Published online Oct 26, 2023.
https://doi.org/10.4142/jvs.23128
Bone marrow stem cells incubated with ellipticine regenerate articular cartilage by attenuating inflammation and cartilage degradation in rabbit model
,1,†
Soyeon Lim,2,†
Kiran D. Bhilare,1,†
Md Jahangir Alam,1
Baicheng Chen,1
Ajay Vijayakumar,1
Hakyoung Yoon,1
Chang Won Kang,1
and Jong-Hoon Kim1
Abstract
Background
Ellipticine (Ellip.) was recently reported to have beneficial effects on the differentiation of adipose-derived stem cells into mature chondrocyte-like cells. On the other hand, no practical results have been derived from the transplantation of bone marrow stem cells (BMSCs) in a rabbit osteoarthritis (OA) model.
Objectives
This study examined whether autologous BMSCs incubated with ellipticine (Ellip.+BMSCs) could regenerate articular cartilage in rabbit OA, a model similar to degenerative arthritis in human beings.
Methods
A portion of rabbit articular cartilage was surgically removed, and Ellip.+BMSCs were transplanted into the lesion area. After two and four weeks of treatment, the serum levels of proinflammatory cytokines, i.e., tumor necrosis factor α (TNF-α) and prostaglandin E2 (PGE2), were analyzed, while macroscopic and micro-computed tomography (CT) evaluations were conducted to determine the intensity of cartilage degeneration. Furthermore, immuno-blotting was performed to evaluate the mitogen-activated protein kinases, PI3K/Akt, and nuclear factor-κB (NF-κB) signaling in rabbit OA models. Histological staining was used to confirm the change in the pattern of collagen and proteoglycan in the articular cartilage matrix.
Results
The transplantation of Ellip.+BMSCs elicited a chondroprotective effect by reducing the inflammatory factors (TNF-α, PGE2) in a time-dependent manner. Macroscopic observations, micro-CT, and histological staining revealed articular cartilage regeneration with the downregulation of matrix-metallo proteinases (MMPs), preventing articular cartilage degradation. Furthermore, histological observations confirmed a significant boost in the production of chondrocytes, collagen, and proteoglycan compared to the control group. Western blotting data revealed the downregulation of the p38, PI3K-Akt, and NF-κB inflammatory pathways to attenuate inflammation.
Conclusions
The transplantation of Ellip.+BMSCs normalized the OA condition by boosting the recovery of degenerated articular cartilage and inhibiting the catabolic signaling pathway.
INTRODUCTION
Osteoarthritis (OA) of the knee is a chronic joint disorder characterized by the degeneration of articular cartilage and extracellular matrix (ECM) destruction, ultimately leading to the loss of knee joint function. Articular cartilage undergoes structural and biochemical-related changes associated with aging, which impair its biomechanical functions [1]. Senescence-dependent changes in the cartilage include decreased chondrocyte proliferation and collagen density that lead to a compromised biosynthetic function [2]. With age, more senescent cells accumulate in the body and secrete inflammatory cytokines, chemokines, and proteases [3]. The constant renewal of cellular components occurs in vascular tissues with high cellular turnover rates to counter this [4]. With age, however, the regeneration and renewal ability of chondrocytes decreases because of stem cell exhaustion phenomena [5]. In addition, OA-related loss of cartilage homeostasis causes the continued destruction of ECM components, such as proteoglycan and collagen, leading to pain and inflammation in elderly people [6].
