Dose- and sex-dependent effects on umbilical cord-derived mesenchymal stem cell efficacy in regeneration of a
full-thickness tendon defect in a rat model

Ji-Hye Yea; Chris Hyunchul Jo

doi:10.5397/cise.2024.00626

Clin Shoulder Elb > Volume 28(1); 2025 > Article

Yea and Jo: Dose- and sex-dependent effects on umbilical cord-derived mesenchymal stem cell efficacy in regeneration of a full-thickness tendon defect in a rat model

Original Article

Clinics in Shoulder and Elbow 2025; 28(1): 49-59.

Published online: February 17, 2025

DOI: https://doi.org/10.5397/cise.2024.00626

Dose- and sex-dependent effects on umbilical cord-derived mesenchymal stem cell efficacy in regeneration of a full-thickness tendon defect in a rat model

Ji-Hye Yea^1,^2,³

, Chris Hyunchul Jo^3,⁴

¹Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA

²Institute for Cell Engineering, Johns Hopkins University, Baltimore, MD, USA

³Department of Translational Medicine, Seoul National University College of Medicine, Seoul, Korea

⁴Department of Orthopedic Surgery, SMG-SNU Boramae Medical Center, Seoul National University College of Medicine, Seoul, Korea

Correspondence: Chris Hyunchul Jo Department of Orthopedic Surgery, SMG-SNU Boramae Medical Center, Seoul National University College of Medicine, 20 Boramae-ro 5-gil, Dongjak-gu, Seoul 07061, Korea.
Tel: +82-2-870-2315, Fax: +82-2-870-3864, E-mail: chrisjo@snu.ac.kr

Received: August 13, 2024 Revised: November 21, 2024 Accepted: December 2, 2024

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Mesenchymal stem cells (MSCs) have shown potential in regenerative medicine. In the present study the effects of MSC dosage and recipient sex on tendon regeneration were evaluated.

Methods

A full-thickness tendon defect (FTTD) was created on supraspinatus tendons (SSTs) of rats and cryoprotective solution (CPS) and MSCs (0.05, 0.1 and 0.5 million MSCs [M-MSC] for female groups and 1.0 M-MSC for both female and male groups) were applied. After 2 and 4 weeks, macroscopic and histological evaluations were performed.

Results

Total macroscopic scores were improved in all MSC groups compared with the CPS group, with no significant differences among the MSC groups. Furthermore, all MSC groups had lower total degenerative scores than the CPS group; however, only 0.1 M-MSC, 0.5 M-MSC, and 1 M-MSC groups showed significantly improved hyalinization compared with the CPS group at 4 weeks. Collagen organization and coherence were higher in all MSC groups than in the CPS group at both 2 and 4 weeks; however, 0.5 M-MSC and 1 M-MSC groups scored better than the 0.05 M-MSC group at 4 weeks. Heterotopic matrix analysis revealed smaller glycosaminoglycan (GAG)-rich areas in the 0.1 M-MSC, 0.5 M-MSC, and 1 M-MSC groups compared with the CPS group at 4 weeks. Overall, macroscopic and histological evaluations were not significantly different between female and male groups except for GAG-rich area.

Conclusions

The MSC dosage affected collagen and heterotopic matrix formation in a FTTD rat model; however, the efficacy of MSCs (1.0 M dose) in collagen regeneration was not affected based on the sex of the recipient.

Level of evidence

Key Words: Shoulder pain; Tendon regeneration; Mesenchymal stem cells; Sex; Dose

INTRODUCTION

Rotator cuff disease is one of the most common causes of shoulder pain. In the United States, over 4.5 million physician visits and 300,000 operations due to shoulder problems occur annually [1]. Conservative treatments such as rest, physical therapy, and non-steroidal anti-inflammatory drugs [2] have been used for the management of patients with rotator cuff disease; however, approximately 45% of all patients experience persistent symptoms 1 year later [3]. This is attributed to the inability of the current conservative treatments to address fundamental etiology of rotator cuff disease, and limited healing potential of tendons due to their acellular and avascular structure [4]. These challenges have increased research on alternative therapeutic approaches including new biological strategies such as stem cell technology for the regeneration of tendon structure [5].

Umbilical cord (UC)-derived mesenchymal stem cells (MSCs) have attracted attention for their potential in tendon regeneration. These cells are easily obtainable through non-invasive methods and considered a cost-effective source because they are derived from medical waste after delivery [6]. UC MSCs exhibit strong proliferation and self-renewal capacities [7], rendering them suitable for regenerative applications. In previous studies, UC MSCs were shown to improve collagen organization, enhance tendon strength, and promote overall tendon healing in a tendon defect rat model [8].

