Attenuating Cartilage Degeneration in a Low Mechanical Compression Rat Model Through Intra-Articular Injections of Allogeneic Bone Marrow-Derived Mesenchymal Stem Cells

Abstract

Objective

Mechanical stimulation significantly contributes to posttraumatic osteoarthritis (PTOA), a condition that impedes patient recovery following intra-articular injury. Effective treatment options for compression-induced injuries are limited. Bone marrow-derived mesenchymal stem cell (BMSC) implantation has emerged as a potential therapeutic breakthrough for joint diseases. The aim of this study was to attenuate the progression of PTOA induced by cyclic loading and demonstrate the potential effectiveness of BMSCs in a rat model of low mechanical compression.

Design

Using a rat model of compression-induced articular cartilage injury, assessments were conducted 2, 4, and 8 weeks after cyclic compressive loading. The expression of matrix metallopeptidase 13, transforming growth factor-beta 3 (TGF-β3), insulin-like growth factor 1 (IGF-1), and cleaved caspase-3 was evaluated through immunohistochemistry to investigate the mechanistic aspects underlying the prevention of compression-induced injury following BMSCs treatment.

Results

Intra-articular injections of BMSCs significantly improved scores in the OARSI (Osteoarthritis Research Society International) Osteoarthritis Cartilage Histopathology Assessment System and Histological-Histochemical Grading System. This treatment showed positive outcomes in maintaining high relative cell density and reducing proteoglycan loss after cyclic compression-induced injury. The expression patterns of IGF-1 and TGF-β3 provide valuable insights into the presence and distribution of these growth factors in healthy and injured cartilage.

Conclusions

These findings highlight the efficacy of BMSCs treatment in attenuating the advancement of compression-induced injuries, albeit within a limited timeframe.

Keywords

osteoarthritis mesenchymal stem cell stem cell therapy cell injection cartilage

Introduction

Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation, synovitis, subchondral bone sclerosis, and osteophyte formation.¹ Patients with OA typically experience progressive joint pain, stiffness, swelling, limited joint range of motion, and joint deformities. Despite the increasing prevalence of OA in aging populations, the absence of viable disease-modifying medications can be attributed to the complex and chronic nature of the condition. Posttraumatic OA (PTOA) refers to a specific type of OA linked to joint damage and instability caused by physical trauma. The interplay between mechanical loading, exercise, and joint health plays a distinct role in the onset and progression of OA. Excessive mechanical loading is a major contributing factor to the risk of OA.²

Bone marrow-derived mesenchymal stem cells (BMSCs) originate from the stromal compartment of the bone marrow and serve as precursors of connective tissue cells. BMSCs are multipotent progenitor cells that can self-renew and differentiate into several lineages, including adipocytes, osteoblasts, and chondrocytes.³ BMSCs obtained from the bone marrow have been extensively utilized in animal models and certain clinical cases to explore their potential for promoting cartilage formation in the treatment of OA. In addition to their structural contributions to tissue repair, BMSCs exhibit robust immunomodulatory properties and notable anti-inflammatory effects.^4,5 BMSCs release a wide range of bioactive molecules with immunoregulatory and regenerative properties.^6-8 These properties make BMSCs promising candidates for cell therapy for the treatment of OA-related conditions. However, most studies have focused on the application of BMSCs treatment in the advanced stages of OA.⁹

In our previous study, we observed that low mechanical compression resulted in a decline in cell density within the femoral lesion area, exhibiting a sustained decrease over an extended duration.¹⁰ Our study focused on the initial stages of OA, addressing the period preceding irreversible structural damage. Preventive measures against the progression of compression-induced injury in rat models of low mechanical compression remain incompletely understood. Therefore, we hypothesized that intra-articular injection of BMSCs would be effective in hindering the onset of degenerative joint disease or impeding its progression in rats undergoing treatment for compression-induced injury. The primary aim of this study was to evaluate the efficacy of BMSCs in attenuating PTOA progression in a rat model of low mechanical compression.

Methods

Animals

In this study, a group of 69 healthy male Wistar rats, aged 12 weeks (272.0 ± 10 g), was obtained from SHIMIZU Laboratory Supplies Co. Ltd., Kyoto, Japan. Nine rats underwent intact procedures, while 54 rats were used to characterize PTOA progression ( Fig. 1 ). The rats were housed in plastic cages that provided sufficient space for normal cage activity, subjected to a 12-hour day/night cycle, and received ample amounts of standard solid pellet feed and water. Rats were anesthetized using isoflurane gas and subsequently administered a combination of three anesthetic agents (0.375 mg/kg medetomidine, 2.0 mg/kg midazolam, and 2.5 mg/kg butorphanol). After euthanasia of Wistar rats through isoflurane inhalation until unconscious, sternotomy was performed to excise the hearts for sacrificial purposes. Subsequently, the right knees were extracted for detailed analysis.

