For treatment of articular cartilage injury, one of the promising procedures is the transplantation of autologous cultured chondrocytes (1). However, surgical invasion of normal articular cartilage and limited ex vivo expansion of the chondrocytes lead to difficulties in repairing large defects. Mesenchymal stem cells (MSCs) have been a fascinating source for use in regenerative medicine because they can be harvested in a less invasive manner. Moreover, MSCs are easily isolated and expanded, with multipotential capabilities, including chondrogenesis (2, 3).
An MSC is defined as being derived from mesenchymal tissue and having the functional capacity for self-renewal, commonly identified by colony-forming unit fibroblast assay (4) and generation of a number of differentiated progeny (5). Increasing evidence suggests that postnatal stem cells are not exclusive to bone marrow, but also are present in various other tissues. We previously compared MSCs derived from bone marrow, synovium, periosteum, adipose tissue, and muscle, demonstrating that synovium was a better cell source for MSCs with regard to cartilage regeneration, in that synovium-derived MSCs had a greater proliferative capacity and chondrogenic potential (6).
Synovium is a thin layer of tissue that lines the joint space and covers a subsynovium. Depending on its anatomic position, subsynovium comprises either a fibrous or an adipose connective tissue. There have been other reports describing human synovium–derived MSCs; however, details regarding the harvest site of synovium and the histologic characteristics were not mentioned (7, 8). Most likely, synovium with subsynovium was used in these studies, because separation of only the synovium layer from the subsynovial tissue is difficult. Our first aim in the present study was to distinguish MSCs by their sources, that is, synovium with fibrous subsynovium, referred to as fibrous synovium, and synovium with adipose subsynovium, referred as adipose synovium.
MSCs derived from adipose synovium, also commonly called the infrapatellar fat pad, have been reported to have multidifferentiation potential. MSCs of the adipose synovium have been regarded as similar to liposuction-derived cells (9), except that the infrapatellar fat pad is covered with synovium. Our second aim in the present study was to identify whether infrapatellar fat pad–derived cells are more closely related to fibrous synovium–derived cells than to subcutaneous fat–derived cells.
In this study, we collected fibrous synovium, adipose synovium, and subcutaneous fat and performed patient-matched quantitative comparisons of the properties of the 3 MSC populations. The properties examined were surface epitopes, proliferative capacity, cloning efficiency, and chondrogenic, osteogenic, and adipogenic differentiation potentials. The goal of this study was to characterize the suitability of fibrous synovium–derived MSCs as compared with adipose synovium– or subcutaneous fat–derived MSCs for cartilage regeneration, from the standpoint of the properties of each MSC population and the accessibility of the MSC sources.
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- PATIENTS AND METHODS
In this study, we compared fibrous synovium–, adipose synovium–, and subcutaneous fat–derived cells from the perspective of common properties of MSCs. In the 3 populations, epitope profiles of the cells were similar, in that the rate of positivity for CD34 and CD45 (hematopoietic cell markers) was low and the rate of positivity for CD44 (hyaluronan receptor) and CD105 (SH-2) was high. These results coincide with the phenotypic properties of bone marrow–derived MSCs (3, 6, 10, 18, 25).
Interestingly, the rate of STRO-1 positivity in fibrous synovium– and adipose synovium–derived cells was higher than that in subcutaneous fat–derived cells. STRO-1 was originally reported to identify colony-forming osteogenic precursor cells isolated from bone marrow (26) and has shown some promise for use in immunophenotyping MSCs (27). The rate of CD106 (VCAM-1) positivity in fibrous synovium– and adipose synovium–derived cells was 5%, which was higher than that in subcutaneous fat–derived cells (1%). VCAM-1 is a cell-surface glycoprotein that is produced by cytokine-activated endothelium, and is expressed primarily on lining layer cells in synovial tissue (28).