Although MSCs therapies have shown promising results, standardization of several factors is required for effective clinical application. A crucial factor is the MSC dose, which has been selected rather arbitrarily to date [9]. A range of 3 ×10⁵ to 3 ×10⁷ cells has been used although 1 ×10⁶ cells are commonly used in the field of tissue regeneration using animal models [9]. We previously showed administration of 1 ×10⁶ UC MSCs induced beneficial effects of tendon regeneration after full-thickness tendon defect (FTTD) in terms of macroscopic, histological, and biomechanical properties in a rat model. Bone marrow (BM) MSCs and UC blood-derived MSCs also had positive efficacy when using 1 ×10⁶ cells per injured area in the field of tendon regeneration [10,11]. However, the recommended number of MSCs for tendon regeneration, and whether higher doses would confer more benefits, remain unclear. Several researchers reported that patient sex influences the prognosis of rotator cuff disease [12,13]. Hormones such as estrogen could reduce fibroblast proliferation and type I collagen synthesis [13], and female sex is a risk factor for ligament and tendon injuries [14]. Furthermore, females tend to have poor clinical outcomes compared with males in the early stages following rotator cuff repair [12]. Despite known sex differences in tendon healing, the effects of recipient sex on the efficacy of MSCs in tendon regeneration have not yet been reported.

In the present study, the effects of MSC dosage on tendon regeneration and whether the efficacy of MSCs differs based on the sex of the recipient in a FTTD rat model were investigated. We hypothesized the efficacy of MSCs on the regenerationrats per group were sacrificed at of FTTD is dose-dependent and recipient sex-dependent regarding macroscopic and histological properties.

METHODS

Animal experiments were performed following the protocol approved by the Institutional Animal Care and Use Committee (IACUC_2020_0049) of Seoul Metropolitan Government, Seoul Metropolitan Government Seoul National University Boramae Medical Center.

Study Design

A total of 50 adult female Sprague-Dawley rats (12 weeks old, 250–270 g) and 8 adult male Sprague-Dawley rats (12 weeks old, 340–360 g) were assigned to one of the following seven experimental groups: (1) normal group (no procedure performed), (2) cryoprotective solution (CPS) group, (3) 0.05 million MSCs (M-MSC) group, (4) 0.1 M-MSC group, (5) 0.5 M-MSC group, (6) 1.0 M-MSC female group (female-MSC group), and (7) 1.0 M-MSC male group (male-MSC group). Two rats from each group were euthanized immediately after surgery for evaluation of the FTTD appearance. In addition, four rats per group were sacrificed at 2 and 4 weeks postoperatively. The supraspinatus tendon (SST) was harvested for macroscopic and histological analyses [8].

Isolation and Culture of UC MSCs

The Institutional Review Board of Seoul Metropolitan Government Seoul National University Boramae Medical Center approved this study (No. 16-2015-115). Prior to the study, informed consent was obtained from all participants. Human UCs were collected from full-term cesarean deliveries. The UCs were washed two to three times with Dulbecco’s Phosphate-Buffered Saline (Welgene) to eliminate blood residue, after which their length and weight were measured. The UCs were then cut into small cube-shaped explants (2–4 mm each) using surgical scissors.

The explants (1 g) were arranged at consistent intervals on 15-cm culture dishes and allowed to adhere to the dish surface for 60 minutes in a 5% CO₂ incubator at 37 °C with humidified air. A culture medium composed of low-glucose Dulbecco’s modified Eagle medium (LG-DMEM; HyClone) supplemented with 10% fetal bovine serum (HyClone) and antibiotic-antimycotic solution (100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B; Welgene), was carefully added to the dishes. The medium was refreshed twice a week and non-adherent cells were discarded.

Once the cells reached 80% confluency, they were detached by incubation with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (0.05% trypsin, 0.53 mM EDTA; Welgene) for 3 minutes. Tissues were removed using a 100 μm cell strainer (SPL Life Sciences) and the cells were centrifuged at 500 g for 5 minutes at 20 °C before replating at a density of 3 ×10³ cells/cm². The culture medium was changed every 2–3 days and the cells were continuously maintained. The UC MSCs were cultured up to passage 10 before use in experiments and characterized based on their morphology. Growth kinetics, fibroblast colony-forming cells, flow cytometry, and trilineage differentiation assays were performed as previously described [15]. The cells were used in vivo with a CPS (including 10% DMSO; Zenoaq Resource) to represent usage in clinical conditions.