Figure 1.

Overview of the general experimental procedure. (A) Cyclic compressive loading model and BMSCs therapy evaluation flow diagram. (B) A rat underwent knee injury induction through a single overload cycle of femur compression. (C-D) Each rat within the experimental group (intact samples, post-compression [PC] group, PBS-injected [PC + PBS] group, and BMSCs-injected [PC + BMSCs] group) underwent a singular compression session with a total of 60 cycles of cyclic loading and each cycle consisted of an initial preload of 5 N, a peak load of 20 N, speed of 1 mm/s and 10-second rest intervals. The intact group (n = 6) was subjected to euthanasia at the age of 12 weeks without undergoing the compression procedure. As for the experimental group, rats were euthanized at 2-, 4-, and 8-week intervals in each interval group (each group = 6). In addition, the PKH26-labeled BMSCs group was euthanized at the same intervals in each group (each group = 3). BMSCs, bone marrow-derived mesenchymal stem cells; PBS, phosphate-buffered saline.

Femur Compression-Induced Knee Injury

Following the anesthesia protocol, each rat underwent knee injury induction through a single overload cycle of femur compression in accordance with a standardized protocol^11,12 with a flexion angle of approximately 140° ( Fig. 1B ). This marked the compressive injury level in the rats, using a system similar to previous studies.¹⁰ In this study, the specimens subjected to cyclic loading were classified as post-compression (PC) group. Specimens that underwent cyclic loading along with phosphate-buffered saline (PBS) injection were designated as the PC + PBS group, whereas those subjected to cyclic loading and BMSCs injection were assigned to the PC + BMSC group. Each rat in the experimental group underwent a single session of compression involving 60 cycles of loading throughout the study. Each cycle included a 5 N preload, followed by a peak load of 20 N with an approaching speed of 1 mm/s, accompanied by 10-second rest intervals between cycles ( Fig. 1D ). The intact group (N = 6) was sacrificed at the age of 12 weeks without undergoing compression. The rats were euthanized at 2, 4, and 8 weeks (N = 6 in each group) following the implementation of the compression-induced injury model.

Preparation of Bone Marrow-Derived Cells

The bone marrow was extracted from the Wistar rat femur and rinsed with α-minimum essential medium (Gibco, USA) to isolate BMSCs according to a previous study.¹³ Proper disinfection of the skin above the femoral bone of the MSC donor rat (12-week old, male) was performed. Subsequently, an incision was made to isolate the femur and the bone marrow was aspirated from three donor rats using an 18-gauge needle. The collected bone marrow was then combined and suspended in a medium consisting of α-minimum essential medium + GlutaMAX (Gibco, USA), supplemented with 10% fetal bovine serum (Hyclone, USA), 50 U/mL penicillin (Nacalai Tesque Inc., Japan), and 50 μg/mL streptomycin (Nacalai Tesque). The bone marrow cells were seeded onto a 100-mm culture dish and incubated in a humidified atmosphere of 5% CO₂ and 95% air at 37°C for 24 hours. Following the initial 24-hour incubation period, the medium was replaced every 3 days. Cell viability was promptly determined using the trypan blue dye exclusion test. Viability: live cell count/(dead cell count + live cell count) = 123/127 × 100% = 96.85%. In addition, we confirmed that the harvested cells possessed multipotency and the ability to differentiate into osteoblasts, adipocytes, and chondrocytes (Supplementary Fig. 1).

Bone Marrow-Derived Cells Labeling With PHK26 and Tracking In Vitro and In Vivo

Passage four BMSCs were harvested and subjected to red fluorescent dye labeling using PKH26 (Sigma, USA). The experimental procedures strictly adhered to the protocol provided with the dye kit and were performed at room temperature. The labeled BMSCs were visualized using a fluorescence microscope, and cells exhibiting PKH26 staining that correlated with DAPI (4’,6-diamidino-2-phenylindole) staining were enumerated and quantified (Supplementary Fig. 2). The PKH26-labeled rate of BMSCs was 98.2% in vitro. To evaluate the survival and homing ability of PKH26-labeled BMSCs (N = 3), each specimen was collected at 2, 4, and 8 weeks after injection. The images were exported as TIFF files and analyzed using Fiji2 software (National Institutes of Health, USA). Each PKH26-positive particle, characterized by its fluorescence intensity and several adjacent pixels, was extracted from the image at a 20% intensity threshold level.

