Mesenchymal stem cells (MSCs) are attractive as a cell source for regenerative medicine, particularly in the treatment of cartilage injuries (1) and diseases such as osteoarthritis (OA) (2). An increasing number of reports suggest that MSCs can be isolated from various types of adult mesenchymal tissue, such as synovium (3), periosteum (4), skeletal muscle (5), and adipose tissue (6), in addition to bone marrow (7). We previously compared MSCs derived from the various types of mesenchymal tissue in young patients during ligament reconstruction. In this prior study, we found that synovial MSCs retained both a higher capacity for proliferation and a greater chondrogenic potential than did MSCs from other sources (8). In addition, we previously analyzed cells harvested from the fibrous synovium and adipose synovium of both young and elderly donors. Fibrous synovium and adipose synovial cells demonstrated similar self-renewal and differentiation capacities, irrespective of donor age (9). Furthermore, we used synovial MSCs for the repair of cartilage defects in rabbit knees, and the results demonstrated that implanted MSCs differentiated into cartilage appropriate to the microenvironment in vivo (10).
OA is a condition of destruction of articular cartilage in a joint, and the disease progression is associated with varying degrees of synovitis. Mechanical factors will affect the synovium as well as the cartilage, possibly resulting in a different distribution in cellular compartmentalization. A successful outcome from the use of synovial MSCs for cartilage regeneration in the OA knee requires that a sufficient quantity of MSCs be obtained. Therefore, the distribution of MSCs in OA synovium must be determined. The first objective of the present study was to examine whether MSCs are distributed equally or unequally in the synovium of patients with knee OA. Determining the relationship between the number of MSCs and the histologic features of regions of the synovium would help clarify the progenitor, or niche, of synovial MSCs.
In addition, synovial MSCs may alter their properties during in vitro proliferation, and the conditions in which to expand the cells to optimize their potential to differentiate into multiple cell lineages has not yet been defined. Thus, our second objective was to examine the effects of 2 specific variables, preculture period and harvest site, on 2 cell parameters, cell size and surface epitopes, to determine how the chondrogenic differentiation potential of synovial MSCs can vary. This study is directed toward developing a clinically feasible strategy for use of synovial MSCs in the repair of cartilage defects. We thus set out to address this by identifying the optimal parameters for in vitro culture expansion and chondrogenesis of synovial MSCs.
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Although the identity of MSCs has yet to be fully defined (20), we recognize that MSCs are derived from mesenchymal tissue and have the functional capacity both to self-renew and to generate a number of differentiated progeny (21). Since the earliest studies by Friedenstein et al (22), the standard assay used to identify the self-renewal ability of MSCs is the colony-forming–unit fibroblast assay. This assay measures the percent of cells with high replicative capabilities in a culture. Although clonal colonies of MSCs are readily prepared, the colonies rapidly become heterogeneous as they expand (23–25). The synovial cells used in the present study showed both colony-forming ability and multipotentiality, the hallmarks used to define cells as MSCs.
We recently reported that fibrous synovium, harvested from the inner side of the lateral joint capsule, and adipose synovium, harvested from the infrapatellar fat pad, had similar growth and differentiation potentials in young patients with anterior cruciate ligament injury and in elderly patients with OA (9). These findings suggest that adherent colony-forming cells derived from the synovium have a similar proliferation ability and multipotentiality, independent of the location in the knee. In this study, we also demonstrated that the capacity for proliferation and potential for chondrogenesis of synovial MSCs were similar among the different harvest sites in the OA synovium.
CD31, recognized as platelet endothelial cell adhesion molecule 1 (26), has been used as a candidate marker of endothelial cells (26, 27). It has been reported that both von Willebrand factor and CD31 are expressed on the same endothelial cells (28). However, CD31 is strongly expressed on vascular endothelial cells, whereas von Willebrand factor is strongly produced on other types of endothelial cells. In addition, previous studies have used CD31 as a marker of vascular endothelial cells (29), and α-smooth muscle actin has been considered a marker of vascular pericytes, specifically in the synovium (18). Therefore, in our study, we used CD31 to label vascular endothelial cells.