Surgical Procedure

Anesthesia was administered using a combination of Zoletil-Rompun (30 mg/kg and 10 mg/kg, respectively). All procedures were performed on the left shoulder. A 2-cm skin incision was made along the anterolateral border of the acromion. After exposing the SST by detaching the trapezius and deltoid muscles from the acromion, a circular FTTD with a 2 mm-diameter was created 1 mm from the tendon insertion using a Biopsy Punch (BP-20F, Kai Medical Europe). The defect size corresponded to approximately 50% of the tendon width, representing a large but not massive tear based on a previously established method [16]. Equal volumes (10 μL) of CPS and MSCs (0.05 million cells in CPS for the 0.05 M-MSC group, 0.1 million cells in CPS for the 0.1 M-MSC group, 0.5 million cells in CPS for the 0.5 M-MSC group, and 1.0 million cells in CPS for the 1.0 M-MSC group) were intratendinously administered into areas adjacent to both sides of the defect in two divided doses using a 30-G needle. The male group also received an injection of 10 μL containing 1.0 million cells in CPS. Following the injection, the deltoid and trapezius muscles were sutured using a 4-0 Vicryl suture (W9074, Ethicon) and the skin was closed with black silk sutures (SK439, AILee). After the procedure, the animals were allowed unrestricted cage activity [8].

Macroscopic Evaluation of FTTD

At 2 and 4 weeks post-injection, the rats were euthanized in a CO₂ chamber. The SST was collected as well as the humeral head, preserving the muscle intact. For macroscopic evaluation, a modified semi-quantitative scoring system based on the method described by Stoll et al. [17] was used. This system included 12 parameters: tendon rupture, inflammation, tendon surface, neighboring tendon, defect level, defect size, swelling/redness of the tendon, connection to surrounding tissue and slidability, tendon thickness, tendon color, single muscle strain, and transition of the construct to the surrounding healthy tissue. Each parameter was scored on a scale of 0–1, except for swelling/redness (0–2) and tendon thickness (0–3). The total macroscopic score ranged from 0 (indicating a normal tendon) to 15 (indicating the most severe injury) [8,18].

Histological Evaluation of FTTD

Following macroscopic evaluation, the harvested tissues were promptly fixed in 4% (w/v) paraformaldehyde (Merck) for 24 hours followed by decalcification in 10% EDTA (Sigma-Aldrich) for 2 days. After decalcification, the tissues underwent dehydration through a graded ethanol series, defatting in chloroform, and embedding in paraffin blocks. The tissues were then trimmed to the appropriate central region of the tendon and sectioned into 4-μm-thick serial slices.

A randomly chosen slide was stained with hematoxylin and eosin (H&E) and analyzed under a light microscope (U-TVO 63XC; Olympus Corp.). Tendinopathy was assessed on each slide using a previously established semi-quantitative grading scale [19]. The scale included seven parameters: fiber structure, fiber arrangement, rounding of nuclei, variations in cellularity, vascularity, stainability, and hyalinization. Each parameter was scored from 0–3, resulting in a total degeneration score ranging from 0 (normal tendon) to 21 (most severely degenerated).

The integration of structure between the defect and adjacent intact tendon was evaluated at the distal defect-tendon junction using a 0–3 grading system: 0 (no gaps), 1 (recognizable transition), 2 (abrupt transition with splitting/gaps or callus tissue), and 3 (empty defect site) [20]. The presence of heterotopic ossification was assessed when separated, clustered, or bar-shaped foci were observed throughout the tendon structure on H&E-stained slides [21].

Slides were further stained with picrosirius red and analyzed for collagen fiber organization and coherence under circularly polarized light microscopy at ×200 magnification. Collagen organization was quantified as bright white areas of diffracted light on a grayscale (black, 0; white, 255) using ImageJ software with the NII plugin (National Institutes of Health). A higher grayscale value indicated more organized and mature collagen [22]. Collagen fiber coherence, representing the degree of fiber alignment along the major axis, was calculated using the OrientationJ plugin in ImageJ, and the resulting value was multiplied by 100 to determine final coherence [10]. Five regions of interest were analyzed and the mean value was calculated. Detailed procedures are outlined in the in vitro experiments. Cartilage formation was evaluated using slides stained with safranin-O/fast green observed under a light microscope at ×40 magnification. Glycosaminoglycan (GAG)-rich areas were measured using ImageJ software [8,18].

Statistical Analysis

All data are presented as the mean±standard deviation. Statistical analysis was conducted using one-way analysis of variance followed by Bonferroni post-hoc multiple comparison tests. A t-test was used to assess significant differences between the means of the female and male groups. All statistical analyses were performed using IBM SPSS software version 23 (IBM Corp.). A P-value <0.05 was considered statistically significant.