Intra-Articular Injection of BMSCs

After a 3-week period in culture (passages 4-5), the cells were treated with trypsin, rinsed with PBS, and suspended when the cell confluence reached approximately 80%. In the PC + BMSCs group, the rat’s knee joints with compression injuries received a single intra-articular injection of 1 × 10⁶ cells using a 26-gauge needle (each injection equaled 50 μL), while the same amount of PBS was injected into another sample as the PC + PBS group. The needle was inserted behind the inner edge of the patellar ligament and passed through the triangular space formed by the femoral epicondyle. Subsequently, the joint was flexed and extended repeatedly to disperse the cell suspension throughout the intra-articular region. All rats were closely monitored for 2, 4, and 8 weeks, and then sacrificed.

Histological Analysis

At 2, 4, and 8 weeks following treatment, the rats were euthanized, and articular cartilage samples were collected. The collected tissues were fixed with 4% paraformaldehyde for 24 hours and subsequently decalcified for 25 days in a 10% EDTA (ethylenediaminetetraacetic acid) solution at a pH of 7.4. These tissues were then embedded in paraffin and sectioned into 6-μm-thick slices. The serial sections were derived from the lateral compartments with intervals of 100 μm. Sections were then deparaffinized in xylene and rehydrated using a graded series of ethanol washes. Sections were stained with hematoxylin and eosin (H&E) and Safranin O/Fast Green. To assess the degree of cartilage degeneration, the evaluation focused on assessing the most severe injury of the lateral femoral condyle joint using both the Histologic/Histochemical Grading System (HHGS)¹⁴ and the OARSI (Osteoarthritis Research Society International) OA Cartilage Histopathology Assessment System (OOCHAS),¹⁵ and the grade of OA progression was evaluated by double-blind observation (CT and ZZ). Cell counting was performed using the Fiji2 software and images of H&E-stained sections. The relative cell density, representing cells per unit volume, was assessed by quantifying the number of cells within this defined volume using Fiji2 software by employing freehand and area-measurement tools. The determination of the relative cellular density in each region considered both the number of nuclei and the corresponding relative area of the cartilage.

Immunohistochemical Staining Analysis

Immunohistochemistry was used to detect the expression of matrix metallopeptidase 13 (MMP-13), transforming growth factor-beta 3 (TGF-β3), insulin-like growth factor 1 (IGF-1), and cleaved caspase-3 (CCasp3). Initially, the samples were deparaffinized in xylene to remove the paraffin that had permeated the tissue. After deparaffinization, the slides were hydrated using a series of graded alcohol solutions, and the xylene residue was eliminated using 100% ethanol. Subsequently, the sections were subjected to a 30-minute heat treatment at 65°C using a 1:10 diluted HistoVT One solution (Nacalai Tesque, Inc., Japan) and subsequently washed three times with PBS to enable antigen retrieval. Endogenous peroxidases in the tissues were blocked with 0.3% H₂O₂ for 30 minutes. A blocking solution containing 5% normal goat serum was added and incubated for 30 minutes at room temperature (approximately 25°C). The sections were then incubated overnight at 4°C with a specific primary antibody targeting MMP-13 (Abcam, ab39012; diluted at 1:1,000), TGF-β3 (Thermo Fisher Scientific, PA597064; diluted at 1:200), IGF-1 (Thermo Fisher Scientific, 500-3724; diluted at 1:500), and CCasp3 (Asp175) (Cell Signaling Technology, 9661S; diluted at 1:500). After incubation with the primary antibody, a mouse- and rabbit-specific horseradish peroxidase-diaminobenzidine detection immunohistochemical kit (Abcam, USA) was used.

After rinsing, sections were incubated with goat anti-rabbit immunoglobulin G (IgG) for 30 minutes at room temperature. The visualization of antigen-antibody complexes was accomplished using ABC reagent (ABC kit PK-6100, Vector Laboratories, USA) supplemented with 3,3’-diaminobenzidine (DAB kit SK-4105, Vector Laboratories, USA). Finally, the sections were counterstained with hematoxylin. The obtained data were quantified using Fiji2 software. Sections displaying a brown or brownish-yellow appearance were considered positive staining. The assessment of MMP-13, TGF-β3, IGF-1, and CCasp3 results involved the evaluation of immunostaining in positive cells, normalized by the total cell count (positive-stained cell rate).

Statistical Analysis

The statistical analysis was conducted utilizing GraphPad Prism 9 software (GraphPad Inc., USA) for data processing and plotting. All data obtained in this study were presented as the mean ± standard deviation. One-way analysis of variance followed by Tukey’s test was used to compare data among different groups. Statistical significance was determined using a P value of less than 0.05.

Results

PKH26-Labeled Rat Bone Marrow Stem Cells Can Be Tracked After Being Injected into Joints for Up to 8 Weeks

In the intact ( Fig. 2A ) or PC + PBS groups, no cells expressing PKH26 were detected. The presence of implanted BMSCs became increasingly apparent 2 and 4 weeks after injection. Conversely, in the PC + BMSC group, transplanted BMSCs were identified on the surface of the articular cartilage, and scattered cells were observed for up to 8 weeks. Upon scrutiny of the image on the right side depicting BMSCs treatment, PKH26-labeled BMSCs were distinctly discernible ( Fig. 2B ).

