Stem cell therapy has been suggested as a potential regenerative strategy to treat IVD degeneration and GDF6 has been shown to differentiate adipose-derived stem cells (ASCs) into an NP-like phenotype. However, for clinical translation, a delivery system is required to ensure controlled and sustained GDF6 release. This study aimed to investigate the encapsulation of GDF6 inside novel microparticles (MPs) to control delivery and assess the effect of the released GDF6 on NP-like differentiation of human ASCs. GDF6 release from PLGA-PEG-PLGA MPs over 14 days was determined using BCA and ELISA. The effect of MP loading density on collagen gel formation was assessed through SEM and histological staining. ASCs were cultured in collagen hydrogels for 14 days with GDF6 delivered exogenously or via microspheres. ASC differentiation was assessed by qPCR for NP markers, glycosaminoglycan production (DMMB) and immunohistochemistry.Background
Methods
Impaction bone grafting with milled human allograft is the gold standard for replacing lost bone stock during revision hip surgery. Problems surrounding the use of allograft include cost, availability, disease transmission and stem subsidence (usually due to shear failure of the surrounding allograft). Aims. To investigate various polymers for use as substitute allograft. The ideal graft would be a composite with similar mechanical characteristics as allograft, and with the ability to form de novo bone. High and low molecular weight (MW) forms of three different polymers (polylactic acid (PLA), poly (lactic-co-glycolic) acid (PLGA) and polycaprolactone (PCL)) were milled, impacted into discs, and then tested in a custom built shear testing rig, and compared to allograft. A second stage of the experiment involved the addition of skeletal stem cells (SSC) to each of the milled polymers, impaction, 8 days incubation, and then tests for cell viability and number, via fluorostaining and biochemical (WST-1, DNA) assays.Background
Methods
Disease transmission, availability and economic costs of allograft have resulted in significant efforts into finding an allograft alternative for use in impaction bone grafting (IBG). Biotechnology offers the combination of skeletal stem cells (SSC) with biodegradable polymers as a potential solution. Recently polymers have been identified with both structural strength and SSC compatibility that offer the potential for clinical translation. The aim of this study was to assess whether increasing the porosity of one such polymer via super critical CO2 fluid foaming (SCF) enhanced the mechanical and cellular compatibility characteristics for use as an osteogenic alternative to allograft in IBG. High molecular weight PLA scaffolds were produced via traditional (solid block) and SCF (porous) techniques, and the differences characterised using scanning electron microscopy (SEM). The polymers were milled, impacted, and mechanical comparison between traditional vs SCD created scaffolds and allograft controls was made using a custom shear testing rig, as well as a novel agitation test to assess cohesion. Cellular compatibility tests for cell number, viability and osteogenic differentiation using WST-1 assays, fluorostaining and ALP assays were determined following 14 day culture with SSC's.Aims
Methods
Impaction bone grafting with milled human allograft is the gold standard for replacing lost bone stock during revision hip surgery. Problems surrounding the use of allograft include cost, availability, disease transmission and stem subsidence (usually due to shear failure of the surrounding allograft). The aim of this study was to investigate various polymers for use as substitute allograft. The ideal graft would be a composite with similar mechanical characteristics as allograft, and with the ability to form High and low molecular weight (MW) forms of three different polymers (polylactic acid (PLA), poly (lactic co-glycolic) acid (PLGA) and polycaprolactone (PCL)) were milled, impacted into discs, and then tested in a custom built shear testing rig, and compared to allograft. A second stage of the experiment involved the addition of skeletal stem cells (SSC) to each of the milled polymers, impaction, 8 days incubation, and then tests for cell viability and number, via fluorostaining and biochemical (WST-1) assays.Aims