There is vast research-related interest in determining the anatomic location of MSCs within the tissue. In this study, we demonstrated that the number of α-smooth muscle actin–positive vessels and the number of CD31+ cells were strongly correlated with the number of colony-forming cells derived from the synovium. It was reported that the endothelial cells in both rheumatoid arthritis synovium and OA synovium are surrounded with cells positive for the STRO-1 surface protein, one marker of MSCs (29). In bone marrow, the perivascular region has been suggested as the niche for MSCs (30). In addition, microvascular pericytes derived from the retina and aorta were reported to have multipotential differentiation capabilities (31). This suggests that synovial MSCs may also exist in the perivascular niche. Our results, as well as those from other studies, indicate the possibility that MSCs in the synovium arise from committed progenitors that belong to the distinct lineage of vascular pericytes.
We collected synovium from 4 different sites in the knee, and our analyses showed that synovium at the medial outer region contained more CD31+ vascular endothelial cells and more α-smooth muscle actin–positive vessels than were found in the synovium at the suprapatellar pouch, infrapatellar fat pad, and medial inner regions. The femorotibial angle in all patients was >185°, and all patients were diagnosed as having medial compartment OA. The pathogenesis of OA is described as a process of destruction of the articular cartilage by both mechanical and biochemical factors, which is associated with varying degrees of synovitis (32–34). Saito and Koshino compared the distribution of neuropeptides in the synovium of knees with medial compartment OA and showed a higher perivascular neural network in the medial compartment than in the lateral compartment or the suprapatellar pouch (34). In our study, the medial compartment of the OA knees demonstrated this variability in the distribution of vascular endothelial cells and vascular pericytes, which also accounted for the varying distribution of colony-forming cells.
We previously reported that bone marrow–derived MSCs can undergo a time-dependent transition from small cells to large cells, and that the preculture period affects the chondrogenic differentiation potential (11, 12). In this study, we examined 2 variables, preculture period and harvest site, for their effects on 2 parameters, cell size and surface epitopes, to determine how the chondrogenic differentiation potential of synovial MSCs can vary. Similar to previous findings, the preculture period affected chondrogenesis, with an increase in size of the synovium-derived MSCs with increasing preculture period. Anatomic location did not affect chondrogenesis. Of the surface epitopes tested, no relationship was observed between chondrogenic potential and CD31/α-smooth muscle actin–positive staining or expression of CD105, CD166, or STRO-1. In contrast, CD90 expression was predictive of chondrogenesis (Figure 6).
Figure 6. Summary of cell size, surface antigen expression, and chondrogenic potential during the expansion of synovial cells. During clonal expansion of synovial cells over the 21 days of preculture, the cell size increased, while the rate of positive expression of CD90 and the chondrogenic potential of the cells decreased.
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CD90 (Thy-1), which was first recognized as a marker of thymus-derived lymphocytes (35), was detected through a screening of heterologous antisera against mouse leukemia cells. CD90 has also been considered a marker antigen of MSCs (36), although the precise biologic function is not yet clear (37). Fickert et al reported that initial sorting for CD9/CD90/CD166 triple-positive synovial cells revealed the multipotency of these cells (38), which supports our results. In our study, we found that CD90 is an important indicator of the chondrogenic differentiation potential of synovial MSCs.
We have previously quantitatively evaluated the chondrogenic potential of MSCs by pellet wet weight. According to our previous studies, during in vitro chondrogenesis of MSCs, the pellet increased in size, weight, and cartilage matrix synthesis. Conversely, the DNA yield per pellet decreased. Radioactivity per DNA in the cells, assessed by prelabeling with 3H-thymidine, was found to be stable during in vitro chondrogenesis of MSCs (39). These results indicate that the increase in pellet size can be attributed to production of extracellular matrix, and not the proliferation of the cells. We believe that both the size and the weight of the pellet are quantitative indicators of the ability of MSCs to produce chondrogenesis in vitro.