Figure 2.

The assessment of PKH26-labeled BMSCs survival and homing proficiency. (A) Immediate extraction of the intact articular cartilage from the knee. (B) Each specimen (N = 3) was obtained at 2-, 4-, and 8-week post-injection (scale: 500 μm). The deposition of PKH26 red fluorescence-positive donor cells was exclusively observed within the PC + BMSCs groups, and not within the PC and PC + PBS group. Upon closer examination of PC + BMSCs at 2 weeks on the right, it is evident that the irregular contour of the cartilage surface hinders the microscope’s ability to attain comprehensive focal precision regarding the positive cells (stained with red dye) across the entirety of the cartilage specimen. Consequently, this phenomenon results in zones marked by indistinctness (highlighted by the yellow arrow), interspersed with segments displaying enhanced visual clarity (as indicated by the white arrow). BMSCs, bone marrow-derived mesenchymal stem cells; PBS, phosphate-buffered saline; PC, post-compression.

Matched Pair Analysis: The Effect of Cyclic Loading on the Progression of Compression Injury After BMSCs Injection Treatment

Injury progression was observed after the cyclic loading during the early stages of the investigation. Microscopic observations of gross morphological changes in the femoral condyles of the PC + BMSC group revealed subtle cartilage lesions at 2 weeks, slight progression at 4 weeks, and conspicuous cellular demise at the 8-week mark ( Fig. 3A ). In contrast, the cartilage of the femoral condyle in the PC and PC + PBS groups appeared to be more severely affected throughout the study. Significant discernible distinctions in the cartilage area of the complete lesion on the lateral femur were observed between groups that received BMSCs treatment and those that did not. Compared with observations in the intact group, the cell density with BMSC injections did not show a trend of change at 2 weeks, but there was a noticeably lower cell density at 4 weeks after compression. The PC + BMSCs group, PC + PBS group, and PC groups exhibited a distinct pattern of reduced cell density at the 8-week mark ( Fig. 3B ). Moreover, the cell density of the lateral femur decreased and tended to decline dramatically after compression in the PC and PC + PBS groups ( Fig. 3B ).

Fig. 3.

Histological assessments were conducted at 2-, 4-, and 8-week post-cyclic compression test using safranin O and fast green staining. (A) Representative sections depicting interventions, including intact samples, the PC group, PC + PBS group, and PC + BMSCs groups (200× magnification). In the PC group (C and G), PC + PBS group (D and H), and PC + BMSC group (E and I), OA scores showed a significant increase over the 8-week period. (B) Notably, the PC + BMSC group demonstrated a preventive effect on cartilage damage, incurring less injury compared with that in both the PC and PC + PBS groups at 2- to 8-week intervals (PC: P < 0.0001 [C], PC + PBS: P < 0.0001 [D], PC + BMSC: P < 0.0001 [E] in OOCHAS; PC: P < 0.0001 [G], PC + PBS: P < 0.0001 [H], PC + BMSC: P < 0.0001 [I]). Statistically significant differences were observed between the 4- and 8-week time points (PC: P = 0.0018 [C], PC + PBS: P = 0.0036 [D], PC + BMSCs: P < 0.0001 [E] in OOCHAS; PC: P = 0.0101 [G], PC + PBS: P = 0.0009 [H], PC + BMSCs: P = 0.0042 [I] in HHGS), while the 2- and 4-week time points were not statistically significant in the PC + BMSCs group (PC: P = 0.0001 [C], PC + PBS: P = 0.0005 [D], PC + BMSCs: ns [E] in OOCHAS; PC: P < 0.0001 [G], PC + PBS: P < 0.0001 [H], PC + BMSCs: P < 0.0001 [I] in HHGS). Overall, the OOCHAS and HHGS scores indicated mild-to-moderate osteoarthritic changes. Quantification of the cartilage injury progression revealed elevated scores in the PC and PC + PBS groups, with mitigated progression in the PC + BMSCs group. Notably, the treatment effect was most significant at 4 weeks, with the smallest effect observed at 8 weeks. The relative cell density of the lateral femur decreased following compression in the PC group ( Fig. 2B ), whereas the BMSCs treatment group exhibited reduced cell density at 4 weeks, with no discernible trend at 2 weeks. At the 8-week mark, all groups displayed a distinct pattern of lower relative cell density. BMSCs, bone marrow-derived mesenchymal stem cells; PBS, phosphate-buffered saline; PC, post-compression; OOCHAS, Osteoarthritis Cartilage Histopathology Assessment System; HHGS, Histologic/Histochemical Grading System; OA, osteoarthritis.

