Establishment of a preclinical ovine model for tibial segmental bone defect repair by applying bone tissue engineering strategies

Currently, well-established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties, however they are limited in access and availability and associated with donor site morbidity, haemorrhage, risk of infection, insufficient transplant integration, graft devitalisation, and subsequent resorption resulting in decreased mechanical stability. As a result, recent research focuses on the development of alternative therapeutic concepts. The field of tissue engineering has emerged as an important approach to bone regeneration. However, bench to bedside translations are still infrequent as the process towards approval by regulatory bodies is protracted and costly, requiring both comprehensive in vitro and in vivo studies. The subsequent gap between research and clinical translation, hence commercialization, is referred to as the ‘Valley of Death’ and describes a large number of projects and/or ventures that are ceased due to a lack of funding during the transition from product/technology development to regulatory approval and subsequently commercialization. One of the greatest difficulties in bridging the Valley of Death is to develop good manufacturing processes (GMP) and scalable designs and to apply these in pre-clinical studies. In this article, we describe part of the rationale and road map of how our multidisciplinary research team has approached the first steps to translate orthopaedic bone engineering from bench to bedside byestablishing a pre-clinical ovine critical-sized tibial segmental bone defect model and discuss our preliminary data relating to this decisive step.

Enhanced human bone marrow stromal cell affinity for modified poly(L-lactide) surfaces by the upregulation of adhesion molecular genes

To enhance and regulate cell affinity for poly (l-lactic acid) (PLLA) based materials, two hydrophilic ligands, poly (ethylene glycol) (PEG) and poly (l-lysine) (PLL), were used to develop triblock copolymers: methoxy-terminated poly (ethylene glycol)-block-poly (l-lactide)-block-poly (l-lysine) (MPEG-b-PLLA-b-PLL) in order to regulate protein absorption and cell adhesion. Bone marrow stromal cells (BMSCs) were cultured on different composition of MPEG-b-PLLA-b-PLL copolymer films to determine the effect of modified polymer surfaces on BMSC attachment. To understand the molecular mechanism governing the initial cell adhesion on difference polymer surfaces, the mRNA expression of 84 human extracellular matrix (ECM) and adhesion molecules was analysed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). It was found that down regulation of adhesion molecules was responsible for the impaired BMSC attachment on PLLA surface. MPEG-b-PLLA-b-PLL copolymer films improved significantly the cell adhesion and cytoskeleton expression by upregulation of relevant molecule genes significantly. Six adhesion genes (CDH1, ITGL, NCAM1, SGCE, COL16A1, and LAMA3) were most significantly influenced by the modified PLLA surfaces. In summary, polymer surfaces altered adhesion molecule gene expression of BMSCs, which consequently regulated cell initial attachment on modified PLLA surfaces.

Processing of polycaprolactone and polycaprolactone-based copolymers into 3D scaffolds and their cellular responses

Synthetic polymers have attracted much attention in tissue engineering due to their ability to modulate biomechanical properties. This study investigated the feasibility of processing poly(varepsilon-caprolactone) (PCL) homopolymer, PCL-poly(ethylene glycol) (PEG) diblock, and PCL-PEG-PCL triblock copolymers into three-dimensional porous scaffolds. Properties of the various polymers were investigated by dynamic thermal analysis. The scaffolds were manufactured using the desktop robot-based rapid prototyping technique. Gross morphology and internal three-dimensional structure of scaffolds were identified by scanning electron microscopy and micro-computed tomography, which showed excellent fusion at the filament junctions, high uniformity, and complete interconnectivity of pore networks. The influences of process parameters on scaffolds' morphological and mechanical characteristics were studied. Data confirmed that the process parameters directly influenced the pore size, porosity, and, consequently, the mechanical properties of the scaffolds. The in vitro cell culture study was performed to investigate the influence of polymer nature and scaffold architecture on the adhesion of the cells onto the scaffolds using rabbit smooth muscle cells. Light, scanning electron, and confocal laser microscopy showed cell adhesion, proliferation, and extracellular matrix formation on the surface as well as inside the structure of both scaffold groups. The completely interconnected and highly regular honeycomb-like pore morphology supported bridging of the pores via cell-to-cell contact as well as production of extracellular matrix at later time points. The results indicated that the incorporation of hydrophilic PEG into hydrophobic PCL enhanced the overall hydrophilicity and cell culture performance of PCL-PEG copolymer. However, the scaffold architecture did not significantly influence the cell culture performance in this study.

