ABSTRACT
Objective To construct the three-dimensional (3D) fluid model at the physiological level of shear stresses and study the effects of fluid shear stress (FSS) on adhesion, differentiation and mechanical sensitivity of osteoblasts. Methods The MC3T3-E1 osteoblasts cultured on β-tricalcium phosphate (β-TCP) scaffolds were subjected to various FSSs in the perfusion flow chamber for 6 hours to compare cell adhesion in FSS-loading groups and control group. Nitric oxide (NO) and alkaline phosphatase (ALP) were detected to compare mechanical sensitivity and cell differentiation. The FSS magnitude and distributions corresponding to various fluid rates were calculated with nonlinear fluid-structure coupling analysis. Results Cell adhesion rate was up to 74%~81% when the average FSS magnitude was lower than 0.4 Pa, but reduced to 60.22% when the average FSS was 0.41 Pa. The NO production rate reached the maximal concentration after loading for 5 min, then significantly reduced at 15 min, and gradually diminished to none at 30 min. ALP level significantly increased (P0.05) with the increase of shear stress. Conclusions Majority of the cells kept a normal adherence to the scaffold at the physiological level of shear stresses. The mechanical sensitivity of the cells under 3D condition was dependent on the FSS rate, which was consistent with two-dimensional (2D) condition. When the average FSS was lower than 0.304 Pa in the scaffold, FSS could significantly promote cell differentiation, but no significant change in cell differentiation could be found when FSS was higher than 0.304 Pa. The present study is expected to accelerate the realization of bone tissue engineering.
ABSTRACT
Recently, many researches have been carried out on the production of artificial tissue using tissue engineering. However, studies on fat tissue are still insufficient. The purpose of this study was to examine that alginate sponge can be used as a three-dimensional scaffold for the culture of preadipocytes compared with fibroblasts, and that preadipocytes can differentiate into mature adipocytes in this sponge. The 3T3-L1 preadipocytes and the 3T3 fibroblasts were separately cultured in three-dimensional alginate sponge for 14 days. The morphology of cell and sponge, cell proliferation rate, and glycerol phosphate dehydrogenase activity were evaluated at the indicated periods. The results were as follows; 1. The alginate sponge showed a highly porous, well-interconnected pore structure and the sizes of pores were from 100 to 400micrometer. 2. The fibroblasts in sponge exhibited spindle shape with long irregular fibers on the 7th day and there was no oil-red O stained cell until 14 days. However, the preadipocytes in alginate sponge were round and some of cells transformed into mature fat cells which were stained with oil-red O after 14 days. 3. The proliferation rates of preadipocyte group were increased gradually during the culture period, but lower than those of fibroblast group(P< 0.05). 4. The glycerol phosphate dehydrogenase activities of preadipocyte group were significantly higher than those of fibroblast group during the culture period(P< 0.05), and the activities of 14 day-cultured preadipocytes were about 30 times higher than those of 7 day-cultured preadipocytes. The results suggest that alginate sponge, which has fixed shape and porosity, is adequate three-dimensional scaffolds for culture of fibroblast and preadipocyte. In addition, preadipocytes could be well proliferated and differentiated into adipocyte in the alginate sponge.
Subject(s)
Adipocytes , Cell Proliferation , Fibroblasts , Glycerol , Oxidoreductases , Porifera , Porosity , Tissue EngineeringABSTRACT
Chitosan is a biodegradable natural polymer that has been demonstrated its ability to improve wound healing, and calcium metaphosphate(CMP) is a unique class of phosphate minerals having a polymeric structure. In this study, chitosan/CMP and platelet derived growth factor(PDGF-BB) loaded chitosan/CMP sponges were developed, and the effect of the sponges on bone regeneration and their possibility as scaffolds for bone formation by three-dimensional osteoblast culture were examined. PDGF-BB loaded chitosan/CMP sponges were prepared by freeze-drying of a mixture of chitosan solution and CMP powder, and soaking in a PDGF-BB solution. Fabricated sponge retained its 3-dimensional porous structure with 100-200micrometer pores. The release kinetics of PDGF-BB loaded onto the sponge were measured in vitro with 125I-labeled PDGF-BB. In order to examine their possibility as scaffolds for bone formation, fetal rat calvarial osteoblastic cells were isolated, cultured, and seeded into the sponges. The cell-sponge constructs were cultured for 28 days. Cell proliferation, alkaline phosphatase activity were measured at 1, 7, 14 and 28 days, and histologic examination was performed. In order to examine the effect on the healing of bone defect, the sponges were implanted into rat calvarial defects. Rats were sacrificed 2 and 4 weeks after implantation and histologic and histomorphometrical examination were performed. An effective therapeutic concentration of PDGF-BB following a high initial burst release was maintained throughout the examination period. PDGF-BB loaded chitosan/CMP sponges supported the proliferation of seeded osteoblastic cells as well as their differentiation as indicated by high alkaline phosphatase activities. Histologic findings indicated that seeded osteoblastic cells well attached to sponge matrices and proliferated in a multi-layer fashion. In the experiments of implantation in rat calvarial defects, histologic and histomorphometric examination revealed that chitosan/CMP sponge promoted osseous healing as compared to controls. PDGF-BB loaded chitosan/CMP sponge further enhanced bone regeneration. These results suggested that PDGF-BB loaded chitosan/CMP sponge was a feasable scaffolding material to grow osteoblast in a three-dimentional structure for transplantation into a site for bone regeneration.