Ellipticine (Ellip., 5,11-dimethyl-6H-pyrido[4,3-b]carbazole, Fig. 1A) is a naturally available alkaloid obtained mostly from Apocyanaceae plants. The compound has promising anti-malignant activity against leukemia, endometrial, and neuroblastoma cancer cells [7, 8, 9]. Ellip. was recently reported to induce the differentiation of adipose-derived stem cells (ASCs) into chondrocytes through p53-based Sox9 regulation to have a protective effect in an OA murine model [10]. Stem cell therapy for OA is a good alternative to repair articular cartilage [11]. In this connection, bone marrow stem cells (BMSCs) can prevent the destruction of articular cartilage as a remedy for knee joint inflammation by stimulating chondrocyte proliferation on articular cartilage [12, 13]. The activation of nuclear factor-κB (NF-κB)-stimulated proinflammatory mediators (such as cyclooxygenase-2, prostaglandin E2 [PGE2], and inducible nitric oxide synthase [iNOS]) and matrix-metallo proteinases (MMPs) production cause cartilage degradation during knee joint injury, whereas PI3K/Akt signaling plays a crucial role in cartilage destruction [14, 15]. In addition, the p38-mitogen-activated protein kinase (MAPK) pathway showed high expression of MMPs during OA, leading to cartilage damage [16, 17, 18]. The regenerative effects of autologous BMSCs incubated with Ellip. (Ellip.+BMSCs) on rabbit articular cartilage has not been reported. Therefore, this study examined the regenerative effect of transplantation of Ellip.+BMSCs while elucidating the signaling pathways involved with articular cartilage regeneration on a rabbit OA model. The aim was to establish a safe and efficacious therapeutic for the transplantation of an Ellip.+BMSCs in a rabbit OA model and provide clues to the development of stem cell therapy for OA treatment in the future.
Fig. 1
*p < 0.05 and **p < 0.01 when treatment groups are compared with the chondrectomy group.
Morphological evaluation of rabbit’s articular cartilage in treating BMSCs incubated with ellipticine. (A) chemical structure of ellipticine, (B) scores of macroscopic analysis of femoral condyles from damaged cartilage, and (C) chemical structure of ellipticine. The red arrow indicates the defect area of the femoral condyle. The values are the means ± SD.
N/C, normal control; P/C, positive control; BMSC, bone marrow stem cell; Ellip., ellipticine; Ellip.+BMSC, BMSC incubated with ellipticine;.
MATERIALS AND METHODS
Animals
Twelve-week-old white New Zealand female rabbits weighing 3.6 ± 0.3 kg (n = 36) were purchased from Koatech, Korea. The experimental guidelines for the laboratory animals were followed, as mentioned by the International Animal Care and Use Committee of Chonbuk National University. All surgical procedures were performed after seeking approval from the Chonbuk National University Animal Ethical Care and Use Committee (approval No. JBNU 2021-0150). The animal room facility was controlled by a 12 h dark /12 h light cycle at 24 ± 2°C with air conditioning, and the rabbits were served a normal diet and supplied with water ad libitum.
Reagents
Phosphate buffer saline (1 × PBS), Dulbecco's modified eagle medium (DMEM/F12), fetal bovine serum (FBS), and antibiotic-antimycotic solution (1% penicillin and streptomycin) were purchased from Gibco (USA). The PGE2 detection enzyme-linked immunosorbent assay (ELISA) kit was acquired from R&D Systems (USA). The MMP-3 detection ELISA kit was supplied by CUSABIO (China), and the TNF-α detection ELISA kit was obtained from Abcam (USA). The BCA kit used for protein quantification was procured from Thermo Scientific (USA). The primary antibodies against rat NF-κB, p-Akt, and Akt were purchased from Abcam (UK), while the remaining primary antibodies (p-P65, p-P38, and p38) and HRP-conjugated secondary antibodies were obtained from Cell Signaling (USA). The hematoxylin–eosin stain was purchased from Sigma–Aldrich (USA) for histological analysis. The Masson trichrome stain was obtained from Scytek Lab (USA). Safranin O stain was procured from Sigma (USA), and the MMP-1 staining vectastatin ABC kit was supplied by Vector Lab Inc. (USA). All other reagents were high quality (purity ≥ 99.5%) molecular biology grade.