The Exeter Hip. 40 Years of Innovation in Total Hip Arthroplasty

* Propoerties and use of acrylic cement * Design and biomechaniscs of a cemented hip replacement * The science of loosening, lysis and wear * Preparation of patients for surgery * Potential complications and their avoidance * Modern primary surgical techniques and new developments * Complex primary hip replacement and specialist techniques * Outcomes of cemented hip replacement * Principles of revision hip replacement * Basic science of bone grafting in revision surgery * Femoral acetabular impaction bone grafting techniques * Results of revision with bone graft and cement

Sustained release and osteogenic potential of heparan sulfate-doped fibrin glue scaffolds within a rat cranial model

This paper explores the potential therapeutic role of the naturally occurring sugar heparan sulfate (HS) for the augmentation of bone repair. Scaffolds comprising fibrin glue loaded with 5 lg of embryonically derived HS were assessed, firstly as a release-reservoir, and secondly as a scaffold to stimulate bone regeneration in a critical size rat cranial defect. We show HS-loaded scaffolds have a uniform distribution of HS, which was readily released with a typical burst phase, quickly followed by a prolonged delivery lasting several days. Importantly, the released HS contributed to improved wound healing over a 3-month period as determined by microcomputed tomography (lCT) scanning, histology, histomorphometry, and PCR for osteogenic markers. In all cases, only minimal healing was observed after 1 and 3 months in the absence of HS. In contrast, marked healing was observed by 3 months following HS treatment, with nearly full closure of the defect site. PCR analysis showed significant increases in the gene expression of the osteogenic markers Runx2, alkaline phosphatase, and osteopontin in the heparin sulfate group compared with controls. These results further emphasize the important role HS plays in augmenting wound healing, and its successful delivery in a hydrogel provides a novel alternative to autologous bone graft and growth factorbased therapies.

Evaluation of piezoelectric PVDF polymers for use in space environments. III. Comparison of the effects of vacuum UV and gamma radiation

Films of piezoelectric PVDF and P(VDF-TrFE) were exposed to vacuum UV (115-300 nm VUV) and -radiation to investigate how these two forms of radiation affect the chemical, morphological, and piezoelectric properties of the polymers. The extent of crosslinking was almost identical in both polymers after -irradiation, but surprisingly, was significantly higher for the TrFE copolymer after VUV-irradiation. Changes in the melting behavior were also more significant in the TrFE copolymer after VUV-irradiation due to both surface and bulk crosslinking, compared with only surface crosslinking for the PVDF films. The piezoelectric properties (measured using d33 piezoelectric coefficients and D-E hysteresis loops) were unchanged in the PVDF homopolymer, while the TrFE copolymer exhibited more narrow D-E loops after exposure to either - or VUV-radiation. The more severe damage to the TrFE copolymer in comparison with the PVDF homopolymer after VUV-irradiation is explained by different energy deposition characteristics. The short wavelength, highly energetic photons are undoubtedly absorbed in the surface layers of both polymers, and we propose that while the longer wavelength components of the VUV-radiation are absorbed by the bulk of the TrFE copolymer causing crosslinking, they are transmitted harmlessly in the PVDF homopolymer.

Evaluation of piezoelectric PVDF polymers for use in space environments. II. Effects of atomic oxygen and vacuum UV exposure

The effects of atomic oxygen (AO) and vacuum UV radiation simulating low Earth orbit conditions on two commercially available piezoelectric polymer films, poly(vinylidene fluoride) (PVDF) and poly(vinylidene fluoride-trifluoroethylene) P(VDF-TrFE), have been studied. Surface erosion and pattern development are significant for both polymers. Erosion yields were determined as 2.8 � 10�24 cm3/atom for PVDF and 2.5 � 10�24 cm3/atom for P(VDF-TrFE). The piezoelectric properties of the residual material of both polymers were largely unchanged after exposure, although a slight shift in the Curie transition of the P(VDF-TrFE) was observed. A lightly cross-linked network was formed in the copolymer presumably because of penetrating vacuum ultraviolet (VUV) radiation, while the homopolymer remained uncross-linked. These differences were attributed to varying degrees of crystallinity and potentially greater absorption, and hence damage, of VUV radiation in P(VDFTrFE) compared with PVDF.

Evaluation of piezoelectric poly(vinylidene fluoride) polymers for use in space environments. I. Temperature limitations

Smart materials, such as thin-film piezoelectric polymers, are interesting for potential applications on Gossamer spacecraft. This investigation aims to predict the performance and long-term stability of the piezoelectric properties of poly(vinylidene fluoride) (PVDF) and its copolymers under conditions simulating the low-Earthorbit environment. To examine the effects of temperature on the piezoelectric properties of PVDF, poly(vinylidenefluoride-co-trifluoroethylene), and poly(vinylidenefluoride-cohexafluoropropylene), the d33 piezoelectric coefficients were measured up to 160 8C, and the electric displacement/electric field (D–E) hysteresis loops were measured from �80 to þ110 8C. The room-temperature d33 coefficient of PVDF homopolymer films, annealed at 50, 80, and 125 8C, dropped rapidly within a few days of thermal exposure and then remained unchanged. In contrast, the TrFE copolymer exhibited greater thermal stability than the homopolymer, with d33 remaining almost unchanged up to 125 8C. The HFP copolymer exhibited poor retention of d33 at temperatures above 80 8C. In situ D–E loop measurements from �80 to þ110 8C showed that the remanent polarization of the TrFE copolymer was more stable than that of the PVDF homopolymer. D–E hysteresis loop and d33 results were also compared with the deflection of the PVDF homopolymer and TrFE copolymer bimorphs tested over a wide temperature range.

