The effect of mesenchymal populations and vascular endothelial growth factor delivered from biodegradable polymer scaffolds on bone formation.

The capacity to deliver, temporally, bioactive growth factors in combination with appropriate progenitor and stem cells to sites of tissue regeneration promoting angiogenesis and osteogenesis offers therapeutic opportunities in regenerative medicine. We have examined the bone regenerative potential of encapsulated vascular endothelial growth factor (VEGF(165)) biodegradable poly(DL-lactic acid) (PLA) scaffolds created using supercritical CO(2) fluid technology to encapsulate and release solvent-sensitive and thermolabile growth factors in combination with human bone marrow stromal cells (HBMSC) implanted in a mouse femur segmental defect (5 mm) for 4 weeks. HBMSC seeded on VEGF encapsulated PLA scaffolds showed significant bone regeneration in the femur segmental defect compared to the scaffold alone and scaffold seeded with HBMSC as analysed by indices of increased bone volume (BV mm(3)), trabecular number (Tb.N/mm) and reduced trabecular separation (Tb.Sp.mm) in the defect region using micro-computed tomography. Histological examination confirmed significant new bone matrix in the HBMSC seeded VEGF encapsulated scaffold group as evidenced by Sirius red/alcian blue and Goldner's trichrome staining and type I collagen immunocytochemistry expression in comparison to the other groups. These studies demonstrate the ability to deliver, temporally, a combination of VEGF released from scaffolds with seeded HBMSC to sites of bone defects, results in enhanced regeneration of a bone defect.

The application of human bone marrow stromal cells and poly(dl-lactic acid) as a biological bone graft extender in impaction bone grafting.

Concerns over disease transmission, high costs and limited supply have led to interest in synthetic grafts in the field of impaction bone grafting (IBG). Poly(DL-lactic acid) (PLA) grafts are attractive alternatives due to their biocompatibility, established safety and versatile manufacturing process. This study examined the potential of PLA scaffolds augmented with human bone marrow stromal cells (HBMSCs) in IBG. In vitro and in vivo studies were performed on impacted morsellised PLA seeded with HBMSC and compared to PLA alone. In vitro samples were incubated under osteogenic conditions and in vivo samples were implanted subcutaneously into severely compromised immunodeficient mice, for 4 weeks. Biochemical, histological, mechanical and 3D micro-computed tomography analyses were performed. HBMSC viability, biochemical activity and histological evidence of osteogenic cellular differentiation, post-impaction were observed in vitro and in vivo in PLA/HBMSC samples compared to impacted PLA alone. In vitro PLA/HBMSC samples demonstrated evidence of mechanical enhancement over PLA alone. In vivo studies showed a significant increase in new bone and blood vessel formation in the PLA/HBMSC constructs compared to PLA alone. With alternatives to allograft being sought, these studies have demonstrated PLA/HBMSC living composites, to be a potential prospect as a biological bone graft extender for future use in the field of IBG.

Controlling protein release from scaffolds using polymer blends and composites.

We report the development of three protein loaded polymer blend and composite materials that modify the release kinetics of the protein from poly(dl-lactic acid) (P(dl)LA) scaffolds. P(dl)LA has been combined with either poly(ethylene glycol) (PEG), poly(caprolactone) (PCL) microparticles or calcium alginate fibres using supercritical CO(2) (scCO(2)) processing to form single and dual protein release scaffolds. P(dl)LA was blended with the hydrophilic polymer PEG using scCO(2) to increase the water uptake of the resultant scaffold and modify the release kinetics of an encapsulated protein. This was demonstrated by the more rapid release of the protein when compared to the release rate from P(dl)LA only scaffolds. For the P(dl)LA/alginate scaffolds, the protein loaded alginate fibres were processed into porous protein loaded P(dl)LA scaffolds using scCO(2) to produce dual release kinetics from the scaffolds. Protein release from the hydrophilic alginate fibres was more rapid in the initial stages, complementing the slower release from the slower degrading P(dl)LA scaffolds. In contrast, when protein loaded PCL particles were loaded into P(dl)LA scaffolds, the rate of protein release was retarded from the slow degrading PCL phase.