In our study, the pellet weight decreased in conjunction with increasing preculture period. It is indeed possible that beyond a certain number of population doublings, the chondrogenic potential would tend to plateau to a minimum level, so that differences would not be detectable, at least not with the assays used in this study. A similar mechanism can apply to adipogenesis and osteogenesis. For these assays, passage 0 cells were precultured for 7, 14, or 21 days and then replated at a very low density, in order to obtain colonies for the differentiation assays. Thus, cells underwent additional population doublings prior to differentiation. Also, in this case, it is possible that with the increase in population doublings, there was a decrease in the osteogenic and adipogenic differentiation potentials, and this would tend to plateau to a minimum, so that a difference, if any, would be difficult to detect. In other words, minimally expanded cells could have overall greater differentiation potential, which would decrease quickly during culture expansion.
We found variability in pellet weight between donors after in vitro chondrogenesis (Figure 4A, panel b). The harvest site, digestion of the synovium, expansion of the cells, and findings on in vitro chondrogenesis assay were similar between donors. Variability between donors depends on the number of chondroprogenitors and their potential for cartilage matrix synthesis.
Our findings demonstrate that the in vitro chondrogenic potential of MSCs can be affected by preculture conditions. It will be intriguing to examine whether the in vitro results would reflect those obtained in vivo. We are currently investigating the relationship between in vitro and in vivo chondrogenesis. For assessment of in vivo chondrogenesis, undifferentiated MSCs were transplanted into the joints of rabbits with cartilage defects (10), and the chondrogenic potential of the MSCs was evaluated histologically. The results of that study showed that when the difference in pellet weights between the 2 populations was large, this was reflected in the in vivo results.
In the present study, the adipogenic colony-forming efficiency of the synovial MSCs (∼60%) was higher than the calcification colony-forming efficiency (∼30%). We have previously compared MSCs derived from several mesenchymal tissue types and found that the adipogenic colony-forming efficiency was higher than the calcification colony-forming efficiency in MSCs derived from the synovium and adipose tissue. In contrast, the calcification colony-forming efficiency was higher than the adipogenic colony-forming efficiency of MSCs derived from the bone marrow and periosteum (8). These results suggest that the local tissue microenvironment may be directing the “fate” of the MSCs toward a particular lineage (9).
In this study, the preculture period did not affect the adipogenic potential or osteogenic potential of synovial MSCs. We previously reported that the ability of bone marrow–derived MSCs to undergo adipogenesis was higher when the cells were plated at lower densities and cultured for shorter preculture periods (11). We also demonstrated that the adipogenic potential was much higher in synovial MSCs than in bone marrow–derived MSCs (8). Evaluation of adipogenic potential through assessment of the oil red O–positive colony-forming efficiency is a simple method, but its sensitivity may be too low to detect any difference in synovial MSCs. For determination of osteogenic ability, more sensitive methods may be able to detect differences induced by preculture conditions.
The number of MSCs was correlated with the number of CD31/α-smooth muscle actin–positive cells, and these cells were located in different distributions in different areas of the synovium of patients with medial compartment OA. However, the proliferation and differentiation potentials of the MSCs were not affected by harvest site.
MSCs in a shorter preculture period display higher chondrogenic potential, which suggests that the whole synovium in patients with medial compartment knee OA can be used to obtain a high number of MSCs with high chondrogenic potential, provided that the cells are expanded in a minimal culture period. However, since the amount of synovium needed to obtain a sufficient number of MSCs is limited, a longer culture period is required to obtain a higher number of MSCs. Therefore, 2 issues need to be addressed with regard to assessment of synovial MSCs in the preculture period: how to expand the synovial cells to sufficient numbers for clinical relevance, and how to retain the chondrogenic potential of the synovial MSCs to achieve efficacy. In addition, analysis of CD90 expression on synovial MSCs may serve as a useful tool for optimizing chondrogenesis of these cells in cartilage repair.