Resorbable bioceramics are attractive for medical applications such as bone substitution. Biochemical analysis on cells cultured on these biomaterials is vital to predict the impact of the materials in vivo and RNA ex...Resorbable bioceramics are attractive for medical applications such as bone substitution. Biochemical analysis on cells cultured on these biomaterials is vital to predict the impact of the materials in vivo and RNA extraction is an essential step in gene expression study using RT-qPCR. In this study, we describe simple modifications to the TRIzol? RNA extraction protocol widely used in biology and these allow high-yield extraction of RNA from cells on resorbable calcium phosphates. Without the modifications, RNA is trapped in the co-precipitated calcium compounds, rendering TRIzol? extraction method infeasible. Among the modifications, the use of extra TRIzol? to dilute the lysate before the RNA precipitation step is critical for extraction of RNA from porous ?-tricalcium phosphate (?-TCP) discs. We also investigate the rationale behind the undesirable precipitation so as to provide clues about the modifications required for other resorbable materials with high application potential in bone tissue engineering.展开更多
In this study, we use a pluripotent mesenchymal stem cell (MSC) model, C3H/10T1/2, to evaluate three calcium phos-phate (CaP) materials, namely the hydroxyapatite (HA), α-tricalcium phosphate (α-TCP) and β-tricalci...In this study, we use a pluripotent mesenchymal stem cell (MSC) model, C3H/10T1/2, to evaluate three calcium phos-phate (CaP) materials, namely the hydroxyapatite (HA), α-tricalcium phosphate (α-TCP) and β-tricalcium phosphate (β-TCP). 10T1/2 cell was chosen as it has advantages over its counterparts in terms of ease of maintenance, free of ethical concerns and also more reproducible results. ALP enzymatic assay, RT-qPCR, DAPI staining and SEM were employed to assess the osteoinductivity of these materials. A good reference material which also acts as a scientific control is necessary for comparisons of results from different experimental batches and hence other materials such as titanium, Nunclon plastic surface, BD Falcon plastic surface and gold coated porous HA were also tested. The results show that ceramics induce a more sustained osteo-differentiation state as compared with plastics. Inductivity was found to be acting in descending order of strength with HA > β-TCP > α-TCP, which is reversed in terms of their impact on proliferation rate (HA TCP) and in vivo osteoinductivity in terms of incidence and quality of bone described previously (HA > β-TCP > α-TCP). These confirm the suitability of using 10T1/2 cells in cell culture assay of osteoinductivity.展开更多
文摘Resorbable bioceramics are attractive for medical applications such as bone substitution. Biochemical analysis on cells cultured on these biomaterials is vital to predict the impact of the materials in vivo and RNA extraction is an essential step in gene expression study using RT-qPCR. In this study, we describe simple modifications to the TRIzol? RNA extraction protocol widely used in biology and these allow high-yield extraction of RNA from cells on resorbable calcium phosphates. Without the modifications, RNA is trapped in the co-precipitated calcium compounds, rendering TRIzol? extraction method infeasible. Among the modifications, the use of extra TRIzol? to dilute the lysate before the RNA precipitation step is critical for extraction of RNA from porous ?-tricalcium phosphate (?-TCP) discs. We also investigate the rationale behind the undesirable precipitation so as to provide clues about the modifications required for other resorbable materials with high application potential in bone tissue engineering.
文摘In this study, we use a pluripotent mesenchymal stem cell (MSC) model, C3H/10T1/2, to evaluate three calcium phos-phate (CaP) materials, namely the hydroxyapatite (HA), α-tricalcium phosphate (α-TCP) and β-tricalcium phosphate (β-TCP). 10T1/2 cell was chosen as it has advantages over its counterparts in terms of ease of maintenance, free of ethical concerns and also more reproducible results. ALP enzymatic assay, RT-qPCR, DAPI staining and SEM were employed to assess the osteoinductivity of these materials. A good reference material which also acts as a scientific control is necessary for comparisons of results from different experimental batches and hence other materials such as titanium, Nunclon plastic surface, BD Falcon plastic surface and gold coated porous HA were also tested. The results show that ceramics induce a more sustained osteo-differentiation state as compared with plastics. Inductivity was found to be acting in descending order of strength with HA > β-TCP > α-TCP, which is reversed in terms of their impact on proliferation rate (HA TCP) and in vivo osteoinductivity in terms of incidence and quality of bone described previously (HA > β-TCP > α-TCP). These confirm the suitability of using 10T1/2 cells in cell culture assay of osteoinductivity.
