entry into the bloodstream and migration to other injury sites\u0026nbsp;[33].\u003c/p\u003e\n\u003cp\u003eTo improve therapeutic effects, we employed multiple local injections as the administration technique, with an interval of 3 days for the first three injections. Furthermore, we observed that CM-Dil-labeled UC-MSCs did not settle in other visceral organs within the rabbits (Supplementary Material 1), which could be due to the relatively low number of cells injected locally and oligovascularity, as well as the non-traumatic nature of the induced tendon diseases, indicating the safety of UC-MSC treatment.\u003c/p\u003e\n\u003cp\u003eIn our study, we found that UC-MSCs improved the structural stability at the inflammatory site by upregulating Collagen I and Collagen III expression and inhibiting MMP-9. Mature tendons are mainly composed of Collagen I (\u0026gt;95%), but also include a small amount of Collagen III\u0026nbsp;[34]. Wei et al. reported a significant reduction in Collagen I levels after induction of collagenase-I compared to normal tendons\u0026nbsp;[35]. Similarly, our results showed a decrease in Collagen I levels of approximately 70% in the AT group compared to the control group. However, UC-MSC treatment resulted in a 1.8-fold increase in Collagen I expression (Fig 5A \u0026amp; Fig 5B). Additionally, Lee et al. demonstrated that MSCs transplanted into rat tendons survived for at least 12 weeks, differentiated into tendon lineage cells, and secreted type I collagen to enhance tendon repair\u0026nbsp;[36]. Therefore, UC-MSCs may enhance type I collagen production. The degradation of collagen represents the opposite side, where M1 macrophage-promoted inflammatory reactions lead to the production of MMPs and a decrease in type I collagen, resulting in ECM degradation\u0026nbsp;[11]\u0026nbsp;. MMPs play a crucial role in tissue remodeling, and a study on rat flexor tendon healing found that MMP-9 is involved in the early stage of collagen I degradation\u0026nbsp;[37]. In our study, protein and transcription levels of matrix metalloproteinase-9 (MMP-9) demonstrated an increasing trend in the AT group compared to the control group, showing approximately a 4.5-fold increase in expression. However, UC-MSC treatment significantly reduced IL-1\u0026beta; and inhibited MMP-9 activity (Fig 5C), consistent with the results reported by Xu et al.[38]and Wei et al.[35]. We also measured Tenascin C (TNC), a glycoprotein that interacts with collagen fibers to maintain ECM mechanical properties\u0026nbsp;[39]. In the AT group, TNC levels decreased by 3-4 times compared to the control group. However, after UC-MSC treatment, the expression of TNC increased by 40%-50% (Fig 5D). Furthermore, recent studies have suggested that the healing effects of MSCs arise from their paracrine effects, such as secretion of cytokines and growth factors to recruit, proliferate, and differentiate tissue-specific progenitor cells for synthesizing specific matrices\u0026nbsp;[17]\u0026nbsp;.\u003c/p\u003e\n\u003cp\u003eFurther research using other cellular and molecular assays, such as Western blotting,, focusing on the analysis of TGF-\u0026beta;/SMAD2/3 pathway-related collagen protein synthesis and degradation，is warranted to improve our understanding of the mechanisms involved\u0026nbsp;[15]. Although the collagenase-induced tendonitis model partially mimics natural injuries, it still differs from the natural course of the disease. Moreover, rabbit tendon diseases should be differentiated from human tendon injuries. Additionally, a comprehensive evaluation of animal behavior outcomes, including pain response and gait analysis, should be conducted. While we performed four injections of cells, the assessment of tendon healing at different time points was lacking. The timing of cell injection (acute, sub-acute, or chronic stage) influences the therapeutic effect since the local environment undergoes changes during disease progression or acute injury healing. Multiple injections may also create inconvenience for future clinical treatments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study underscores the satisfactory capacity of UC-MSCs transplantation in promoting tendon functional repair in a rabbit model of tendon disease. UC-MSC therapy improves tendon functionality at macroscopic, histological, and biomechanical levels. This process involves upregulation of Collagen I and Collagen III expression, inhibition of MMP-9 to enhance the structural stability of the inflammatory site, and an increase in muscle calcium-binding protein C (TnC) to strengthen muscle fiber contraction.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Hangzhou Medical and Health Science and Technology Project [Grant No. B20210041].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eXiangyi Sun\u003c/strong\u003e : experimental activity, analyzing the results, and writing the draft of the article. \u003cstrong\u003eZhiwei Lin\u003c/strong\u003e and \u003cstrong\u003eJinyang Chen\u003c/strong\u003e : experimental activity. \u003cstrong\u003eZhe Wang\u003c/strong\u003e and \u003cstrong\u003eGuangqi Zhu\u003c/strong\u003e : experimental activity, conceptualization. \u003cstrong\u003eRuchao Long\u003c/strong\u003e : Supervision. \u003cstrong\u003eZhihua Yang\u003c/strong\u003e : conceptualization, supervision, writing, editing the final version of the article, and funding acquisition.All authors are responsible for the overall integrity of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDeclaration of Competing Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe authors declare that there is no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKujala, U.M., Sarna, S. and Kaprio, J. 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Cell Sci., 116, 857-66, http://doi: 10.1242/jcs.00303.\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":"Umbilical cord-derived mesenchymal stem cells (UC-MSCs), Achilles tendonitis, Collagen type I, Fluorescent tracking, Mechanisms","lastPublishedDoi":"10.21203/rs.3.rs-4293359/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4293359/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Achilles tendon rupture is a common tendinopathy.We investigated the therapeutic effects of UC-MSCs on the inflammatory condition, and explored the preliminary mechanisms underlying their role in rabbit Achilles tendon repair.Tendon structure and functional recovery were evaluated through histological assessment, pathology, tissue hydroxyproline (Hyp) content measurement, and biomechanical testing.Then the inflammation and metabolic status of the extracellular matrix, along with potential mechanisms, were assessed through quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemical staining.This study demonstrated the satisfactory ability of UC-MSCs transplantation to promote functional repair of tendon in a rabbit model of tendon diseases. The mechanisms involved include upregulation of Collagen I and Collagen III expression, inhibition of MMP-9, and enhancement of muscle fiber contraction through increased expression of troponin C (TnC), thereby improving the structural stability at the site of inflammation. We concluded that UC-MSCs hold promising potential as an enhancement strategy for MSC-based therapy in tendon diseases.","manuscriptTitle":"Human Umbilical Cord Mesenchymal Stem Cells promote tendon functional repair in a Collagenase-Induced Tendinopathy Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-25 13:45:09","doi":"10.21203/rs.3.rs-4293359/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":"46fc1505-ebe7-48f6-bd07-00f0271e8a55","owner":[],"postedDate":"April 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-01T18:08:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-25 13:45:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4293359","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4293359","identity":"rs-4293359","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}