Methods
Disease transmission, availability and economic costs of allograft have resulted in significant efforts into finding an allograft alternative for use in impaction bone grafting (IBG). Biotechnology offers the combination of skeletal stem cells (SSC) with biodegradable polymers as a potential solution. Recently polymers have been identified with both structural strength and SSC compatibility that offer the potential for clinical translation. The aim of this study was to assess whether increasing the porosity of one such polymer via super critical CO2 dissolution (SCD) enhanced the mechanical and cellular compatibility characteristics for use as an osteogenic alternative to allograft in IBG. High molecular weight PLA scaffolds were produced via traditional (solid block) and SCD (porous) techniques, and the differences characterised using scanning electron microscopy (SEM). The polymers were milled, impacted, and mechanical comparison between traditional vs SCD created scaffolds and allograft controls was made using a custom shear testing rig, as well as a novel agitation test to assess cohesion. Cellular compatibility tests for cell number, viability and osteogenic differentiation using WST-1 assays, fluorostaining and ALP assays were determined following 14 day culture with SSCs. SEM showed increased porosity of the SCD produced PLA scaffolds, with pores between 50-100 micrometres. Shear testing showed the SCD polymer exceeded the shear strength of allograft controls (P<0.001). Agitation testing showed greater cohesion between the particles of the SCD polymer (P<0.05). Cellular studies showed increased cell number, viability and osteogenic differentiation on the SCD polymer compared to traditional polymer (P<0.05) and allograft (P<0.001). The use of supercritical C02 to generate PLA scaffolds significantly improves the cellular compatibility and cohesion compared to traditional non-porous PLA, without substantial loss of mechanical shear strength. The improved characteristics are critical for clinical translation as a potential osteogenic composite for use in impaction bone grafting.
Impaction bone grafting with milled human allograft is the gold standard for replacing lost bone stock during revision hip surgery. Problems surrounding the use of allograft include cost, availability, disease transmission and stem subsidence (usually due to shear failure of the surrounding allograft). The aim of this study was to investigate various polymers for use as substitute allograft. The ideal graft would be a composite with similar mechanical characteristics as allograft, and with the ability to form de novo bone. High and low molecular weight (MW) forms of three different polymers (polylactic acid (PLA), poly (lactic co-glycolic) acid (PLGA) and polycaprolactone (PCL)) were milled, impacted into discs, and then tested in a custom built shear testing rig, and compared to allograft. A second stage of the experiment involved the addition of skeletal stem cells (SSC) to each of the milled polymers, impaction, 8 days incubation, and then tests for cell viability and number, via fluorostaining and biochemical (WST-1) assays. The shear strengths of both high/ low MW PLA, and high/low MW PLGA were significantly higher than those of milled allograft (P<0.001, P<0.001, P<0.005 and P<0.005) but high and low MW PCL was poor to impact, and had significantly lower shear strengths (P<0.005, P<0.001). Fluorostaining showed good cell survival on high MW PLA, high MW PCL and high MW PLGA. These findings were confirmed with WST-1 assays. High MW PLA as well as high MW PLGA performed well both in mechanical testing and cell compatibility studies. These two polymers are good contenders to produce a living composite for use as substitute human allograft in impaction bone grafting, and are currently being optimised for this use via the investigation of different production techniques and in-vivo studies.
