BMSCs are a kind of inducible osteogenic precursor cells, and vitamin C, β-sodium glycerophosphate, and dexamethasone are the essential condition for osteogenic differentiation of BMSCs[9]. Vitamin C promotes BMSCs to synthesize collagen, contributing to calcification, and regulates ALP activity and non-collagen matrix protein synthesis[9]. β-sodium glycerophosphate can provide phosphate ions for the functional motion of osteogenic cells, serves as ALP substrate to induce and activate ALP, promotes organic phosphorus transformation into inorganic phosphorus, as well as calcium salt deposition and calcification, and therefore is the essential condition of induced mineralized nodules of BMSCs[10-12]. Maniatopoulos et al[10] reported that mineralized nodules formed only under the condition of dexamethasone addition into BMSCs cultured in vitro and that the formation time, shape, and amounts of mineralized modules are dependent on dexamethasone use time and dose. Dexamethasone at some a concentration greatly enhances ALP activity of BMSCs, regulates osteoblast-like cells to synthesize insulin-like growth factors, increases the synthesis of type I collage and osteopontin messenger RNA, and induces osteoblastic transformation of BMSCs[13-14].
In the present study, induced and non-induced cells exhibited similar growth curves, i.e., typical “S” shape. BMSCs reached a growth peak at about 7 days and induced cells peaked at 8 days. After induction, the proliferative capacity of BMSCs is slightly decreased. This occurs because dexamethasone inhibits cellular proliferation while it promotes osteogenic differentiation of BMSCs and enhances ALP activity. Cell sheet engineering is a novel technique that uses temperature-responsive culture dish to harvest and transfer seed cells. Yamada et al found that temperature-responsive culture dishes can be made by covalently bonding temperature-responsive polymer, i.e., poly(N-isopropylacrylamide) (PIPAAm), to the surface of common polyethylene culture dish[15]. When BMSCs in the temperature-responsive culture dishes are cooled below the critical temperature 32 ℃, they are gradually detached from the surface of culture flask and form complete BMSC sheets. Under the condition of 37 ℃, 5% CO2 saturated humidity, the surface of culture medium is hydrophobic, and BMSCs can adhere, expand, and proliferate on the surface of temperature-responsive culture dish. Under the condition of
20 ℃, 5% CO2 saturated humidity, PIPAAm becomes from hydrophobic to hydrophilic, and a hydration shell forms between culture dish surface and cultured cells, thus cells would shed off from the surface of culture medium and form cell sheets. Evidence exists that when the thickness of temperature-responsive polymer ranges 15-20 nm, cells can proliferate on the surface of culture dish, but temperature-responsive polymer with a thickness of over 30 nm would influence cell adhesion and proliferation[16].
BMSCs harvested by cell sheet engineering appear to be a laminar structure containing extracellular matrix. BMSC sheets retain important cell surface proteins, such as ion channel and connectin, which enables better intercellular signal transmission and keep functional coordination[17]. Cells in the BMSC sheets are compactly arranged, similar to osteoblast arrangement during the formation of natural bone; at the same time, the stress produced by cell sheet contraction regulates the polarization arrangement of BMSCs[18]. With mineral deposition and calcification surrounding the cells, a lamellar bone-like structure forms. Within a time period after implantation of BMSC sheets into recipient site, the necessary nutrition is primarily from the nutritional ingredient diffused from peripheral tissue fluid; the thickness of cell sheets should be less than 80 µm to avoid cellular apoptosis[19]. Biodegradable scaffold materials are wrapped with homotypic multi-layer BMSC sheets and then implanted into recipient site, which would contribute to the formation of tissue-engineered bone with lamellar bone-like structure. The present study isolated, cultured seed cells BMSCs, and successfully prepared BMSC sheets, providing good evidence for construction of functional tissue-engineered bone. Density gradient centrifugation is a technique that enables effective harvest of BMSCs and induced osteogenic induction. Cell sheet engineering for harvesting and transferring BMSCs avoids the cell damage caused by trypsin digestion, retains massive extracellular matrix, and greatly enhances cell availability and the activity of transferred cells. Cell sheet engineering in conjunction with bone tissue engineering are promising to construct large blocks of tissue-engineered bone containing lamellar bone-like structure.
