The production of living, tissue engineered, bone graft from progenitor cells using nanotechnology

The principal aim of this research was the development of a technique (based upon the effects of nanoscale topography) that facilitates the in vitro expansion of bone graft for subsequent implantation. Nanoscale topography increases the bioactivity of a material and stimulates specific responses at the molecular level (third generation biomaterial properties). Nanoscale topography thus confers these third generation properties upon biomaterials that are otherwise first generation (bioinert) or second generation (bioresorbable or bioactive) in nature. Two topographies (nanopits and nanoislands) were embossed into the clinically licensed bioresorbable polymer Polycaprolactone (PCL). A protocol was developed which enabled three dimensional cell culture using double-sided embossing of substrates, seeding of both sides, and vertical positioning of the substrates during cell culture. Human bone marrow was harvested and the mononuclear cell fraction culture expanded. These human bone marrow cells (HBMCs) were used for cellular analysis of substrate bioactivity. In addition, acellular analysis of substrate patterning and degradation was also performed. The osteogenic behaviour (and cell line specificity) was demonstrated using alizarin red staining, immunohistochemistry, real-time polymerase chain reaction (rtPCR), and quantitative PCR (qPCR). The osteogenicity of PCL was increased by the presence of nanotopography, and by the incorporation of hydroxyapatite (HA) into the PCL forming a hydroxyapatite-PCL composite (HAPCL). The performance of these substrates was compared to exposure to bone morphogenic protein 2 (BMP2), and the use of osteogenic media. The protocol from shim production to bone marrow harvesting and vertical cell culture on nanoembossed PCL has been shown to be reproducible and potentially applicable to economical larger scale production