Injectable scaffolds which also deliver cells and bioactive molecules to augment bone healing overcome many of the limitations associated with current bone graft substitutes. The aim of this study was to develop and test a novel injectable scaffold that self-assembles isothermically in situ to form a biodegradable porous osteoconductive material, and to assess the viability of human mesenchymal stem cells (hMSC) seeded onto the scaffold. Rheological assessment was performed on three different molecular weights (Mw) of poly(lactic-co-glycolic acid) (PLGA) (26kDa, 53kDa and 92kDa) combined with differing ratios of polyethylene glycol (PEG) to control the temperature required for scaffold self-assembly. The strength (MPa) and stiffness (Young's Modulus) patterns of the scaffolds were assessed in compression. The cell viability, proliferation and distribution patterns of hMSCs seeded within the scaffold were assessed through various assays (Alamar Blue), confocal microscopy and micro-CT. The hMSC differentiation in osteogenic media was characterised by the identification of specific bone formation markers (e.g. alkaline phosphatase).Background/Study Aim
Methods
Open fractures occur with an annual incidence of 11.5 per 100,000 (6900 pa in UK). Infection rates, even with intravenous broad-spectrum antibiotics, remain as high as 22%. For this reason necessary bone grafting is usually delayed until soft-tissue cover of the bone injury is achieved. A biodegradable bone graft that released sustained high concentrations of antibiotics and encouraged osteogenesis, that could be implanted safely on the day of injury would reduce infection rates and avoid reoperation and secondary grafting. The non –union rate (approx 350 pa in UK) should also be reduced. Such a graft, consisting of a PLA/PGA co –polymer and containing antibiotics, is under development and here we report assessment of spectrum and duration of antimicrobial activity and effect of addition of antibiotics on mechanical properties. Varying concentrations of gentamicin, colistin, clindamycin and trimethoprim, singly and in combination, were added to the copolymer and test pieces were made. These were then tested using an established method (SPTT) which determines degree and duration of antimicrobial activity as well as risk of emerging resistance. Test bacteria were Staphylococcus epidermidis, Staphylococcus aureus, MRSA and Escherichia coli. Mechanical properties (compressive strength and porosity) were determined using established methods.Introduction
Methods
Impaction bone grafting with morsellised allograft is a recognized technique to reconstitute loss of bone stock often encountered during revision hip surgery. Concerns over disease transmission, high costs and limited supply has led to interest in synthetic grafts. Poly (lactic acid) (PLA) grafts are attractive to the tissue engineering community as a consequence of their biocompatibility, ease of processing into three-dimensional structures, their established safety as suture materials and the versatility that they offer for producing chemically defined substrates for bone graft matrices. This study set out to examine the potential of PLA scaffolds augmented with human bone marrow stromal cells in impaction bone grafting (IBG).
The use of designer scaffolds to deliver biologically active osteogenic growth factors such as recombinant human bone morphogenetic protein-2 (rhBMP-2) to the sites of tissue regeneration in for example orthopaedics, has tremendous therapeutic implications. The aims of this study were to generate biomimetic biodegradable porous osteogenic scaffolds using a supercritical fluid process to encapsulate rhBMP-2, and to examine the ability of the scaffolds to promote human osteoprogenitor differentiation and bone formation in vitro and in vivo. The rhBMP-2 encapsulated in Poly(-lactic acid) (PLA) scaffolds (100ng/mg PLA) were generated using an innovative supercritical fluid mixing method. The bioactivity of rhBMP-2 encapsulated PLA scaffolds were confirmed by induction of the C2C12 promyoblast cell line into the osteogenic lineage as detected by alkaline phosphatase expression. No induction of alkaline phosphatase-positive cells was observed using blank scaffolds. BMP-2 released from encapsulated constructs promoted adhesion, migration, expansion and differentiation of human osteoprogenitor cells on 3-D scaffolds. Enhanced matrix synthesis and cell differentiation on growth factor encapsulated scaffolds was observed following culture of human osteoprogenitors on explants of chick femoral bone wedge defects in an ex vivo model of bone formation developed using the chick chorioallantoic membrane model. In vivo studies using diffusion chamber implantation and subcutaneous implantation of human osteoprogenitors on rhBMP-2 encapsulated scaffolds showed morphologic evidence of new bone matrix and cartilage formation in athymic mice as assessed by x-ray analysis, immunocytochemistry and birefringence. These studies provide evidence of controlled release of BMP-2 from biodegradable polymer scaffolds initiating new bone formation in vivo. The generation of 3-D biomimetic structures incorporating osteoinductive factors such as BMP-2 indicates their potential for de novo bone formation that exploits cell-matrix interactions and, significantly, realistic delivery protocols for growth factors in musculo-skeletal tissue engineering.