Experimental design
Thirty-six rabbits were randomly divided into the following six groups (n = 6): 1) normal control (N/C), articular cartilage was open and closed; 2) chondrectomy (represented as Chondrectomy), articular cartilage on the femoral condyle of the rabbit knee joint was destroyed as the OA control with no treatment; 3) collagen control (represented as the positive control, P/C), commercial collagen sheet treatment after chondrectomy; 4) BMSCs alone, treatment with BMSCs (1 × 106/mL) intra-articular injection after chondrectomy; 5) Ellip alone, treatment with Ellip. (1.0 μM) intra-articular injection after chondrectomy; and 6) Ellip.+BMSCs, transplantation with BMSCs (1 × 106/mL) pre-incubated with Ellip. (1.0 μM). The dose and duration of treatment were decided according to the previous study [10].
Isolation of BMSC and incubation with ellipticine
Experimental rabbits were anesthetized with isoflurane. Both femurs were collected and stored on cold PBS. The heparinized BMSCs were obtained from the femur using a syringe plunger and centrifuged at 400 × g for 6 min. After RBC lysis, the BMSCs were plated in 75 cm2 cell cultured flasks in DMEM media supplemented with 10% FBS and 1% antibiotics at 37°C with 5% CO2 for 24 h. 1.0 × 106 cells were seeded into a six-well culture plate with 1.0 μM Ellip. for 24 h. After incubation, the Ellip.-treated BMSCs were collected by washing the culture plate with PBS three times, followed by transplantation on the defect site of the femoral condyle.
OA rabbit modeling
The procedure was followed as reported previously [17]. Briefly, the knee joints were exposed by medial para-patellar incision, and the articular cartilage destruction was produced in both femoral condyles using an electrical trephine (2 mm in depth and 5 mm in diameter). The skin was closed using a 4-0 Nylon suture. After two weeks of surgery, the inter-articular transplantation of autologous BMSCs was performed for the respective groups except for the P/C group in which collagen was implanted on the day of surgery. The rabbits were euthanized after two and four weeks, and the experimental samples were collected.
Swelling measurements of the knee joint
Before the rabbits were euthanized at the aforementioned time intervals, the swelling condition of the knee was evaluated by measuring the knee joint thickness using commercially available Vernier calipers.
Macroscopic assessment
Macroscopic analysis was carried out to evaluate the effect of the treatment on the damaged femoral condyles. The articular cartilage destruction was measured using a four-point scale, as described elsewhere [18]: destruction that spread to the subchondral bone (score 4); destruction that spread into the deep layers (score 3); destruction spread out into the superficial or central layers only (score 2); negligible fibrillation or a minor yellowish discoloration of the surface (score 1); normal-appearing surface (score 0). A higher score indicated severe damage to the articular cartilage of the rabbit femoral condyle.
Micro-computed tomography (CT) analysis
After a macroscopic examination, a micro-CT evaluation was performed to detect new cartilage tissue formation on the damaged area, as mentioned previously [17]. Briefly, the femoral condyles previously fixed in 10% buffered formaldehyde were used for 360° micro-CT scanning (SKYSCAN 1076, Belgium). The unique defect area was assessed to evaluate the region of interest, while 3D femoral condyle images were recorded with the help of CTvox software (Sky Scan Company).
ELISA
For serum collection, blood samples obtained from the respective groups were centrifuged at 3,000 rpm for 10 min. The serum was transferred to a fresh tube and stored at −20°C for future ELISA analysis. The serum levels of MMP-3, TNF-α, and PGE2 were measured according to the manufacturer’s protocol. All the evaluations were performed in triplicate.
Western blot analysis
Post-euthanization, the cartilage was triturated under −70°C conditions to isolate the total protein, as mentioned previously [17]. After protein estimation using a BCA kit, the respective protein samples were used for immunoblotting. The samples were incubated overnight at 4°C with the primary antibodies against NF-κB (1:500), p-Akt (1:1,000), and Akt (1:500) from Abcam (UK) and p-P65 (1:1,000), p-P38 (1:800), and p38 (1:1,000), the HRP-conjugated secondary antibodies were incubated for 2h at RT. The final bands were visualized using a Lumi pico solution, and final blot images were captured using an ImageQuant LAS-500 image analyzer (GE Healthcare, Sweden). The target bands were quantified using Image J software (ver 1.49).
