Effect of Biphasic Calcium Phosphate Treated with Vascular Endothelial Growth Factor on Osteogenesis and Angiogenesis Gene Expression In Vitro

Abstract

Biodegradable biphasic calcium phosphate (BCP) scaffold holds tremendous potential for bone tissue engineering. It elicits response from cells such as endothelial cells (ECs) that are similar to those elicited by bone. ECs promote bone regeneration by stimulating both neovascularisation and osteogenesis. For an effective coupling of angiogenesis and osteogenesis, vascular endothelial growth factor (VEGF) is required. The aim of this work is to study the effectiveness of VEGF-added-BCP on the expression of osteogenesis and angiogenesis genes in ECs. Commercially obtained rat aortic ECs were cultured in the endothelial-cell growth medium. The cells were treated with three different modalities: BCP- only, VEGF- only, and VEGF-added-BCP. The optimal BCP and VEGF concentrations were determined. The cells were harvested at four different time intervals (day 3, day 7, day 10 and day 14) and were subjected to RNA isolation using RNA extraction kit (analytikjena, Germany). This was followed by performing reverse transcriptase-PCR (RT-PCR) (Qiagen, Germany) to amplify the osteogenesis and angiogenesis-regulated genes. The RT-PCR products were then electrophoresed. The gel image was captured using Image Analyser. Suitable concentration of BCP was 10mg/ml while optimal VEGF was 15ng/ml. Angiogenesis and osteogenesis genes were clearly expressed in ECs in response to treatments. Angiogenesis gene (VEGF) was highly expressed by VEGFonly treatment but showed some changes with added BCP. Osteogenesis genes (BMP-2, ALP, OC and OPN) were shown to be positively affected by both BCP and VEGF. Some genes were expressed at an earlier time interval compared to the other genes depending on the type of treatments. BCP-only treatment induced high expression of early regulating osteogenesis genes (BMP-2 and OPN). Mineralized gene markers (ALP and OC) were however, highly expressed with VEGF-added-BCP treatment. Combination of BCP and VEGF modality on ECs was suggested to initiate osteogenesis and angiogenesis related gene expressions earlier than the other modalities.