Histologically, the PC and PC + PBS injection groups exhibited severe cartilage defects and reduced safranin O staining intensity, whereas the PC + BMSC group showed fewer cartilage defects and decreased proteoglycan loss after cyclic compression ( Fig. 3A ). Comparatively, the PC + BMSC group demonstrated lower scores in both OOCHAS (Fig. 3C –F) and HHGS (Fig. 3G –J) when compared with those of the PC and PC + PBS groups. Although both groups experienced greater cartilage loss after 8 weeks of the compression test, the BMSCs treatment group still displayed significantly lower OOCHAS ( Fig. 3F ) and HHGS scores ( Fig. 3J ).

Reduction of Apoptosis in Cartilage Through BMSCs Injection

Based on our observations, at 2 weeks, both the PC and PC + PBS groups demonstrated a significant upregulation in CCasp3 expression within the cartilage tissue compared with that of the PC + BMSC group ( Fig. 4A ), and a decline in chondrocyte presence PC resulted in an insignificantly low CCasp3 expression at the 8-week interval ( Fig. 4B ). CCasp3 expression levels continued to rise in the PC and PC + PBS groups yet remained significantly lower in the PC + BMSC group ( Fig. 4B ). Chondrocytes were notably diminished in both the PC and PC + PBS groups compared with those in the PC + BMSC group at 8 weeks. However, there was no significant difference in the expression levels because a considerable number of cells had already undergone apoptosis, rendering the assessment of CCasp3 expression inconclusive ( Fig. 4A ). These findings highlight the ability of BMSCs injections to mitigate apoptosis.

Figure 4.

Immunohistochemical analysis for CCasp3 in cartilage: (A) Specimens representing distinct conditions (PC, PC + PBS, and PC + BMSCs) were collected at 2-, 4-, and 8-week post-compression-induced testing, and the analysis centered on assessing CCasp3 expression within the lateral femoral condyle (scale: 100 μm). (B) The proportion of CCasp3-positive cells (n = 6) was assessed. The impact of BMSCs injection treatment was systematically examined at 2 weeks (PC:PC + BMSCs → P < 0.0001; PC + PBS:PC + BMSCs → P = 0.0001), 4 weeks (PC:PC + BMSCs → P < 0.0001; PC + PBS:PC + BMSCs → P < 0.0001), and 8 weeks (PC:PC + BMSCs → ns; PC + PBS:PC + BMSCs → P = 0.0116). BMSCs, bone marrow-derived mesenchymal stem cells; PBS, phosphate-buffered saline; PC, post-compression; CCasp3, cleaved caspase-3.

Suppressive Effect of BMSCs Injection Treatment on MMP-13 Expression

When investigating the effects of BMSCs injection in our Wistar rat compression injury model, upregulation of MMP-13 expression was evident following compression, and subsequent injection of BMSCs resulted in a substantial reduction observed at both 2- and 4-week intervals ( Fig. 5A ). The expression of MMP-13 in the compression injury area of the PC + BMSCs group was significantly lower than that in both the PC and PC + PBS groups at the 2- and 4-week intervals ( Fig. 5B ). At the 8-week mark ( Fig. 5B ), cell death following compression resulted in insignificant MMP-13 expression.

Figure 5.

Immunohistochemical analysis for MMP-13 in cartilage. (A) Representative specimens (PC, PC + PBS, and PC + BMSCs) collected at 2, 4, and 8 weeks after the compression-induced testing were evaluated for MMP-13 expression within the lateral femoral condyle (scale: 100 μm). (B) The proportion of MMP-13 positive cells (n = 6) was assessed. The effects of BMSCs injection treatment were evaluated across intervals of 2 weeks (PC:PC + BMSCs → P < 0.0001; PC + PBS:PC + BMSCs → P < .0001), 4 weeks (PC:PC + BMSCs → P = 0.0152; PC + PBS:PC + BMSCs → P = 0.0167), and 8 weeks (PC:PC + BMSCs → ns; PC + PBS: PC + BMSCs → ns). BMSCs, bone marrow-derived mesenchymal stem cells; PBS, phosphate-buffered saline; PC, post-compression; MMP-13, matrix metallopeptidase 13.