Histological analysis
The pathophysiological changes were evaluated using the sliced cartilage specimens from the eroded area for histochemical staining with different dyes, as reported elsewhere [17]. Hematoxylin-eosin (H/E), Masson trichrome, and Safranin O staining were performed to determine the total chondrocytes, collagen, and proteoglycan content of the articular cartilage, respectively. MMP-1 expression was detected by overnight incubation with the primary MMP-1 antibody (1:200) at 4°C, followed by secondary antibody incubation for 1 h at RT. All histological staining images and MMP-1 intensity were detected using an inverted contrast microscope (Zeiss Corporation, Germany) at a 200× fixed magnification.
Statistical analysis
All experimental results are expressed as the mean ± SD. Statistical analyses were evaluated using the variance (ANOVA) followed by Tukey’s multiple comparison tests. A statistically significant relationship was defined as p < 0.05 unless indicated otherwise. All the experiments were performed in triplicate, and the mean value was reported.
RESULTS
Ellip.+BMSCs prevented cartilage defect and surface discontinuity
The irregular and reddish articular cartilage area was larger at four weeks than at two weeks in the chondrectomy group. In contrast, the N/C group showed regular cartilage with null erosion to either of the femoral condyles. As shown in Fig. 1A, treatment with BMSCs, Ellip., Ellip.+BMSCs, and P/C could recover the cartilage damage in the femoral condyle. At four weeks, the Ellip.+BMSCs group had the lowest cartilage damage score (Fig. 1B) compared to other treatment groups. Therefore, the Ellip.-treated BMSC transplantation led to the significant formation of new hyaline cartilage on the degenerated articular surface in a time-dependent manner.
Ellip.+BMSCs ameliorated articular cartilage
The chondrectomy group showed severe destruction of the femoral condyle gross structure, indicating that the femoral bone lost its regular structure due to bone tissue necrosis during the experimental tenure (Fig. 2A). In contrast, BMSCs, Ellip.+BMSCs, and P/C group exhibited the significant recovery of degraded articular cartilage in the femoral condyle. At four weeks, the micro CT images of the Ellip.+BMSCs group were similar to normal bone structure because they had the lowest CT damage score compared to other treatment groups (Fig. 2B).
Fig. 2
*p < 0.05 and **p < 0.01 when the treatment groups are compared to the chondrectomy group.
Visualization of the rabbit’s articular cartilage in treating BMSC incubated with ellipticine. (A) micro-CT 3D analysis was used to evaluate the severity of damaged articular cartilage from femoral condyles at four and eight weeks and (B) scores of micro-CT analysis. The defect area of the femoral condyle is marked by red color. The values are the means ± SD.
N/C, normal control; P/C, positive control; BMSC, bone marrow stem cell; Ellip., ellipticine; Ellip.+BMSC, BMSC incubated with ellipticine; CT, computed tomography;.
Elip.+BMSCs alleviated knee joint inflammation
Inflammation is a protective immune response to any injury or infection characterized by heat, redness, swelling, and pain at the pathological site. The present study showed that both knee areas had more swelling, as observed from the measurement of joint thickness. The chondrectomy and treatment groups expressed opposite trends to each other except for the N/C group (Fig. 3A). In addition, the proinflammatory cytokine TNF-α (Fig. 3B) and degradation biomarker PGE2 (Fig. 3C) levels in the serum were downregulated significantly compared to the chondrectomy group in a time-dependent manner. During bone degeneration, the PGE2 cytokine (interleukin [IL]-1) upregulates MMP production (catabolic signal) that further degrades the ECM of articular cartilage [19]. Thus, the Ellip.+BMSCs group displayed the highest anti-inflammatory response by inhibiting the levels of MMP-3 (Fig. 3D) and other inflammatory cytokines.
Fig. 3
*p < 0.05 and **p < 0.01 when treatment groups are compared with the chondrectomy group.
Alteration of pro-inflammatory levels on treating BMSC incubated with ellipticine. (A) knee thickness evaluation, (B, C, and D) represent TNF-α, PGE2, and MMP-3 serum concentration, respectively, as identified using ELISA analysis. The values are the means ± SD.