In contrast, the rate of CD10 positivity in subcutaneous fat–derived cells was 40%, which was higher than that in fibrous synovium– and adipose synovium–derived cells (10%). CD10 is also known as common acute lymphocytic leukemia antigen or human membrane–associated neutral endopeptidase. Colter et al previously demonstrated that single-cell–derived colonies of bone marrow–derived MSCs contained 3 morphologically distinct cell types: large flat cells, small spindle-shaped cells, and extremely small, rapidly dividing cells, and showed that samples enriched for the small and extremely small cells had a greater ability for multipotential differentiation than did samples enriched for the large cells. Those authors found that CD10 was a negative marker for small and extremely small cells (18). In this study, fibrous synovium– and adipose synovium–derived cells expressed lower levels of CD10, showed increased proliferation, and had higher chondrogenic and osteogenic potential than did subcutaneous fat–derived cells, which seems to support the findings in bone marrow–derived MSCs reported by Colter et al.
Several reports have described MSCs derived from human adipose tissue. To harvest the cells, great amounts of liposuction tissue were collected, digested with collagenase, separated by stromal–vascular fraction, and expanded (29–31). The processed lipoaspirate-derived cells contaminate endothelial, smooth muscle, and pericyte cell populations (31). In our study, subcutaneous fat–derived cells lacked robust chondrogenic activity. We collected only an ∼100-mg fat tissue, and after digestion, the cells were plated without gradient separation. The quantity of tissue and procedure for fractionation may explain the difference in properties observed in the adipose-derived cells in this study.
MSCs derived from the infrapatellar fat pad (adipose synovium) have also been described (9, 32). Dragoo et al (9) regarded infrapatellar fat pad–derived cells as adipose-derived cells; however, the infrapatellar fat pad is composed of synovium and subsynovial adipose tissues. In our study, the results from morphologic study of infrapatellar fat pad tissue, morphologic study of expanded cells, examination of surface epitopes, and studies of the proliferation, colony-forming efficiency, and chondrogenetic and osteogenetic potential indicate that adipose synovium–derived cells are more similar to fibrous synovium–derived cells than to subcutaneous fat–derived cells.
We evaluated the properties of MSCs in the 3 populations in young and elderly donors separately, because we had initial concerns that synovium-derived MSCs from elderly donors might lack the ability for expansion and differentiation. There are several reports describing the influence of aging on the properties of bone marrow–derived MSCs, and this topic remains controversial. Some studies have shown that aging does not affect colony-forming efficiency (33–35), adipogenesis (34), and calcification (33, 34, 36). In contrast, others have reported that aging affects the proliferative capacity at passage 1 (36) as well as the chondrogenic (36), osteogenic (35, 37), and adipogenic (36) differentiation ability of bone marrow–derived MSCs.
Our previous study indicated no obvious differences between bone marrow–derived MSCs from young and elderly donors in terms of the yields of cells at passage 0, the colony-forming efficiency at passage 0, surface-cell antigens, and chondrocyte, adipocyte, and osteoblast differentiation potentials. However, the proliferative ability of passage 1 cells decreased with age, with results observed as a decrease in cell numbers per colony (10). Apparently, this discrepancy can be attributable to the differences in donor status, site, differentiation protocol, and evaluation method. For example, D'Ippolito et al demonstrated that the number of MSCs with osteogenic potential decreased during aging. They collected bone marrow from vertebral bodies, switched to osteogenic medium 1 day after plating, and evaluated osteogenic potential as the ratios of alkaline phosphatase–positive colonies (35). We collected bone marrow from the proximal tibiae, switched osteogenic medium 14 days after plating, and evaluated osteogenic potential by the ratios of alizarin red–positive colonies (10).