RESULTS

Macroscopic Evaluation of FTTD

At 2 weeks, all MSC groups had lower total macroscopic score and the 0.1 M-MSC group, 0.5 M-MSC group, and 1.0 M-MSC group values were significantly lower (0.1 M-MSC group: 8.75±0.50, 0.5 M-MSC group: 8.25±0.96, and 1.0 M-MSC group: 8.50±1.29) than the CPS group (11.25±0.96; P=0.024, P=0.010, P=0.002, and P=0.004 respectively). In detail, the scores of surface, defect level, swelling/redness, and tendon thickness were at least 0.5 points lower in the MSC groups than in the CPS group. After 5 weeks, scores in the MSC groups decreased further (0.05 M-MSC group: 7.75±1.50, 0.1 M-MSC group: 7.25±1.71, 0.5 M-MSC group: 6.75±0.50, and 1.0 M-MSC group: 6.25±1.71) than in the CPS group (12.00±0.82; P=0.002 for the 0.05 M-MSC group and P<0.001 for the rest of MSC groups). The scores of surface, defect level, swelling/redness, connecting surrounding tissue and slidability, and tendon thickness were at least 0.5 points lower in the MSC groups than in the CPS group (Fig. 1A-C). However, significant difference was not observed among MSC groups and between female and male groups (8.50±1.29 at 2 weeks and 6.25±1.71 at 4 weeks) (Fig. 1B-D).

Histological Evaluation of FTTD

In the total degeneration score, significant difference was not observed between MSC groups and CPS group at 2 weeks. However, after 4 weeks, all MSC groups had a significantly lower total degeneration score (0.05 M-MSC group: 11.25±1.50, 0.1 M-MSC group: 10.00±1.83, 0.5 M-MSC group: 9.25±2.06, and 1.0 M-MSC group: 8.25±2.22) than the CPS group (19.75±1.26; all P<0.001). Significant differences were observed in fiber structure, variations in cellularity, rounding of the nuclei, and decreased stainability; however, significant difference was not found among MSC groups. Hyalinization in the MSC groups was significantly lower than in the CPS group in 0.1 M-MSC, 0.5 M-MSC, and 1.0 M-MSC groups (P=0.007 for the 0.1 M-MSC group and P=0.001 for both 0.05 M-MSC and 1.0 M-MSC groups) (Fig. 2A and B). Conversely, significant difference was not observed in total degeneration score between female and male groups (14.25±2.63 at 2 weeks and 8.00±1.83 at 4 weeks) (Fig. 3A and 2A).

For integration of structure at the distal part, significant difference was not observed among MSC and CPS groups at 2 weeks. However, after 4 weeks, the scores were significantly reduced in all MSC groups (0.05 M-MSC group: 2.00±0.00, 0.1 M-MSC group: 1.75±0.50, 0.5 M-MSC group: 1.50±0.58, and 1.0 M-MSC group: 1.50±0.58, respectively) compared with the CPS group (3.00±0.00; tendon defect sites were still empty; P=0.029 for the 0.05 M-MSC group, P=0.004 for the 0.01 M-MSC group, P=0.001 for the 0.5 M-MSC group, and P=0.001 for the 1.0 M-MSC group) (Fig. 2A and C). However, significant difference was not observed among MSC groups and between female and male groups (2.25±0.50 at 2 weeks and 1.50±0.58 at 4 weeks) (Fig. 2A and 3B).

Heterotopic ossification was not observed in any group at any time point (Fig. 2A). Collagen organization values were significantly higher in all MSC groups than in the CPS group at 2 weeks (CPS group: 9.63±2.61, 0.1 M-MSC group: 31.46±4.85, 0.5 M-MSC group: 33.50±6.56, and 1.0 M-MSC group: 37.04±9.02; P=0.002 for the 0.05 M-MSC group, P=0.001 for the 0.1 M-MSC group, and P<0.001 for the rest of MSC groups, respectively). At 4 weeks, the collagen organization values were significantly higher in all M-MSC groups than in the CPS group (CPS group: 22.39±5.82, 0.05 M-MSC group: 58.50±6.84, 0.1 M-MSC group: 77.98±7.35, 0.5 M-MSC group: 91.13±8.46, and 1.0 M-MSC group: 92.13±13.83; P=0.001 for the 0.05 M-MSC group and P<0.001 for the rest of the MSC groups). The 0.5 M-MSC and 1.0 M-MSC groups had significantly improved collagen organization compared with the 0.05 M-MSC group at 4 weeks (P=0.007 in the 0.5 M-MSC group and P=0.005 for the 1.0 M-MSC group). However, significant difference was not found between female and male groups at 2 and 4 weeks (37.04±9.02 at 2 weeks and 92.13±13.83 at 4 weeks) (Fig. 4A-C).