The Expression of TGF-β3 Demonstrates Downregulation With BMSCs Treatment at an Early Stage

The predominant localization of TGF-β3 expression within the cartilage was intracellular. At 2 weeks post-compression, a significant increase in the expression of TGF-β3 was evident at the cartilage wound site, whereas BMSCs treatment exhibited comparatively lower expression ( Fig. 6A ). By the 4th week, positive staining for growth factors was observed in chondrocytes at the periphery of the cartilage wound, coinciding with cellular death. The population of cartilage cells expressing immunostaining for TGF-β3 peaked at 2 weeks and gradually declined thereafter, with discernible levels persisting until the 4th week. A decrease in TGF-β3 expression was observed between weeks 4 and 8 ( Fig. 6B ). After the 8-week interval, a decline in chondrocyte presence PC resulted in an insignificantly low TGF-β3 expression ( Fig. 6B ). In addition, the discernible brownish staining observed in the PC and PC + PBS groups at 8 weeks may indicate chondrocyte loss.

Figure 6.

Immunohistochemical analysis of TGF-β3 in cartilage. (A) Specimens representing distinct conditions (PC, PC + PBS, and PC + BMSCs) were collected at 2-, 4-, and 8-week post-compression-induced testing, and the analysis centered on assessing TGF-β3 expression within the lateral femoral condyle (scale: 100 μm). (B) The proportion of TGF-β3-positive cells (n = 6) was assessed. The impact of BMSCs injection treatment was systematically examined at 2 weeks (PC:PC + BMSCs → P < 0.0001; PC + PBS:PC + BMSCs → P < 0.0001), 4 weeks (PC:PC + BMSCs → ns; PC + PBS:PC + BMSCs → ns), and 8 weeks (PC:PC + BMSCs → ns; PC + PBS:PC + BMSCs → ns). BMSCs, bone marrow-derived mesenchymal stem cells; PBS, phosphate-buffered saline; PC, post-compression; TGF-β3, transforming growth factor beta-3.

Temporal Dynamics of IGF-1 Expression in Response to Cyclic Compressive Loading and MSCs Therapy

As illustrated in Figure 7 , the intact group exhibited a baseline expression of IGF-1. Under cyclic compressive loading at week 2, a noticeable augmentation in the staining area of IGF-1 was observed compared with that in the intact group ( Fig. 7A ). However, a marginal downregulation was noted in the PC + BMSCs group when compared with that in both the PC and PC + PBS groups ( Fig. 7B ). By week 4, which coincided with the onset of cell death, the expression declined, and this trend became more pronounced at week 8. Consequently, at the 8-week time point, the expression of IGF-1 in the PC + BMSC group exhibited less deviation than that in the intact group, suggesting a potential return to the steady-state level. In contrast, both the PC and PC + PBS groups were downregulated compared with the steady-state levels ( Fig. 7B ).

Figure 7.

Immunohistochemical analysis of IGF-1 in cartilage. (A) Specimens representing distinct conditions (PC, PC + PBS, and PC + BMSCs) were systematically collected at 2-, 4-, and 8-week post-compression-induced testing, and the analysis centered on assessing IGF-1 expression within the lateral femoral condyle (scale: 100 μm). (B) The proportion of IGF-1-positive cells (n = 6) was evaluated. The systematic investigation into the influence of BMSCs injection treatment on our Wistar rat compression injury model unfolded across three time points: 2 weeks (PC:PC + BMSCs → ns; PC + PBS:PC + BMSCs → ns), 4 weeks (PC:PC + BMSCs → P = 0.0317; PC + PBS:PC + BMSCs → ns), and 8 weeks (PC:PC + BMSCs → P < 0.0001; PC + PBS:PC + BMSCs → P < 0.0001). BMSCs, bone marrow-derived mesenchymal stem cells; PBS, phosphate-buffered saline; PC, post-compression; IGF-1, insulin-like growth factor 1.

Discussion

In this study, we investigated the therapeutic potential of BMSCs in mitigating cartilage damage induced by a single overload cycle of femoral compression in a rat model of degenerative joint disease. The in vivo study revealed the persistence of transplanted PKH26-labeled BMSCs in the articular cartilage for up to 8 weeks. Hence, this assay serves as a valuable tool for identifying the homing sites of transplanted BMSCs. In addition, it enables the detection of successful BMSCs injections into the knee joint capsule. BMSCs injection treatment significantly attenuated the cartilage injury caused by cyclic compressive loading. However, a marked decrease in cell density within the lateral articular cartilage was observed at the 8-week intervals. BMSCs treatment attenuated compression-induced injury, with the optimal effect observed within the 4-week timeframe. To comprehensively elucidate the clinical responses and effects of BMSCs, further research is required that includes rats that receive regular injections.