Oreffo et al demonstrated a significant decrease in the ratio of alkaline phosphatase–positive colonies in elderly donors with osteoporosis as compared with young donors, whereas there was no difference in this ratio between elderly donors with osteoarthritis and young donors (37). Murphy et al demonstrated a significant reduction in chondrogenic and adipogenic activity of bone marrow–derived MSCs from elderly osteoarthritis patients compared with those from younger donors (36). Their chondrogenic medium did not contain bone morphogenetic proteins, whereas ours included bone morphogenetic protein 2, which enhanced the in vitro chondrogenesis of MSCs (13). Furthermore, they evaluated chondrogenic potential by the amount of GAG standardized to DNA content, while we compared cartilage pellet weight. With regard to adipogenesis, Murphy et al plated MSCs at high density, differentiated the MSCs into adipocytes, and quantitated nile red fluorescence standardized to 4′,6-diamidino-2-phenylindole, whereas we plated at low density, formed cell colonies, differentiated cells into adipocytes, and evaluated oil red O–positive colony rates.
This study showed no remarkable differences between young and elderly donors in terms of the proliferative ability and colony-forming efficiency of the cells at passage 1, or the chondrocyte, osteoblast, and adipocyte differentiation potential in each MSC population derived from fibrous synovium, adipose synovium, and subcutaneous fat. Contrary to our initial predictions, the nucleated cell number per tissue weight in the fibrous synovium of elderly donors was larger than that in young donors. Revell et al reported that synovium from elderly patients with osteoarthritis is likely to be fibrous (38), and our results were consistent with this observation (as shown in Figure 1C).
Osteoarthritis comprises a common, age-related heterogeneous group of disorders that are characterized pathologically by focal areas of loss of articular cartilage in synovial joints, associated with varying degrees of osteophyte formation, subchondral bone changes, and synovitis (39). The secondary inflammation in the synovium could alter its cellular composition and could be responsible for changes to the stem cell populations. Detailed pathologic investigation of synovium will be important for clarifying the role of the disease and the role of aging in the characteristics of stem cells derived from the synovium. This general information will be valuable for comparison with other studies.
An age-related decrease in the chondrogenic differentiation potential has been reported in rabbit fibrous synovium (40) and periosteum (41) in an ex vivo organ culture (42). In contrast, De Bari et al demonstrated that the chondrogenic potential of synovium-derived cells was independent of donor age (7), which is similar to our result. These findings suggest that aging may affect the chondrogenic differentiation potential in organ culture but does not affect the cells expanded in vitro.
We demonstrated that both fibrous synovium– and adipose synovium–derived MSCs had a better chondrogenic capacity than did subcutaneous fat–derived MSCs. Given this difference, important biologic questions are raised, and we have developed 2 hypotheses: 1) The observed differences between synovium and subcutaneous fat may be due to differences in the number of ancestral MSCs, or alternatively, 2) the observed differences in chondrogenesis could have arisen as a result of different MSC propensities to follow a chondrogenic pathway, suggesting that the local tissue microenvironment may be directing the “fate” of the MSCs toward a particular lineage.
We also demonstrated that synovium-derived MSCs had a higher osteogenetic ability than did adipose-derived MSCs. Furthermore, single-cell–derived cultures as well as mixed synovial cells showed higher colony-forming efficiency and expansion ability than did those from adipose tissue. These findings may implicate the role of the local tissue microenvironment in directing the “fate” of the MSCs. The adipogenic capacity of MSCs derived from the synovium and those derived from the subcutaneous fat was similar, which would support the first hypothesis described above. However, MSCs derived from adipose tissue could have been preconditioned via the microenvironment, and thus slightly predisposed toward an adipocyte lineage, which would be consistent with the second hypothesis.
The important consideration in tissue engineering is to harvest the greatest amount of MSCs with the highest potential while minimizing the amounts of mesenchymal tissues needed, resulting in less-invasive treatments. Fibrous synovium– and adipose synovium–derived MSCs were similar in terms of their cell morphologic features, epitope profiles, colony-forming efficiency, chondrogenesis, osteogenesis, and adipogenesis potentials. The nucleated cell number per tissue weight was higher in fibrous synovium than in adipose synovium, which may be an advantage of fibrous synovium. However, adipose synovium cells also have an advantage due to their high chondrogenic potential and accessibility, in that sufficient amounts of adipose synovium can be harvested with possibly fewer complications. We therefore conclude that both fibrous synovium and adipose synovium are suitable MSC sources for cartilage regeneration.