Bioactive coatings on 3D printed scaffolds for bone regeneration: Use of Laponite® to deliver BMP-2 in an ovine femoral condyle defect model.

Biomaterial-based approaches for bone regeneration seek to explore alternative strategies to repair non-healing fractures and critical-sized bone defects. Fracture non-union occurs due to a number of factors resulting in the formation of bone defects. Rigorous evaluation of the biomaterials in relevant models and assessment of their potential to translate towards clinical use is vital. Large animal experimentation can be used to model fracture non-union while scaling-up materials for clinical use. Growth factors modulate cell phenotype, behaviour and initiate signalling pathways leading to changes in matrix deposition and tissue formation. Bone morphogenetic protein-2 (BMP-2) is a potent osteogenic growth factor, with a rapid clearance time in vivo necessitating clinical use at a high dose, with potential deleterious side-effects. The current studies have examined the potential for Laponite® nanoclay coated poly(caprolactone) trimethacrylate (PCL-TMA900) scaffolds to bind BMP-2 for enhanced osteoinduction in a large animal critical-sized bone defect. An ovine femoral condyle defect model confirmed PCL-TMA900 scaffolds coated with Laponite®/BMP-2 produced significant bone formation compared to the uncoated PCL-TMA 900 scaffold in vivo, assessed by micro-computed tomography (µCT) and histology. This indicated the ability of Laponite® to deliver the bioactive BMP-2 on the PCL-TMA900 scaffold. Bone formed around the Laponite®/BMP-2 coated PCL-TMA900 scaffold, with no erroneous bone formation observed away from the scaffold material confirming localisation of BMP-2 delivery. The current studies demonstrate the ability of a nanoclay to localise and deliver bioactive BMP-2 within a tailored octet-truss scaffold for efficacious bone defect repair in a large animal model with significant implications for translation to the clinic.

Evolving applications of the egg: chorioallantoic membrane assay and ex vivo organotypic culture of materials for bone tissue engineering.

The chick chorioallantoic membrane model has been around for over a century, applied in angiogenic, oncology, dental and xenograft research. Despite its often perceived archaic, redolent history, the chorioallantoic membrane assay offers new and exciting opportunities for material and growth factor evaluation in bone tissue engineering. Currently, superior/improved experimental methodology for the chorioallantoic membrane assay are difficult to identify, given an absence of scientific consensus in defining experimental approaches, including timing of inoculation with materials and the analysis of results. In addition, critically, regulatory and welfare issues impact upon experimental designs. Given such disparate points, this review details recent research using the ex vivo chorioallantoic membrane assay and the ex vivo organotypic culture to advance the field of bone tissue engineering, and highlights potential areas of improvement for their application based on recent developments within our group and the tissue engineering field.

Harnessing Human Decellularized Blood Vessel Matrices and Cellular Construct Implants to Promote Bone Healing in an Ex Vivo Organotypic Bone Defect Model.

Decellularized matrices offer a beneficial substitute for biomimetic scaffolds in tissue engineering. The current study examines the potential of decellularized placental vessel sleeves (PVS) as a periosteal protective sleeve to enhance bone regeneration in embryonic day 18 chick femurs contained within the PVS and cultured organotypically over a 10 day period. The femurs are inserted into decellularized biocompatibility-tested PVS and maintained in an organotypic culture for a period of 10 days. In femurs containing decellularized PVS, a significant increase in bone volume (p < 0.001) is evident, demonstrated by microcomputed tomography (µCT) compared to femurs without PVS. Histological and immunohistological analyses reveal extensive integration of decellularized PVS with the bone periosteum, and enhanced conservation of bone architecture within the PVS. In addition, the expressions of hypoxia inducible factor-1 alpha (HIF-1α), type II collagen (COL-II), and proteoglycans are observed, indicating a possible repair mechanism via a cartilaginous stage of the bone tissue within the sleeve. The use of decellularized matrices like PVS offers a promising therapeutic strategy in surgical tissue replacement, promoting biocompatibility and architecture of the tissue as well as a factor-rich niche environment with negligible immunogenicity.