The collagen fiber coherence scores were significantly higher in all MSC groups than in the CPS group at both 2 and 4 weeks (at 2 weeks, CPS group: 14.63±1.82, 0.05 M-MSC group: 24.29±4.01, 0.1 M-MSC group: 26.24±1.81, 0.5 M-MSC group: 29.37±2.00, and 1.0 M-MSC group: 28.99±3.05; P=0.002 for the 0.05 M-MSC group and P<0.001 for the rest of the MSC groups (at 4 weeks, CPS group: 16.96±6.02, 0.05 M-MSC group: 58.50±6.84, 0.1 M-MSC group: 32.87±3.89, 0.5 M-MSC group: 38.46±8.46, and 1.0 M-MSC group: 37.95±2.50; P=0.001 for the 0.05 M-MSC and P<0.001 for the rest of the MSC groups). However, significant difference was not observed between female and male groups (28.98±3.05 at 2 weeks and 37.95±2.50 at 4 weeks) (Fig. 4A, D, and E).

When heterotopic matrix formation was evaluated, significant difference was not observed in the GAG-rich area between MSC groups and CPS group at 2 weeks. However, after 4 weeks, the GAG-rich area was significantly smaller in the 0.1 M-MSC, 0.5 M-MSC, and 1.0 M-MSC groups (253.00±284.00 mm², 259.00±99.00 mm², and 200.00±67.00 mm², respectively) than in the CPS group (1,048.00±552.00 mm²; P=0.043 for the 0.01 M-MSC group, P=0.006 for the 0.05 M-MSC group, P=0.007 for the 0.5 M-MSC group, and P=0.003 for the 1.0 M-MSC group. Regarding sex, the male group had significantly smaller GAG-rich area than the female group (50.53±58.26 mm²) (Fig. 5).

DISCUSSION

There were several important findings in the present study. Total macroscopic scores were significantly reduced in all MSC groups compared with the CPS group; however, significant difference was not observed among MSC groups. In histological evaluation, all MSC groups had lower total degenerative scores than the CPS group at 4 weeks; however, hyalinization was significantly improved only in the 0.1 M-MSC, 0.5 M-MSC, and 1 M-MSC groups compared with the CPS group. Collagen organization and collagen fiber coherence in all MSC groups was higher than in the CPS group at both 2 and 4 weeks, and among the MSCs groups, 0.5 M-MSC and 1.0 M-MSC groups had significantly improved scores compared with the 0.05 M-MSC group at 4 weeks. In heterotopic matrix formation, 0.1 M-MSC, 0.5 M-MSC, and 1.0 M-MSC groups had a smaller GAG-rich area than the CPS group at 4 weeks. In all macroscopic and histological evaluations, significant differences were not observed between female and male groups except for GAG-rich area. Taken together, these results indicated that MSCs could improve regeneration of FTTD of rotator cuff tendon, and collagen and heterotopic matrix formation in various aspects of tendon healing was affected based on MSC dosage; however, the efficacy of MSCs was sex independent.

For effective clinical application of MSC therapy, several factors should be considered including the method of administration, the type of carrier, and MSC dosage [23,24]. In a previous research study, local injection of MSCs was reportedly safer than systemic injection and showed a positive effect in horses for flexor tendonitis over a 24-month period [25]. Similarly, in the present study, MSCs were administered locally through intratendinous injection in a defective tendon and CPS used as a carrier to mimic clinical conditions for an “off-the-shelf” use. In the present study, 5 ×10⁴ MSCs had less effect on collagen organization and fiber coherence and had no beneficial effect in heterotopic matrix formation compared with the other amounts of MSCs. In addition, more than 1 ×10⁵ MSCs did not show a statistically significant difference among groups, but difference in the mean values was observed. In patients with lateral epicondylosis, 1 ×10⁷ adipose-derived (AD) MSCs induced earlier pain alleviation and faster plateauing of the functional scores than 1 ×10⁶ AD MSCs; however, all MSC doses could improve elbow pain, performance and structural defects after 52 weeks [25]. Based on the human equivalent dose (HED) formula in the Food and Drug Administration guidelines, HED (mg/kg)=animal dose (mg/kg)×(animal weight in kg/human weight in kg), 1 ×10⁷ MSCs in a human is approximately 1 ×10⁵ MSCs in a rat, and 1 ×10⁶ in human is approximately 1 ×10⁴ in a rat [26]. Similarly, the results of the present study indicated a specific MSC dose is needed for optimal efficacy of MSCs. In a previous study in which the efficacy of low and high MSC doses in a rotator cuff defect rabbit model was investigated, MSC efficacy was reportedly dose-independent [27]; however, 1 ×10⁶ was used for the low dose and 2 ×10⁶ for the high dose and the differences between low and high dose was insufficient to conclude the efficacy of MSCs is dose-dependent. Furthermore, the authors did not quantify the histological properties of regenerated tendon structure, rendering determination of precise differences associated with dose difficult. Therefore, we proposed a specific MSC dosage is required for optimized efficacy of MSCs and this dose may vary depending on the location and severity of injury and type of disease; large size defect or severe damage may need higher doses of MSCs than mild injuries.