The initiation of apoptosis is marked by the activation of caspase-3, a critical effector in the apoptotic pathway.^16,17 As observed, the levels of its cleaved form (CCasp3) increase substantially, indicating its role in orchestrating the dismantling of cellular components. The elevated CCasp3 levels during this phase align with the enzyme’s function in executing apoptosis by degrading essential cellular substrates.¹⁷ The observed reduction in caspase-3 expression following BMSC treatment can be attributed to the multifaceted anti-apoptotic mechanisms inherent to BMSCs.¹⁸ These cells exert paracrine effects, secreting growth factors and cytokines that inhibit apoptotic pathways, modulate immune responses to mitigate inflammation-induced apoptosis, and promote the upregulation of anti-apoptotic proteins.¹⁹ In addition, BMSCs alleviate oxidative stress by neutralizing reactive oxygen species and support tissue regeneration, both of which contribute to the decreased activation of CCasp3.²⁰ Collectively, these actions enhance cell survival and reduce apoptosis in treated tissues. Once the cell has been dismantled and the apoptotic process is nearly complete, the substrates of caspase-3 are largely degraded, marking the cell’s irreversible commitment to death.²¹ Following the cell’s demise, the levels of CCasp3 diminish. This reduction occurs because there is no longer a need for the enzyme’s activity; the apoptotic process is complete, and the enzyme itself may be degraded or rendered inactive.

Furthermore, a study has brought to attention MMP-13 as a principal enzyme implicated in the degradation of cartilage, with its predominant expression localized within connective tissue.²² Notably, MMP-13 exhibits specificity for type II collagen, proteoglycans, type IV and IX collagen, osteonectin, and perlecan in the cartilage matrix.²³ Clinical investigations have revealed increased MMP-13 expression in patients with articular cartilage degradation, underscoring its putative involvement in this degenerative process.^24,25 In our experimental endeavor, we established a rodent model of lateral femur injury induced by mechanical compression, followed by administration of BMSCs. The expression pattern of MMP-13 in compression-induced rats subjected to BMSCs intervention exhibited a discernible reduction at 2- and 4-week intervals post-surgery ( Fig. 5B ). Intriguingly, the therapeutic regimen involving BMSCs injection notably ameliorated the increased expression of MMP-13, which was attributed to mechanical compression stress on the chondrocytes (Fig. 5A–B). Nevertheless, at the 8-week time point, a reduction in the chondrocyte population subsequent to compression transpired, resulting in nominal expression levels of MMP-13. In addition, at weeks 2, 4, and 8, the HHGS and OOCHAS grading scores indicated a deceleration in the progression of lesions in the PC + BMSC group compared with that in the PC + PBS group (Fig. 3F and J). Collectively, these compelling findings strongly suggest that BMSCs hold promise for slowing disease progression. Furthermore, histological analysis revealed that BMSCs treatment effectively inhibited cell death and significantly prevented a decline in relative cell density ( Fig. 3B ).

Finally, we investigated the expression levels of TGF-β3 and IGF-1 in cartilage. TGF-β3 has been implicated in both protective and potentially detrimental capacities within the context of OA.^26,27 Its effects may vary based on the disease stage and specific microenvironment within the joint. In the early stages of OA, TGF-β3 is thought to have protective effects.^26,28 It may contribute to cartilage repair mechanisms by promoting the migration and proliferation of chondrocytes, which are responsible for maintaining cartilage.²⁶ TGF-β3 influences the synthesis of extracellular matrix components, including proteoglycans, by chondrocytes.²⁹ Based on our findings, there was a notable increase in TGF-β3 expression at the cartilage wound site, with BMSCs treatment showing comparatively lower expression at week 2 ( Fig. 6B ). BMSCs may exert their therapeutic effects through mechanisms other than, or in addition to, TGF-β3 upregulation. For instance, they may be involved in promoting tissue regeneration, modulating inflammation, or influencing other factors critical to the healing process. Consequently, BMSCs treatment may contribute to the prevention of cartilage damage, as evidenced by lower expression levels of TGF-β3. In cases of chondrocyte apoptosis, an associated inflammatory response may occur, and TGF-β3, known for its anti-inflammatory properties,³⁰ might play a role in modulating the inflammatory response within the joint. Although TGF-β3 is not directly implicated in chondrocyte death, its upregulation may serve as a compensatory or regulatory mechanism for counteracting inflammation and facilitating tissue repair. Moreover, IGF-1 stimulates the synthesis of extracellular matrix components and supports the overall health and resilience of cartilage tissues.³¹ Based on our results, there could be upregulation as a component of the reparative response ( Fig. 7A ) aimed at facilitating cartilage healing. Notably, the PC + BMSC group exhibited less pronounced expression, suggesting a potential preventive effect against cartilage degeneration. Nevertheless, in the advanced stages of injury or degeneration, the efficacy of IGF-1 may diminish ( Fig. 7B ).

