A fine structure study on osteogenesis of mesenchymal stem cells cultured with a 3D collagen scaffold

We cultured human bone-derived mesenchymal stem cell(MSC)s (HMS0014:Yub621b) on a 3D collagen gel scaffold (Cellmatrix Type I-A) under non-inducing and inducing conditions in vitro. Conventional transmission electron microscopy (TEM) was performed for studying turnover of the MSCs and extracellular matrix (ECM) scaffold. The non-induced MSCs were fibroblast-like cells containing a variable number of membranous organelles and paraplasmic vesicles, and were characterised by elongated cell processes to ensheath reticular fibres; the Cellmatrix gel formed an interwoven fibrillar network in the ECM. Under inducing condition, MSCs were osteoinduced to differentiate into plurimorphic Ob-like cells extending thin elongated processes to make a cellular meshwork in the ECM scaffold. The fine structure study identified that osteoconduction was initiated with contact osteogenesis on the Cellmatrix collagen networks. A temporo-spatial replacement of fine Cellmatrix fibrils by collagen type I fibrils, and matrix vesicleand appositional collagen-mediated mineralisation were found in the ECM scaffold. Results from this study suggest that mature HMS0014 Ob-like cells actively regulate the microenvironment to deposit osteoid tissue by the guided bone regeneration method (GBR) concepts. Introduction Many previous in vitro studies of culturing mesenchymal stem cell(MSC)s on the titanium (Ti) dental implant (IP) have shown that MSCs differentiated into osteogeneic osteoblast (Ob)-like cells, which commenced osteoconduction in direct bone-to-IP contact (BIC) formation at substrate surfaces of the IP under induction conditions. The mature Ob-like cells were secretory cells exhibiting early bone cell phenotype and late-stage osteogeneic differentiation [1-3]. In a recent scanning electron microscopy (SEM) study, we have observed that small polygonal human bone-derived HMS0014:Yub621b (fibroblastlike MSC, bone, human; Riken BRC, Tsukuba, Japan) cells proliferated and differentiated into large flat and pleomorphic cells sending out many cellular processes to attach on titanium (Ti) discs within 180 min culture under osteoinduction [4]. Findings of our histochemical studies revealed that Ob-like HMS0014 cells were activated to initiate osteogenic response since day 1 and thereby mineralising tissue being markedly deposited with calcification loci between days 7 and 14 of experiment. In addition, we recognised that ECM mineralisation significantly progressed in particular in 3D cultures [1,4-6]. The establishment of BIC interfaces was osteoinduction of the pre-Ob cells followed by cement line sedimentation and appositional hard tissue growth to induce osteogenesis phenomena at the substrate surface of the implant [2,7-11]. Furthermore, there have many studies revealed that seeding osteogenic MSCs on 3D scaffold or IPs coated with collagen could enhance cell-biomaterial interactions to promote the growth, differentiation and osteoconduction on Ti dental IPs [1,5,7,1215]. We preliminary estimated that Cellmatrix Type I-A fibrillar component formed an interwoven scaffold network for hard tissue formation by MSCs to surround Ti-IP test samples [11]. In the present study, therefore, we performed a transmission electron microscopy (TEM) examination of HMS0014: Yub621b cells 3D-cultured with Cellmatrix Type I-A gel in vitro, and focused on turnover in Ob-like cells and mineralisation of the ECM scaffold. Materials and methods Immature HMS0014:Yub621b (fibroblast-like MSCs, bone, human; Riken BRC, Tsukuba, Japan) cells were filtrated, centrifuged, and then were incubated and maintained in POWEREDBY10 (GlycoTechnica Ltd., Sapporo, Japan) supplemented with 1% antibiotic-antimycotic agent (100units/mL penicillin + 100μg/mL streptomycin; Nacalai Tesque, Kyoto, Japan) in cell culture 75cm2 flasks (TPP, Switzerland) at 37°C in humid air with 5% CO2 for 72h. Tissue engineering of MSCs on 3D collagen gel scaffold 100mm plastic tissue culture dishes (IWAKI, Tokyo, Japan) were paved with Cellmatrix Type I-A gel (base layer), paved with a layer of cell clusters containing immature HMS0014 cells in Cellmatrix Type I-A collagen gel (top layer, cell layer), and then overlaid with POWEREDBY10 (added with ascorbic acid+βglycerophosphate+dexametazone; overlay medium) hence the cells were induced to different into osteogeneic Ob-like cells on the Cellmatrix scaffold in a 3D microenvironment (1×106cells/mL; humid 5% CO2/37°C, for 21 days) according to the “Collagen Gel Embedded Correspondence to: Michiko Nakatsuka, Department of Oral Anatomy, Osaka Dental University, Osaka, Japan, Tel: +81-(0)72-864-3053; Fax: +81(0)72-864-3153; E-mail: [email protected]