In previous studies, females were reported more susceptible to tendon damage than males [12,28]. Females have weak tendon hypertrophy responses to habitual exercise, a lower tendon collagen synthesis, and diminished mechanical strength after acute exercise [28]. Thus, the influence of sex should be considered in MSC therapy. However, the efficacy of MSCs was reported sex-independent in some research studies; intra-articular injection of AD MSCs was safe with no toxic effects in both female and male groups of severe combined immunodeficiency mice at day 14 and day 89 and significant differences were not observed between groups in osteoarthritis disease [29]. In a traumatic muscle injury model, BM MSCs induced muscle regeneration in both females and males and sex-related difference was not found in extent of fibrous tissue development [30]. Furthermore, in cardiac injury, recipient sex had no influence on the efficacy of skeletal muscle-derived SCs in the structural regeneration or angiogenesis in a rat model [31]. Consistent with these studies, results of the present study showed no significant differences in the efficacy of MSCs between female and male groups on tendon regeneration except for evaluation of the GAG-rich area. The observed differences in GAG-rich areas are considered to be associated with the extent of injury rather than sex. The female group sustained more severe injuries than the male group when subjected to the same size skin punch which was likely due to the smaller size of SST in females [8]. In addition, females are more prone to tendon disease than males [12]. Consequently, we cautiously suggest that differences in injury severity may have contributed to the differences in GAG-rich areas between female and male groups.

This study had several limitations. First, the sex differences were only compared at a 1.0 M dose of MSCs, thus, drawing definitive conclusions regarding the effects of different MSC doses challenging. Further studies are needed to investigate whether differences exist in tendon regeneration between male and female subjects across various MSC dosages. Second, the regeneration of defective tendon was only evaluated at 2 and 4 weeks after injecting MSCs, which was a relatively short period to fully assess the efficacy of MSCs. Therefore, long-term follow-up studies are necessary to establish the sustained efficacy of MSCs on tendon regeneration.

CONCLUSIONS

The dosage of MSCs affected collagen and heterotopic matrix formation in a FTTD rat model; however, the efficacy of MSCs (1.0 M dose) in collagen regeneration was not affected based on the sex of the recipient.

NOTES

Author contributions

Conceptualization: CHJ. Data curation: JHY. Formal analysis: JHY. Funding acquisition: CHJ. Investigation: CHJ. Methodology: JHY. Supervision: CHJ. Visualization: JHY. Writing – original draft: JHY. Writing – review & editing: CHJ. All authors read and agreed to the published version of the manuscript.

Conflict of interest

Chris Hyunchul Jo owns shares of AcesoStem Biostrategies Inc. No other potential conflicts of interest relevant to this article were reported.

Chris Hyunchul Jo is an editorial board member of the journal but was not involved in the peer reviewer selection, evaluation, or decision process of this article. No other potential conflicts of interest relevant to this article were reported.

Funding

This research was supported by a grant (NRF-2015M3A9E6028412) from the Bio & Medical Technology Development Program, a grant (NRF-2017R1A2B2010995) from the Basic Science Research Program of the National Research Foundation of Korea, and a grant (HI20C0386) from the Korea Health Industry Development Institute (KHIDI).

Data availability

Contact the corresponding author for data availability.

Acknowledgments

We thank InJa Kim and Gayoung Sym for supporting this study.

Fig. 1.

Surgery procedure and macroscopic evaluation of regenerated tendons at 2 and 4 weeks after injection with cryoprotective solution (CPS) and mesenchymal stem cells (MSCs). (A) The surgical procedure of full-thickness tendon defect (FTTD) and intratendinous injection of CPS and MSCs. (B) Macroscopic appearance of the supraspinatus tendons (SSTs). (C) The total macroscopic score of regenerated tendons based on dosage. (D) The total macroscopic score of regenerated tendons based on sex. Bar charts represent mean±standard deviation. IST: infraspinatus tendon. *Statistical significance, P<0.05.

Fig. 2.