Development of specific collagen scaffolds to support the osteogenic and chondrogenic differentiation of human bone marrow stromal cells.

Type I Collagen matrices of defined porosity, incorporating carbonate substituted hydroxyapatite (HA) crystals, were assessed for their ability to support osteo- and chondrogenic differentiation of human bone marrow stromal cells (HBMSCs). Collagen-HA composite scaffolds supported the osteogenic differentiation of HBMSCs both in vitro and in vivo as demonstrated by histological and micro-CT analyses indicating the extensive penetration of alkaline phosphatase expressing cells and new matrix synthesis with localised areas immunologically positive for osteocalcin. In vivo, extensive new osteoid formation of implant origin was observed in the areas of vasculature. Chondrogenic matrix synthesis was evidenced in the peripheral regions of pure collagen systems by an abundance of Sox9 expressing chondrocytes embedded within a proteoglycan and collagen II rich ECM. The introduction of microchannels to the scaffold architecture was seen to enhance chondrogenesis. Tissue specific gene expression and corresponding matrix synthesis indicate that collagen matrices support the growth and differentiation of HBMSCs and suggest the potential of this platform for understanding the ECM cues necessary for osteogenesis and chondrogenesis.

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.

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 Chorioallantoic Membrane Assay for Biomaterial Testing in Tissue Engineering: A Short-Term In Vivo Preclinical Model.

The fields of regenerative medicine and tissue engineering offer significant promise to address the urgent unmet need for therapeutic strategies in a number of debilitating conditions, diseases, and tissue needs of an aging population. Critically, the safety and efficacy of these pioneering strategies need to be assessed before clinical application, often necessitating animal research as a prerequisite. The growing number of newly developed potential treatments, together with the ethical concerns involved in the application of in vivo studies, requires the implementation of alternative models to facilitate such screening of new treatments. The present review examines the current in vitro and in vivo models of preclinical research with particular emphasis on the chorioallantoic membrane (CAM) assay as a minimally invasive, short-term in vivo alternative. Traditionally used as an angiogenic assay, the CAM of the developing chick embryo provides a noninnervated rapidly growing vascular bed, which can serve as a surrogate blood supply for organ culture, and hence a platform for biomaterial testing. This review offers an overview of the CAM assay and its applications in biomedicine as an in vivo model for organ culture and angiogenesis. Moreover, the application of imaging techniques (magnetic resonance imaging, microcomputed tomography, fluorescence labeling for tracking) will be discussed for the evaluation of biomaterials cultured on the CAM. Finally, an overview of the CAM assay methodology will be provided to facilitate the adoption of this technique across laboratories and the regenerative medicine community, and thus aid the reduction, replacement, and refinement of animal experiments in research.

In Vivo Assessment of Bone Regeneration in Alginate/Bone ECM Hydrogels with Incorporated Skeletal Stem Cells and Single Growth Factors.

The current study has investigated the use of decellularised, demineralised bone extracellular matrix (ECM) hydrogel constructs for in vivo tissue mineralisation and bone formation. Stro-1-enriched human bone marrow stromal cells were incorporated together with select growth factors including VEGF, TGF-ß3, BMP-2, PTHrP and VitD3, to augment bone formation, and mixed with alginate for structural support. Growth factors were delivered through fast (non-osteogenic factors) and slow (osteogenic factors) release PLGA microparticles. Constructs of 5 mm length were implanted in vivo for 28 days within mice. Dense tissue assessed by micro-CT correlated with histologically assessed mineralised bone formation in all constructs. Exogenous growth factor addition did not enhance bone formation further compared to alginate/bone ECM (ALG/ECM) hydrogels alone. UV irradiation reduced bone formation through degradation of intrinsic growth factors within the bone ECM component and possibly also ECM cross-linking. BMP-2 and VitD3 rescued osteogenic induction. ALG/ECM hydrogels appeared highly osteoinductive and delivery of angiogenic or chondrogenic growth factors led to altered bone formation. All constructs demonstrated extensive host tissue invasion and vascularisation aiding integration and implant longevity. The proposed hydrogel system functioned without the need for growth factor incorporation or an exogenous inducible cell source. Optimal growth factor concentrations and spatiotemporal release profiles require further assessment, as the bone ECM component may suffer batch variability between donor materials. In summary, ALG/ECM hydrogels provide a versatile biomaterial scaffold for utilisation within regenerative medicine which may be tailored, ultimately, to form the tissue of choice through incorporation of select growth factors.