N/C, normal control; P/C, positive control; BMSC, bone marrow stem cell; Ellip., ellipticine; Ellip.+BMSC, BMSC incubated with ellipticine; TNF-α, tumor necrosis factor α; PGE2, prostaglandin E2; MMP-3, matrix-metallo proteinase 3; ELISA, enzyme-linked immunosorbent assay.
Ellip.+BMSCs downregulated MAPK, PI3K/Akt and NF-κB signaling pathway
This study targeted effector kinases, such as MAPK, PI3K/Akt, and NF-κB associated with metabolic and biochemical pathways during the pathogenesis of osteoarthritic bone damage (Fig. 4A). The chondrectomy group displayed the phosphorylation-induced activation of p38 (MAPK), p65 (NF-κB), and Akt in a time-dependent manner. On the other hand, the Ellip.+BMSCs group had the most significant inhibition of this signaling pathway compared to the control group (Fig. 4B-D), particularly at four weeks.
Fig. 4
*p < 0.05 and **p < 0.01 when treatment groups were compared to the chondrectomy group.
Effect of BMSC incubated with ellipticine on western blot was implemented to evaluate the protein level of MAPK, PI3K/Akt, and NF-κB relative signaling pathway, p-P38, p38, p-Akt, Akt, and p-P65, NF-κB. The values are the means ± SD.
N/C, normal control; P/C, positive control; BMSC, bone marrow stem cell; Ellip., ellipticine; Ellip.+BMSC, BMSC incubated with ellipticine; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-κB;
Ellip.+BMSCs prevented collagen degradation
H/E staining showed that the transplantation of Ellip.+BMSCs on the rabbit femoral condyle displayed a significant number of newly formed chondrocytes on the damaged area (Fig. 5) at two and four weeks. H/E staining showed a decrease in the articular cartilage of the chondrectomy group (Fig. 5A(b)) compared to the N/C group (Fig. 5A(a)). In contrast, the Ellip.+BMSCs treated group had more chondrocyte-like cells in the regenerated area with cartilage lacunae (Fig. 5A(f)), similar to the N/C group, especially at four weeks. On the other hand, the P/C group, BMSC group, and Ellip. Group showed no significant difference from the chondrectomy group (Fig. 5A(c-e)). Next, the efficacy of Ellip.+BMSCs was determined by evaluating the structural integrity of the rabbit articular cartilage after Safranin O staining. After two and four weeks, the articular cartilage exhibited severe pathological osteoarthritic changes characterized by Safranin O loss in the chondrectomy group (Fig. 5B(b)) compared to the N/C group (Fig. 5B(a)), as indicated by the arrow. The rabbit cartilage with Ellip.+BMSCs exhibited less Safranin O loss and a higher subchondral plate thickness compared to the P/C group, BMSC group, and Ellip. Group (Fig. 5B(c-e)). In particular, the femoral condyle in the Ellip.+BMSCs group showed the best recovery, as shown in Fig. 5B(f).
Fig. 5
Histologic analysis on the rabbit femoral condyle. A Hematoxylene-eosin, B safranin O, and C masson trichrome stains were shown in segments of articular cartilage from femoral condyle at 2 and 4 weeks (all fixed magnification × 200, scale bar = 200 μm).
N/C, normal control; P/C, positive control; BMSC, bone marrow stem cell; Ellip., ellipticine; Ellip.+BMSC, BMSC incubated with ellipticine; H/E, hematoxylin-eosin;
Masson’s staining usually shows the distribution of collagens, and the typical hyaline-like matrix for fibrous connective tissue is stained light green or blue. On the other hand, the chondrectomy group (Fig. 5C(b)) had few chondrocyte-like cells in the cartilage area compared to the N/C group, as indicated by the arrows (Fig. 5C(a)). By contrast, the regenerated cartilage areas were stained blue at two weeks, indicating that more types I or III collagen were newly secreted in the Ellip.+BMSCs group (Fig. 5C(f)) compared to the P/C, BMSC, and Ellip. Groups (Fig. 5C(c-e)) at two and four weeks. These results indicate successful hyaline cartilage regeneration compared to the chondrectomy group, which expressed poor collagen and destructed cartilage. These results were also similar to the western blotting data in the present study.