Histological evaluation of regenerated tendon at 2 and 4 weeks after injection with cryoprotective solution (CPS) and mesenchymal stem cells (MSCs). (A) H&E staining of the regenerated tendon (Magnification; Normal, FTTD: ×40 and CPS, 0.05 M-MSC, 0.1 M-MSC, 0.5 M-MSC, 1.0 M-MSC, Male-MSC: ×100). (B) Total degeneration score and detailed parameters (fiber structure, fiber arrangement, rounding of nuclei, variations in cellularity, vascularity, stainability, and hyalinization) of regenerated tendons based on dosage. (C) Integration of structure at the distal site based on dosage. Bar charts represent mean±standard deviation. FTTD: full-thickness tendon defect. *Statistical significance, P<0.05.

Fig. 3.

Histological evaluation of regenerated tendon at 2 and 4 weeks after mesenchymal stem cells (MSCs) injection in female and male groups. (A) The total degeneration score and detailed parameters (fiber structure, fiber arrangement, rounding of nuclei, variations in cellularity, vascularity, stainability, and hyalinization) of regenerated tendons based on sex (B) Integration of structure at distal site of regenerated tendons based on sex. Bar charts represent mean±standard deviation. FTTD: full-thickness tendon defect. *Statistical significance, P<0.05.

Fig. 4.

Quantification of collagen matrix changes in the regenerated tendon at 2 and 4 weeks after injection with cryoprotective solution (CPS) and mesenchymal stem cells (MSCs). (A) Picrosirius red staining of regenerated tendons (Magnification; Normal, FTTD: ×40 and CPS, 0.05 M-MSC, 0.1 M-MSC, 0.5 M-MSC, 1.0 M-MSC, Male-MSC: ×100). (B) Collagen organization of regenerated tendons based on dosage. (C) Collagen fiber coherence of regenerated tendons based on dosage. (D) Collagen organization of regenerated tendons based on sex. (E) Collagen fiber coherence of regenerated tendons based on sex. Bar charts represent mean±standard deviation; statistical significance, *P<0.050. FTTD: full-thickness tendon defect.

Fig. 5.

Quantification of glycosaminoglycan (GAG)-rich area in the regenerated tendons at 2 and 4 weeks after injection with cryoprotective solution (CPS) and mesenchymal stem cells (MSCs). (A) Saf-O staining of regenerated tendons (Magnification; Normal, FTTD: ×40 and CPS, 0.05 M-MSC, 0.1 M-MSC, 0.5 M-MSC, 1.0 M-MSC, Male-MSC: ×100). (B) GAG-rich area of regenerated tendons based on dosage. (C) GAG-rich area of regenerated tendons based on sex. Bar charts represent mean±standard deviation. FTTD: full-thickness tendon defect. *Statistical significance, P<0.05.

REFERENCES

1. Abat F, Alfredson H, Cucchiarini M, et al. Current trends in tendinopathy: consensus of the ESSKA basic science committee. Part I: biology, biomechanics, anatomy and an exercise-based approach. J Exp Orthop 2017;4:18.

2. Karasuyama M, Gotoh M, Tahara K, et al. Clinical results of conservative management in patients with full-thickness rotator cuff tear: a meta-analysis. Clin Shoulder Elb 2020;23:86–93.

3. van der Windt DA, Koes BW, Boeke AJ, Devillé W, De Jong BA, Bouter LM. Shoulder disorders in general practice: prognostic indicators of outcome. Br J Gen Pract 1996;46:519–23.

4. Schneider M, Angele P, Järvinen TA, Docheva D. Rescue plan for Achilles: therapeutics steering the fate and functions of stem cells in tendon wound healing. Adv Drug Deliv Rev 2018;129:352–75.

5. Liu XN, Yang CJ, Kim JE, et al. Enhanced tendon-to-bone healing of chronic rotator cuff tears by bone marrow aspirate concentrate in a rabbit model. Clin Orthop Surg 2018;10:99–110.

6. Troyer DL, Weiss ML. Wharton’s jelly-derived cells are a primitive stromal cell population. Stem Cells 2008;26:591–9.

7. Beeravolu N, Khan I, McKee C, et al. Isolation and comparative analysis of potential stem/progenitor cells from different regions of human umbilical cord. Stem Cell Res 2016;16:696–711.

8. Yea JH, Park JK, Kim IJ, Sym G, Bae TS, Jo CH. Regeneration of a full-thickness defect of rotator cuff tendon with freshly thawed umbilical cord-derived mesenchymal stem cells in a rat model. Stem Cell Res Ther 2020;11:387.

9. Tang XL, Rokosh G, Sanganalmath SK, et al. Effects of intracoronary infusion of escalating doses of cardiac stem cells in rats with acute myocardial infarction. Circ Heart Fail 2015;8:757–65.

