John J. A. Barry

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 Goldners 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.

Supercritical carbon dioxide: putting the fizz into biomaterials

This paper describes recent progress made in the use of high pressure or supercritical fluids to process polymers into three-dimensional tissue engineering scaffolds. Three current examples are highlighted: foaming of acrylates for use in cartilage tissue engineering; plasticization and encapsulation of bioactive species into biodegradable polyesters for bone tissue engineering; and a novel laser sintering process used to fabricate three-dimensional biodegradable polyester structures from particles prepared via a supercritical route.

Biocompatibility and osteogenic potential of human fetal femur-derived cells on surface selective laser sintered scaffolds

For optimal bone regeneration, scaffolds need to fit anatomically into the requisite bone defects and, ideally, augment cell growth and differentiation. In this study we evaluated novel computationally designed surface selective laser sintering (SSLS) scaffolds for their biocompatibility as templates, in vitro and in vivo, for human fetal femur-derived cell viability, growth and osteogenesis. Fetal femur-derived cells were successfully cultured on SSLS-poly(d,l)-lactic acid (SSLS-PLA) scaffolds expressing alkaline phosphatase activity after 7days. Cell proliferation, ingrowth, Alcian blue/Sirius red and type I collagen positive staining of matrix deposition were observed for fetal femur-derived cells cultured on SSLS-PLA scaffolds in vitro and in vivo. SSLS-PLA scaffolds and SSLS-PLA scaffolds seeded with fetal femur-derived cells implanted into a murine critical-sized femur segmental defect model aided the regeneration of the bone defect. SSLS techniques allow fabrication of biocompatible/biodegradable scaffolds, computationally designed to fit any defect, providing a template for cell osteogenesis in vitro and in vivo.

Supercritical carbon dioxide foaming of elastomer/heterocyclic methacrylate blends as scaffolds for tissue engineering

This study reports on the supercritical carbon dioxide (scCO2) foaming of rubber toughened heterocyclic methacrylates for potential applications as non-degradable scaffolds in tissue repair and engineering. Porous blends of styrene–isoprene–styrene copolymer elastomer (SIS) and tetrahydrofurfuryl methacrylate (THFMA) at three SIS/THFMA compositions that ranged from methacrylate to elastomer rich were foamed and characterised in terms of their morphological, mechanical and biological properties. The results showed that the foaming factor (FF) was dependent on blend composition and the foaming conditions demonstrating that the process was tuneable. A greater FF, resulting in higher open and total porosities, was obtained for THFMA rich formulations, which were demonstrated by a predominantly open pore structure. Quasi-static and dynamic mechanical analysis (DMA) showed that the foamed SIS/THFMA blends gave distinct behaviours according to their compositions which were in the range of mechanical properties of soft tissues. The loss modulus and mechanical loss tangent through DMA gave two transition regions associated with the glass transition temperatures of poly(THFMA) and polystyrene components in the blends, along with a reduction in storage modulus. Cell adhesion and spreading in terms of neuroblastoma (human neuron-like SH-SY5Y) cells and ovine meniscal chondrocytes were demonstrated for scaffolds with THFMA rich formulations confirming their suitability for tissue engineering.

In vitro study of hydroxyapatite-based photocurable polymer composites prepared by laser stereolithography and supercritical fluid extraction

The fabrication of three-dimensional (3-D) structures using computer-controlled ultraviolet (UV) photopolymerization of acrylates (laser stereolithography) often results in the trapping of residual unreacted monomer and initiator. These residuals can leach from the finished structure and affect the biological response of cells and tissues. Thus the potential applications of these structures for tissue engineering have not been fully realized. In this paper we demonstrate that conventional post-lithography treatments followed by processing in the environmentally benign solvent, supercritical carbon dioxide (scCO(2)), dramatically increased biocompatibility. The scCO(2) processing of pure polyacrylate and polyacrylate/hydroxyapatite composite structures extracts residuals from all structures including those that had received full conventional post-lithography treatment (acetone washing/UV drying). Human osteoblast cells seeded on the extracted surfaces of these structures demonstrated increased cell attachment and proliferation on the scCO(2)-treated materials.

