In vitro chondrogenesis of human synovium-derived mesenchymal stem cells: Optimal condition and comparison with bone marrow-derived cells

Abstract

There are increasing reports that mesenchymal stem cells (MSCs) are present in various tissues other than bone marrow, including synovium. Here we investigated the optimal conditions for in vitro chondrogenesis of human synovium-derived MSCs and compared these cells with bone marrow-derived MSCs, especially in terms of their chondrogenesis potential. Synovium and bone marrow were harvested from six donors during knee operations for ligament injuries. Digested synovium cells or nucleated cells from bone marrow were expanded clonally. A pellet culture system was used for chondrogenesis, and the best combination of up to three cytokines of the seven assessed. Synovium-derived MSCs plated at a lower density expanded more rapidly. Contrary to previous reports, a combination of TGFbeta and dexamethasone was not sufficient to induce chondrogenesis. However, addition of BMP2 to TGFbeta and dexamethasone dramatically increased cartilage pellet size and the synthesis of cartilage matrix. The cartilage pellets were also analyzed by electron microscopy and immunohistology. DNA content per pellet decreased during chondrogenesis, indicating the pellet increased its size through the accumulation of newly synthesized extracellular matrix. Sequential chondrogenic gene expression was demonstrated by RT-PCR. Synovium-derived MSCs looked similar to the bone marrow-derived MSCs in their surface epitopes and proliferation potential; however, cartilage pellets from synovium were significantly larger than those from bone marrow in patient-matched comparisons. We demonstrated that the combination of TGFbeta, dexamethasone, and BMP2 was optimal for in vitro chondrogenesis of synovium-derived MSCs and that the synovium-derived MSCs have a greater chondrogenesis potential than bone marrow-derived MSCs.

Methylation status of CpG islands in the promoter regions of signature genes during chondrogenesis of human synovium–derived mesenchymal stem cells

“…Pellets prepared in the same way were placed in 6-12 test tubes on standing Styrofoam, and the medium was changed every 3-4 days until day 21, and genomic DNA was then isolated from each pellet. Chondrocytic differentiation was ascertained both by histologic analysis and by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis of pellets cultured in parallel, as described previously (22,24).…”

Cell-based Meniscal Tissue Engineering: A Case for Synoviocytes

“…Scaffolds may further be defined as ''smart scaffolds'' if they carry with them some type of signal that can direct appropriate tissue formation either from the implanted cell type or from the surrounding resident cellular population. With respect to the delivery of synoviocytes for cartilage (hyaline or fibrocartilage) engineering, a number of different systems have been investigated including hydrogels (alginate [53,66] collagen [51,63,94], and gellan [28]), pellet cultures [63,80], micromasses [3,66], small intestinal submucosa (SIS) [84], hyaluronan-based scaffolds (Hyaff-111; FAB, Abano Terme, Padova, Italy) [56], polyglycolic acid (PGA) [67,74], PGA/poly (L) lactic acid (PLLA) combinations [34], and scaffold-free cell infusions [44,60]. With relatively few studies directly comparing delivery methods, elucidating an optimal carrier or scaffold from the literature alone is difficult.…”

“…Not surprisingly, with three-dimensional culturing systems, SMSCs become more chondrogenic both in appearance and in production of extracellular matrix constituents versus using two-dimensional systems [66]. The earliest demonstration of this was from the examination of synovial cell pellet culture systems [56,72,80]. However, the surgical applicability of small spheroid pellets is somewhat limited for many large chondral applications.…”

“…Where Sakimura et al [74] has found a combination of the two growth factors results in higher production of glycosaminoglycans from SMSCs cultured on PGA, Bilgen et al [12] found no such advantage. Also frequently investigated is the use of the isoform TGF-b3, which, in combination with other factors, is a powerful chondrogenic stimulus; a large body of work has now started to use a cocktail of TGF-b3 with bone morphogenic proteins (BMPs) and the synthetic glucocorticoid dexamethasone to consistently promote synovial-based chondroid matrix production [50,72,80,94].…”

“…However, the initial cell growth media used by those investigators contained 10% FBS, the BMP-2 concentration used was lower, and the chondrogenic media they used contained dexamethasone [28]. Despite the controversy surrounding the concomitant use of the glucocorticoid dexamethasone and BMPs, numerous recent attempts to use SMSCs for articular cartilage engineering have used the combination of BMP-2, TGF-b3, and dexamethasone as a chondrogenic media in which to immerse three-dimensionally cultured synoviocytes, whether in pellets or collagen gels, all with promising results [50,63,80,94]. Because most of these strategies are geared toward the production of a hyalinelike articular cartilage, typical outcome measures include signaling or production of collagen Type II, aggrecan, and the activation of SOX-9.…”