Regenerative medicine, resource and regulation: lessons learned from the remedi project.

The successful commercialization of regenerative medicine products provides a unique challenge to the manufacturer owing to a lack of suitable investment/business models and a constantly evolving regulatory framework. The resultant slow translation of scientific discovery into safe and clinically efficacious therapies is preventing many potential products from reaching the market. This is despite of the need for new therapies that may reduce the burden on the world's healthcare systems and address the desperate need for replacement tissues and organs. The collaborative Engineering and Physical Sciences Research Council (EPSRC)-funded remedi project was devised to take a holistic but manufacturing-led approach to the challenge of translational regenerative medicine in the UK. Through strategic collaborations and discussions with industry and other academic partners, many of the positive and negative issues surrounding business and regulatory success have been documented to provide a remedi-led perspective on the management of risk in business and the elucidation of the regulatory pathways, and how the two are inherently linked. This article represents the findings from these discussions with key stakeholders and the research into best business and regulatory practices.

The effect of the delivery of vascular endothelial growth factor and bone morphogenic protein-2 to osteoprogenitor cell populations on bone formation.

Regenerating bone tissue involves complex, temporal and coordinated signal cascades of which bone morphogenic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF(165)) play a prominent role. The aim of this study was to determine if the delivery of human bone marrow stromal cells (HBMSC) seeded onto VEGF(165)/BMP-2 releasing composite scaffolds could enhance the bone regenerative capability in a critical sized femur defect. Alginate-VEGF(165)/P(DL)LA-BMP-2 scaffolds were fabricated using a supercritical CO(2) mixing technique and an alginate entrapment protocol. Increased release of VEGF(165) (750.4+/-596.8 rho g/ml) compared to BMP-2 (136.9+/-123.4 r hog/ml) was observed after 7-days in culture. Thereafter, up till 28 days, an increased rate of release of BMP-2 compared to VEGF(165) was observed. The alginate-VEGF(165)/P(DL)LA-BMP-2+HBMSC group showed a significant increase in the quantity of regenerated bone compared to the alginate-VEGF(165)/P(DL)LA-BMP-2 and alginate/P(DL)LA groups respectively in a critical sized femur defect study as indices measured by microCT. Histological examination confirmed significant new endochondral bone matrix in the HBMSC seeded alginate-VEGF(165)/P(DL)LA-BMP-2 defect group in comparison to the other groups. These studies demonstrate the ability to deliver a combination of HBMSC with angiogenic and osteogenic factors released from biodegradable scaffold composites enhances the repair and regeneration of critical sized bone defects.

Mammalian cell survival and processing in supercritical CO(2).

We demonstrate that mammalian cells can survive for 5 min within high-pressure CO(2)(.) Cell survival was investigated by exposing a range of mammalian cell types to supercritical CO(2) (scCO(2)) (35 degrees C, 74 bar; 1 bar = 100 kPa) for increasing exposure and depressurization times. The myoblastic C2C12 cell line, 3T3 fibroblasts, chondrocytes, and hepatocytes all displayed appreciable but varying metabolic activity with exposure times up to 1 min. With depressurization times of 4 min, cell population metabolic activity was >/=70% of the control population. Based on survival data, we developed a single-step scCO(2) technique for the rapid production of biodegradable poly(dl-lactic acid) scaffolds containing mammalian cells. By using optimum cell-survival conditions, scCO(2) was used to process poly(dl-lactic acid) containing a cell suspension, and, upon pressure release, a polymer sponge containing viable mammalian cells was formed. Cell functionality was demonstrated by retention of an osteogenic response to bone morphogenetic protein-2 in C2C12 cells. A gene microarray analysis showed no statistically significant changes in gene expression across 4,418 genes by a single-class t test. A significance analysis of microarrays revealed only eight genes that were down-regulated based on a delta value of 1.0125 and a false detection rate of 0.