Development of in vivo μCT evaluation of neovascularisation in tissue engineered bone constructs

Due to an increasing aging population the need for innovative approaches to aid skeletal repair and reconstruction is a significant socio-economic increasing problem. The emerging discipline of tissue engineering has sort to augment the growth and repair of bone loss particularly in areas of trauma, degeneration and revision surgery. However, the initiation and development of a fully functional vascular network are critical for bioengineered bone to repair large osseous defects, whether the material is osteosynthetic (poly (d,l)-lactic acid, PLA) or natural bone allograft. Quantification and three-dimensional visualization of new vessel networks remain a problem in bone tissue engineering constructs. A novel technique utilising a radio-opaque dye and micro-computed tomography (μCT) has been developed and applied to study angiogenesis in an impaction bone graft model. Tissue-engineered constructs combining human bone marrow stromal cells (HBMSC) with natural allograft and synthetic grafts (PLA) were impacted and implanted into the subcutis of MF-1 nu/nu mice for a period of 28 days. Microfil consisting of radio-opaque polymer was perfused through the mice and scanned using a Bench Top CT system for micro-computed tomography. Analysis of three-dimensional μCT reconstructions demonstrated an increase in vessel volume and vessel number in the impacted scaffolds/HBMC compared to scaffolds alone. Vessel volume: allograft/HBMSC = 0.57 mm3 ± 0.19; allograft = 0.04 mm3 ± 0.04; PLA/HBMSC = 1.19 mm3 ± 0.31; and PLA = 0.12 mm3 ± 0.01. Penetrating vessel number: allograft/HBMSC = 22.33 ± 3.21; allograft = 3.67 ± 1.153; PLA/HBMSC = 32.67 ± 8.33; and PLA = 7.67 ± 3.06. Type 1 collagen and von Willebrand factor immunohistochemistry in scaffold/HBMSC constructs indicated the osteogenic cell phenotype, and new blood vessel formation respectively. Contrast-enhanced 3D reconstructions facilitated the visualization and quantification of neovascularisation. This novel technique has been used to demonstrate neovascularisation in impacted tissue engineered constructs providing a facile approach with wide experimental application.