Laser technologies for fabricating individual implants and matrices for tissue engineering

This paper presents the results of work on the development and modification of laser methods of rapid prototyping and fabrication of individual implants and matrices for tissue engineering. A technology has been developed for shaping mineral-polymer bioactive structures, based on laser photopolymerization of a liquid mixture of polyfunctional acrylic oligomers with microparticles of hydroxyapatite. A method is proposed and developed for surface-selective laser sintering, making it possible to sinter polymeric microparticles without melting them completely, but only submelting their surface. This made it possible to obtain bioactive polymeric matrices based on bioresorbable aliphatic polyesters.

Incorporation of proteins within alginate fibre‐based scaffolds using a post‐fabrication entrapment method

In this study, a physical entrapment process was explored for the incorporation of proteins within preformed fibrous alginates and the release profile was tuned by varying the processing parameters. The entrapment process was carried out in a series of aqueous solutions at room temperature and involved pre‐swelling of the fibrous alginate within a Na+‐rich solution, followed by exposure to the protein of choice and entrapping it by re‐establishing cross‐links of alginate with BaCl2. Entrapment and release of fluorescein isothiocyanate‐labelled bovine serum albumin (FITC‐BSA), a model protein, was studied. It was found that a sustained release of the incorporated protein in cell culture medium for about 6 days was achieved. The main factors determining the release profile included the NaCl/CaCl2 ratio in the pre‐swelling solution, protein concentration, and the exposure time. To retard protein release, alginate fibres with entrapped FITC‐BSA were processed together with poly(d, l‐lactide) (PDLLA) into porous alginate fibre/PDLLA composites using supercritical CO2. In this manner, release of the protein for up to 3 months was achieved.

Biodegradable Scaffolds for Tissue Engineering Fabricated by Surface Selective Laser Sintering

Novel Surface Selective Laser Sintering (SSLS) technique enable precise fabrication of complicated 3D composite biodegradable scaffolds from thermosensitive polylactic and polylactic-co-glycolic acids and even retain bioactivity of incorporated enzymes. The presence of carbon black (CB) nanoparticles in SSLS structures raised concerns about their toxicity and biocompatibility. In present paper we studied this by diverse in vitro analysis using 3T3 fibroblasts, ovine meniscal chondrocytes and C2C12 myoblast cell cultures. All cells “readily” attached to and proliferated on CB containing surfaces. The abundance of live cells spreading out and covering the entire SSLS porous structures confirms their high biocompatibility. Moreover, C2C12 cells in the presence of morphogenetic protein rhBMP-2 have shown strong shift in differentiation pathway from myoblastic to osteoblastic type. These promising results encouraged us to further development of SSLS methodology targeted to custom-designed biodegradable scaffolds and implant fabrication.

Laser rapid prototyping for tissue engineering and regeneration

In present paper we describe the development of advanced laser stereolithography (LS) methodology based on photopolymerisation of a new liquid mixture of polyfunctional acrylic monomers and osteoinductive hydroxyapatite powder. Supercritical carbon dioxide treatment of LS samples introduced both surface and bulk microporosity for enhanced primary cell attachment and to remove toxic additives improving biocompatibility of the materials. The results of in vitro tests comprising human osteoblast cells attachment, spreading and proliferation on the implants demonstrate low level of their cytotoxicity and high level of biocompatibility. We also present a novel Surface Selective Laser Sintering (SSLS) technique for biodegradable polymer scaffolds fabrication from thermosensitive poly(D,L-lactic) - and poly(D,L-lactic-co-glycolic) acids - polymers, which have a wide spread occurrence in biomedical applications. Unlike conventional Selective Laser Sintering (where the powder particles melt because of their volumetric absorption of the laser radiation), in SSLS initiation of the sintering occur due to near IR laser beam absorption by a small amount (<0.1wt.%) of biocompatible carbon black microparticles uniformly distributed along the polymer powder surface. This technique enable to prevent any significant changes in polymer initial structures and even incorporate bioactive enzymes into the samples. The results of our in vitro studies using 3T3 fibroblast, C2C12 myoblast and ovine meniscal chondrocyte cells cultures hold great promise for use of produced scaffolds and developed technique in tissue engineering.