Ellip.+BMSC prevented ECM degradation
During OA, (MMP-1 plays a major role in damaging condyle cartilage. The present study investigated the MMP-1 levels using immunohistochemistry staining at the injured articular cartilage. The chondrectomy group (Fig. 6B) exhibited boosted levels of MMP-1 cells in the superficial, middle, and deep layers of femoral bone cartilage as compared to the N/C group (Fig. 6A) after two and four weeks, as indicated by arrows. By contrast, the articular cartilage in the Ellip.+BMSCs group displayed relatively reduced cartilage damage with fewer MMP-1 positive cells (Fig. 6F) after two and four weeks. On the other hand, even if there was a difference in cartilage regeneration, the BMSC and Ellip. Groups showed no significant differences in MMP-1 cells in the layers of the femoral bone cartilage in (Fig. 6D-E) compared to the chondrectomy group after two and four weeks. Furthermore, the P/C group (Fig. 6C) showed significant cartilage regeneration efficacy compared to the chondrectomy group, but the MMP-1 cells were not reduced significantly compared to the Ellip.+BMSCs group after two and four weeks. Overall, transplantation of Ellip.+BMSCs resulted in the lowest levels of MMPs to boost articular regeneration in the rabbit OA model.
Fig. 6
Effects of BMSCs incubated with Ellipticine on immunohistological staining for MMP-1 expression on articular cartilage from femoral condyle at two and four weeks (all fixed magnification × 200, scale bar = 200 μm).
N/C, normal control; P/C, positive control; BMSC, bone marrow stem cell; Ellip., ellipticine; Ellip.+BMSC, BMSC incubated with ellipticine; MMP-1, matrix-metallo proteinase 1;
DISCUSSION
OA is a joint bone disease that arises due to the destruction or degeneration of joint articular cartilage [20]. Therefore, a novel management system needs to be established to prevent bone damage and limit the progression of OA. One such therapy entails using bone marrow pluripotent stem cells to mitigate damage due to cartilage tissue destruction [21, 22]. A previous study reported that the Ellip. Treatment with ASCs induces a significant amount of type II collagen and Aggrecan levels, acting as a chondrocyte differentiation molecule in the murine model [10]. On the other hand, in vivo data regarding its adherence to cartilage has not been reported. The novelty of the present study lies in demonstrating that Ellip.+BMSCs could provide a chondroprotective effect to improve the OA condition by downregulating the inflammatory signaling pathway in a surgically induced OA rabbit model.
This paper reported three main findings related to the role of transplantation of Ellip.+BMSCs for articular regeneration. First, the transplantation of Ellip.+BMSCs significantly attenuated inflammation in a time-dependent manner. During degenerative OA progression, proinflammatory cytokine (i.e., TNF-α) plays a major role in developing inflammation and early pain in the milieu of cartilage tissue [23]. On the other hand, the Ellip.+BMSCs treatment group alleviated knee joint swelling and attenuated the secretion of TNF-α and PGE2 (Fig. 3A-C), which are the hallmarks of OA-associated pain and inflammation. Hence, transplantation of Ellip.+BMSCs could be a potential therapy for treating inflammation against OA progression. A second important proof of cartilage regeneration was that the transplantation of Ellip.+BMSCs could significantly restore the articular chondrocytes compared to the chondrectomy group. Previous studies reported that PGE2 boosts the secretion of MMPs to inhibit cartilage matrix synthesis and reduces collagen in ECM, causing further damage to the articular cartilage during the bone disorder [19]. Nevertheless, the transplantation of Ellip.+BMSCs increased articular cartilage regeneration by suppressing MMP-1 and MMP-3 collagenase secretion (Figs. 3D and 6).