Strategies for cell manipulation and skeletal tissue engineering using high-throughput polymer blend formulation and microarray techniques.

A combination of high-throughput material formulation and microarray techniques were synergistically applied for the efficient analysis of the biological functionality of 135 binary polymer blends. This allowed the identification of cell-compatible biopolymers permissive for human skeletal stem cell growth in both in vitro and in vivo applications. The blended polymeric materials were developed from commercially available, inexpensive and well characterised biodegradable polymers, which on their own lacked both the structural requirements of a scaffold material and, critically, the ability to facilitate cell growth. Blends identified here proved excellent templates for cell attachment, and in addition, a number of blends displayed remarkable bone-like architecture and facilitated bone regeneration by providing 3D biomimetic scaffolds for skeletal cell growth and osteogenic differentiation. This study demonstrates a unique strategy to generate and identify innovative materials with widespread application in cell biology as well as offering a new reparative platform strategy applicable to skeletal tissues.

Formation of a human-derived fat tissue layer in P(DL)LGA hollow fibre scaffolds for adipocyte tissue engineering.

Development of adipose tissue-engineering strategies, where human bone marrow stromal cells (HBMSC) are combined with three-dimensional scaffolds, is likely to prove valuable for soft tissue restoration. In this study, we assessed the function of poly(DL-lactide-co-glycolide) (P(DL)LGA) hollow fibres in facilitating the development of HBMSC-derived adipocytes for advancement of an associated adipocyte layer. The large surface area of 75:25 P(DL)LGA fibres facilitated the rapid generation of extensive adipocyte aggregates from an undifferentiated HBMSC monolayer, where the fat-laden cells stained positive with Oil Red O and expressed the adipocyte marker, fatty acid binding protein 3 (FABP3). Following implantation subcutaneously in severely compromised immunodeficient mice, the adipogenic phenotype of the PLGA-adipocyte graft was maintained for up to 56 days. Confocal microscopy showed associated LipidTOX Deep Red neutral lipid staining in an (FL)P(DL)LGA fibre-adipocyte graft after 56 days, critical evidence demonstrating maintenance of the adipocyte phenotype in the subcutaneous graft. To support adipose tissue advancement in a defined volume, the P(DL)LGA-adipocyte scaffold was encapsulated within alginate/chitosan hydrogel capsules (typical diameters, 4.0 mm). In a 28-day in vivo trial in immunodeficient mice, clusters of the capsules were maintained at the subcutaneous site. An adipocyte tissue layer advancing within the surrounding hydrogel was demonstrated.

Augmentation of skeletal tissue formation in impaction bone grafting using vaterite microsphere biocomposites.

The development of particulate bone void fillers with added biological function to augment skeletal tissue formation will lead to improved efficacy in bone replacement surgery. We demonstrate the potential for vaterite microsphere biocomposites to augment bone matrix formation within an in vivo model for impaction bone grafting seeded with human bone marrow stromal cells. In vitro tests demonstrate the significance of vaterite microspheres in the activation and promotion of 3D skeletal tissue formation. Further in vitro experiments using functionalized microspheres with surface integrated RGD peptide activate co-cultured skeletal populations in pellets and promote secretion of extracellular matrix collagens and human osteocalcin. Specific temporal release of entrapped RNase A was successfully demonstrated using these specialized microspheres with integrated magnetic beads, which physically disrupted the inorganic macrostructure. These studies demonstrate that bio-inspired calcium carbonate microspheres augment in vivo bone formation in impaction bone grafting. Such microspheres with added biological functionality offer innovative therapeutic approaches to activate skeletal populations and enhance bone formation with reparative implications for hard tissues.

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.