Based on our findings, a solitary BMSCs injection appears to be effective in stalling the advancement of compression-induced injuries over a brief duration. Hence, the administration of regular BMSCs injections might be a better approach for effectively decelerating disease progression, particularly for achieving long-term prevention. However, this approach has some limitations. In the context of intra-articular injections of allogeneic BMSCs in a rat model, these limitations include the potential for immune responses directed against the introduced cells owing to their allogeneic (non-self) origin. This immunological response has the potential to compromise the therapeutic efficacy of treatment, which is attributed to heightened immune reactivity. Furthermore, the transient retention of infused cells within the joint cavity could impose constraints on their sustained therapeutic effect, such as interactions with native tissues and microenvironmental factors. There is also the possibility of uneven cellular distribution after injection into the joint, and the absence of controlled cellular differentiation after injection may have implications for achieving the desired therapeutic outcomes. Future studies could explore methods to reduce or prevent immune reactions against allogeneic BMSCs, such as using immunosuppressive agents, genetically modifying the cells to reduce their immunogenicity, or using autologous cells instead of allogeneic cells. In addition, exploring strategies to achieve a more uniform distribution of cells post-injection could be advantageous. This might involve refining injection techniques, incorporating imaging guidance during the procedure, or developing advanced delivery systems that facilitate even dispersal of cells. Finally, one notable limitation of this study is the difficulty in diagnosing early-stage OA in humans. In this model, BMSCs were utilized as a treatment for cartilage degeneration induced by mechanical compression injury, and their effectiveness is being assessed as an acute intervention immediately following injury. However, the challenge of diagnosing early-stage OA in humans complicates the determination of the optimal timing for this treatment.

Conclusions

In conclusion, our study validates the efficacy of BMSCs injection as a short-term approach to alleviate compression injury. Comprehensive analyses of cartilage histology, relative cell density, and cartilage grading scores (OOCHAS and HHGS) consistently supported the use of BMSC injections. It is crucial to interpret the expression levels of IGF-1 and TGF-β3 in consideration of the specific injury, disease stage, and local microenvironment within the joint. This effect was particularly discernible in the regulation of MMP-13, a key mediator of cartilage degradation, and CCasp-3, which is involved in apoptosis. Collectively, these observations suggest that the administration of BMSCs injections holds promise for attenuating the progression of PTOA induced by low mechanical compression.

Supplemental Material

sj-jpg-1-car-10.1177_19476035241301291 – Supplemental material for Attenuating Cartilage Degeneration in a Low Mechanical Compression Rat Model Through Intra-Articular Injections of Allogeneic Bone Marrow-Derived Mesenchymal Stem Cells

Supplemental material, sj-jpg-1-car-10.1177_19476035241301291 for Attenuating Cartilage Degeneration in a Low Mechanical Compression Rat Model Through Intra-Articular Injections of Allogeneic Bone Marrow-Derived Mesenchymal Stem Cells by Chia Tai, Akira Ito, Zixi Zhao, Hiroshi Kuroki and Tomoki Aoyama in CARTILAGE

Supplemental Material

sj-jpg-2-car-10.1177_19476035241301291 – Supplemental material for Attenuating Cartilage Degeneration in a Low Mechanical Compression Rat Model Through Intra-Articular Injections of Allogeneic Bone Marrow-Derived Mesenchymal Stem Cells

Supplemental material, sj-jpg-2-car-10.1177_19476035241301291 for Attenuating Cartilage Degeneration in a Low Mechanical Compression Rat Model Through Intra-Articular Injections of Allogeneic Bone Marrow-Derived Mesenchymal Stem Cells by Chia Tai, Akira Ito, Zixi Zhao, Hiroshi Kuroki and Tomoki Aoyama in CARTILAGE

Footnotes

Author Contributions

CT and AI conceived and planned the experiments, supervised the project, and analyzed the data. CT took the lead in writing this manuscript. CT and ZZ conducted the experiments and analyzed the data. CT, AI, ZZ, HK, and TA contributed significantly to the revision process. HK provided the financial support for this study. All the authors have thoroughly reviewed and approved the final version of the manuscript for publication.

Acknowledgments and Funding

The authors thank Editage () for the English language editing. They express their gratitude to the members of Kuroki Laboratory for their valuable discussions and guidance. This work was partially supported by the JSPS KAKENHI grants (JP21H03302 and JP23H03245).

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethics Approval and Consent to Participate

All animal experiments were approved by the Ethics Committee for Laboratory Animal Use of the Animal Research Committee of Kyoto University (approval number: Medkyo21576) for the study titled “Treatment Intervention Study for Cartilage Injury of Rat Knee Joint,” granted approval on September 13, 2021. The experimental design, analysis, and reporting adhered to the ARRIVE guidelines ().

ORCID iDs

Chia Tai

Akira Ito

Zixi Zhao

Availability of Data and Material

The data supporting the findings of this study are available within the article and the Supplementary Information. All other raw data are accessible upon reasonable request from the corresponding author.

Supplementary material for this article is available on the Cartilage website at .

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Supplementary Material

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