The third body of evidence strengthening the regenerative role of Ellip.+BMSCs for OA was evident from the micro-CT and histology experimental data. A well-organized cartilaginous matrix and knee articular cartilage were observed because of the formation of new hyaline tissue. The femoral condyle defect that was observed in the chondrectomy group was rejuvenated to normal articular cartilage in the Ellip.+BMSCs treatment group with a high number of chondrocytes and superior cartilage surface (Figs. 1, 2, and 5A). Furthermore, a high proteoglycan and collagen content in the defective cartilage area on the femoral condyle indicated superior regeneration on account of Ellip.+BMSCs treatment compared to other treatment groups, but the chondrectomy group showed only a few cells (Fig. 5B and C). With this evidence, the present study suggests that the Ellip.+BMSCs treatment ameliorates the defective cartilage on condyle in the rabbit model.
The effector kinases of the major NF-κB signaling pathway during OA were targeted to elucidate the mechanistic role of Ellip.+BMSCs behind articular chondrocyte regeneration. These kinases upregulate the gene expression of several inflammatory cytokines (TNF-α, PGE2) and pro-apoptotic genes (caspase-3 and bax) [21, 22, 23]. A previous study reported that several MAP kinases have a crucial role during the development of OA [16, 24]. The regenerative potential of Ellip.+BMSCs can be attributed to the reduced p38 (MAPK) pathway in chondrocytes (Fig. 4). The phosphorylation-induced activation of the PI3K/Akt axis causes chondrocyte cell apoptosis, leading to cartilage destruction [25, 26]. The present study showed that Ellip.+BMSCs attenuated the in vivo stimulation of p-P65, p-P38, and p-Akt kinases (Fig. 4B-D). Therefore, the probable role behind the chondroprotective effect of Ellip.+BMSCs was the inhibition of intracellular NF-κB, MAPKs, and PI3K/Akt signaling pathways in chondrocyte cells of the articular cartilage. Moreover, the pathological symptoms of chondrectomy on the femoral condyle share many common characteristic features observed in a degenerative OA. Therefore, the transplantation of Ellip.+BMSCs might also serve as a novel preventive strategy against degenerative cartilage disease.
IL-1β acts alone or with other inflammatory cytokines, such as IL-6, TNF-α, PGE2, iNOS, COX-2, aggrecanase, and a few MMPs to catalyze cartilage collapse during OA progression by inducing senescence and chondrocyte apoptosis [27, 28, 29]. A major shortcoming of the present study was the lack of such cytokines/marker expression data, which will be addressed in future studies.
This study confirmed the effects of Ellip.+BMSCs in a rabbit OA model. The Ellip.+BMSCs treatment could inhibit MMP expression and ameliorate proteoglycan and collagen type II in the ECM to regenerate the articular cartilage tissue in the defect area by regulating the MAPK, PI3K/Akt, and NF-κB signaling pathways. Therefore, the present results strongly suggest a novel therapeutic role of Ellip.+BMSCs transplantation for OA treatment that needs to be clinically verified in the future.
Funding:This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &Welfare, Republic of Korea (grant number: HI18C0661). This research was also supported by a grant from the Basic Science Research Program administered through the National Research Foundation of Korea (NRF) and funded by the Ministry of Education (2019R1A6A1A03033084).
Conflict of Interest:The authors declare no conflicts of interest.
Author Contributions:
Conceptualization: Kim JH, Kang CW.
Data curation: Hossain MA, Kim JH.
Formal analysis: Hossain MA, Kim JH, Lim S.
Investigation: Hossain MA, Alam MJ.
Methodology: Hossain HA, Kim JH.
Project administration: Kim JH.
Resources: Kim JH.
Software: Hossain MA, Kim JH.
Supervision: Kim JH.
Validation: Alam MJ, Chen B.
Visualization: Hossain MA, Chen B, Yoon H.
Writing - original draft: Hossain MA, Bhilare KD, Kim JH.
Writing - review & editing: Bhilare KD, Vijayakumar A, Kim JH.
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MeSH Terms
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ORCID IDs
Funding Information
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Korea Health Industry Development Institute
HI18C0661
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National Research Foundation of Korea
2019R1A6A1A03033084