10. Degen RM, Carbone A, Carballo C, et al. The effect of purified human bone marrow-derived mesenchymal stem cells on rotator cuff tendon healing in an athymic rat. Arthroscopy 2016;32:2435–43.

11. Kwon DR, Park GY, Lee SC. Treatment of full-thickness rotator cuff tendon tear using umbilical cord blood-derived mesenchymal stem cells and polydeoxyribonucleotides in a rabbit model. Stem Cells Int 2018;2018:7146384.

12. Cho CH, Ye HU, Jung JW, Lee YK. Gender affects early postoperative outcomes of rotator cuff repair. Clin Orthop Surg 2015;7:234–40.

13. Yu WD, Panossian V, Hatch JD, Liu SH, Finerman GA. Combined effects of estrogen and progesterone on the anterior cruciate ligament. Clin Orthop Relat Res 2001;(383):268–81.

14. Jones BH, Bovee MW, Harris JM 3rd, Cowan DN. Intrinsic risk factors for exercise-related injuries among male and female army trainees. Am J Sports Med 1993;21:705–10.

15. Jo CH, Kim OS, Park EY, et al. Fetal mesenchymal stem cells derived from human umbilical cord sustain primitive characteristics during extensive expansion. Cell Tissue Res 2008;334:423–33.

16. Carpenter JE, Thomopoulos S, Flanagan CL, DeBano CM, Soslowsky LJ. Rotator cuff defect healing: a biomechanical and histologic analysis in an animal model. J Shoulder Elbow Surg 1998;7:599–605.

17. Stoll C, John T, Conrad C, et al. Healing parameters in a rabbit partial tendon defect following tenocyte/biomaterial implantation. Biomaterials 2011;32:4806–15.

18. Yea JH, Bae TS, Kim BJ, Cho YW, Jo CH. 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 2020;114:104–16.

19. Jo CH, Shin WH, Park JW, Shin JS, Kim JE. Degree of tendon degeneration and stage of rotator cuff disease. Knee Surg Sports Traumatol Arthrosc 2017;25:2100–8.

20. Meier Bürgisser G, Calcagni M, Bachmann E, et al. Rabbit Achilles tendon full transection model: wound healing, adhesion formation and biomechanics at 3, 6 and 12 weeks post-surgery. Biol Open 2016;5:1324–33.

21. Kraus TM, Imhoff FB, Reinert J, et al. Stem cells and bFGF in tendon healing: effects of lentiviral gene transfer and long-term follow-up in a rat Achilles tendon defect model. BMC Musculoskelet Disord 2016;17:148.

22. Zhao S, Zhao J, Dong S, et al. Biological augmentation of rotator cuff repair using bFGF-loaded electrospun poly(lactide-co-glycolide) fibrous membranes. Int J Nanomedicine 2014;9:2373–85.

23. Yong KW, Wan Safwani WK, Xu F, Wan Abas WA, Choi JR, Pingguan-Murphy B. Cryopreservation of human mesenchymal stem cells for clinical applications: current methods and challenges. Biopreserv Biobank 2015;13:231–9.

24. Moll G, Geißler S, Catar R, et al. Cryopreserved or fresh mesenchymal stromal cells: only a matter of taste or key to unleash the full clinical potential of MSC therapy. Adv Exp Med Biol 2016;951:77–98.

25. Lee SY, Kim W, Lim C, Chung SG. Treatment of lateral epicondylosis by using allogeneic adipose-derived mesenchymal stem cells: a pilot study. Stem Cells 2015;33:2995–3005.

26. Shin JW, Seol IC, Son CG. Interpretation of animal dose and human equivalent dose for drug development. J Korean Orient Med 2010;31:1–7.

27. Kwon DR, Park GY, Lee SC. Regenerative effects of mesenchymal stem cells by dosage in a chronic rotator cuff tendon tear in a rabbit model. Regen Med 2019;14:1001–12.

28. Magnusson SP, Hansen M, Langberg H, et al. The adaptability of tendon to loading differs in men and women. Int J Exp Pathol 2007;88:237–40.

29. Toupet K, Maumus M, Peyrafitte JA, et al. Long-term detection of human adipose-derived mesenchymal stem cells after intraarticular injection in SCID mice. Arthritis Rheum 2013;65:1786–94.

30. von Roth P, Duda GN, Radojewski P, Preininger B, Perka C, Winkler T. Mesenchymal stem cell therapy following muscle trauma leads to improved muscular regeneration in both male and female rats. Gend Med 2012;9:129–36.

31. Drowley L, Okada M, Payne TR, et al. Sex of muscle stem cells does not influence potency for cardiac cell therapy. Cell Transplant 2009;18:1137–46.