ABSTRACT
Nowadays, bone constructs elaborated according to tissue engineering principles are being regarded as an ideal choice for the reconstruction of segmental bone defects. In this study, proliferation and bone differentiation of marrow-derived mesenchymal stem cells [MSCs] were compared in different composite scaffolds containing varying morphologies of nano hydroxyapatite [nHAP]. Needle nHAP/PLLA [poly [L-lactide acid]], spherical nHAP/ PLLA and rod nHAP/PLLA scaffolds were prepared and 3D cultures of passaged-3 rat MSCs were established using the scaffolds. The loading of the cells onto the scaffold internal spaces was confirmed with microscopy and their proliferation was determined by MTT assay. To compare the osteogenic differentiation of the cells on the scaffold surfaces, osteogenic 3D cultures were established and kept for 21 days. At the end of this period culture mineralization and relative bone-related gene expression were quantified using the alizarin red quantification assay and semi quantitative RT-PCR analysis respectively. ANOVA was used to compare the data. According to the MTT assays, cells adhered to all the studied scaffold surfaces tended to proliferate. In this respect the microenvironment provided by the needle nHAP/ PLLA appeared much better than that of either the spherical or rod nHAP/PLLA scaffolds [P<0.05]. Similarly, mineralization was observed to be heavier for the needle nHAP/PLLA scaffold compared to the two other composite scaffolds. In addition, the relative expression of coll I, osteocalcin, runx2 and ALP genes all appeared to be significantly higher in the cells cultivated on needle nHAP/PLLA scaffolds versus their spherical and rod counterparts. Overall, needle nHAP/PLLA scaffolds appear to provide the most appropriate matrix for producing bone construct using MSCs
ABSTRACT
The use of co-culture cells as well as extra cellular matrix are among those strategies that have been employed to direct mesenchymal stem cell [MSC] bone differentiation in culture. In this regard, there is no study considering the effects of Matrigel on mesenchymal stem cell [MSC] in vitro bone differentiation. This was the subject of the present study. Human passaged-3 MSCs isolated from the marrow aspirates were seeded on either Matrigel or conventional polystyrene plastic surfaces [as control] for 10 days. To compare the cell proliferation in two cultures, the cell numbers were determined during the cultivation period. For bone differentiation, the confluent cultures from either group were provided with osteogenic medium and incubated for 21 days during which the alkaline phosphates [ALP] activity, culture mineralization and the expression of some bone-related genes were quantified and statistically compared. MTT assay indicated that Matrigel-cultivated cells underwent statistically less proliferation than polystyrene-cultivated cells [P<0.05]. Regarding the osteogenic differentiation, ALP activity was significantly high in Matrigel versus plastic cultures. Calcium deposition in Matrigel cultures tended to be significantly extensive compared with that of control cultures [2.533 +/- 0.017 versus 0.607 +/- 0.09 mM]. Furthermore, according to the semi-quantitative RT-PCR analysis, compared with polystyrene plastic surface, Matrigel seemed to provide a microenvironment in which human MSC expressed osteocalcin and collagen I genes in a significantly higher level. Collectively it seems that Matrigel could be considered as an appropriate matrix for MSC osteogenic differentiation
Subject(s)
Humans , Proteoglycans , Mesenchymal Stem Cells , Cell Proliferation , In Vitro TechniquesABSTRACT
Bones constructed by tissue engineering are being considered as valuable materials to be used for regeneration of large defects in natural bone. In an attempt to prepare a new bone construct, in this study, proliferation and bone differentiation of marrow-derived mesenchymal stem cells [MSCs] on our recently developed composite scaffolds of nano-, micro-hydroxyapatite/poly[l-lactic acid] were compared with pure poly[l-lactic acid] scaffolds. For this purpose, some passaged-3 rat MSCs were three-dimensionally cultivated on the scaffold surfaces and their morphology was observed with scanning electron microscopy. Cell proliferations on different scaffolds were examined by MTT assays. Osteogenic cultures were established with the scaffolds loaded with MSCs for 21 days at the end of which culture mineralization; the cell alkaline phosphatase [ALP] Level and the relative expression of selected bone specific genes were quantified and compared to each other. Our results indicated that the cells having been adhered and assumed spherical morphology were able to proliferate in all studied scaffolds. The microenvironment provided by nano-scaffolds appeared much better medium than those of micro-scaffolds and pure PLLA [P < 0.05]. The osteogenesis assays indicated to the superiority of nanoscaffolds in supporting MSCs undergoing bone differentiation, which was reflected in high cellular ALP levels, increased bone-related gene expression and enhanced culture mineralization. Collectively, the bone construct prepared with nano-hydroxyapatite/ poly [llactic acid] scaffold and proliferated MSCs would be suitable candidate for use in bone regenerative medicine