Grafted fluoropolymers as supports for solid-phase organic chemistry : preparation and characterization

Solid-phase organic chemistry has rapidly expanded in the last decade, and, as a consequence, so has the need for the development of supports that can withstand the extreme conditions required to facilitate some reactions. The authors here prepare a thermally stable, grafted fluoropolymer support (see Figure for an example) in three solvents, and found that the penetration of the graft was greatest in dichloromethane.

Modulation of dermal microvascular endithelial cell responses to growth factors and haemostatic factors in the presence of vitronectin

In order to effect permanent closure in burns patients suffering from full thickness wounds, replacing their skin via split thickness autografting, is essential. Dermal substitutes in conjunction with widely meshed split thickness autografts (+/- cultured keratinocytes) reduce scarring at the donor and recipient sites of burns patients by reducing demand for autologous skin (both surface area and thickness), without compromising dermal delivery at the wound face. Tissue engineered products such as Integra consist of a dermal template which is rapidly remodelled to form a neodermis, at which time the temporary silicone outer layer is removed and replaced with autologous split thickness skin. Whilst provision of a thick tissue engineered dermis at full thickness burn sites reduces scarring, it is hampered by delays in vascularisation which results in clinical failure. The ultimate success of any skin graft product is dependent upon a number of basic factors including adherence, haemostasis and in the case of viable tissue grafts, success is ultimately dependent upon restoration of a normal blood supply, and hence this study. Ultimately, the goal of this research is to improve the therapeutic properties of tissue replacements, through impregnation with growth factors aimed at stimulating migration and proliferation of microvascular endothelial cells into the donor tissue post grafting. For the purpose of my masters, the aim was to evaluate the responsiveness of a dermal microvascular endothelial cell line to growth factors and haemostatic factors, in the presence of the glycoprotein vitronectin. Vitronectin formed the backbone for my hypothesis and research due to its association with both epithelial and, more specifically, endothelial migration and proliferation. Early work using a platform technology referred to as VitroGro (Tissue Therapies Ltd), which is comprised of vitronectin bound BP5/IGF-1, aided keratinocyte proliferation. I hypothesised that this result would translate to another epithelium - endothelium. VitroGro had no effect on endothelial proliferation or migration. Vitronectin increases the presence of Fibroblast Growth Factor (FGF) and Vascular Endothelial Growth Factor (VEGF) receptors, enhancing cell responsiveness to their respective ligands. So, although Human Microvascular Endothelial Cell line 1 (HMEC-1) VEGF receptor expression is generally low, it was hypothesised that exposure to vitronectin would up-regulate this receptor. HMEC-1 migration, but not proliferation, was enhanced by vitronectin bound VEGF, as well as vitronectin bound Epidermal Growth Factor (EGF), both of which could be used to stimulate microvascular endothelial cell migration for the purpose of transplantation. In addition to vitronectin's synergy with various growth factors, it has also been shown to play a role in haemostasis. Vitronectin binds thrombin-antithrombin III (TAT) to form a trimeric complex that takes on many of the attributes of vitronectin, such as heparin affinity, which results in its adherence to endothelium via heparan sulfate proteoglycans (HSP), followed by unaltered transcytosis through the endothelium, and ultimately its removal from the circulation. This has been documented as a mechanism designed to remove thrombin from the circulation. Equally, it could be argued that it is a mechanism for delivering vitronectin to the matrix. My results show that matrix-bound vitronectin dramatically alters the effect that conformationally altered antithrombin three (cATIII) has on proliferation of microvascular endothelial cells. cATIII stimulates HMEC-1 proliferation in the presence of matrix-bound vitronectin, as opposed to inhibiting proliferation in its absence. Binding vitronectin to tissues and organs prior to transplant, in the presence of cATIII, will have a profound effect on microvascular infiltration of the graft, by preventing occlusion of existing vessels whilst stimulating migration and proliferation of endothelium within the tissue.

An electrospun polycaprolactone–collagen membrane for the resurfacing of cartilage defects

A polycaprolactone (PCL)–collagen electrospun mesh is proposed as a novel alternative to the conventional periosteal graft in autologous chondrocyte implantation. This is the first known attempt in designing a cartilage resurfacing membrane using a mechanically resilient PCL mesh with a weight-average molecular weight of 139 300 that is enhanced with bioactive collagen. PCL–collagen 10, 20 and 40% electrospun meshes (Coll-10, Coll-20 and Coll-40) were evaluated and it was discovered that the retention of surface collagen could only be achieved in Coll-20 and Coll-40. Furthermore Coll-20 was stiffer and stronger than Coll-40 and it satisfied the mechanical demands at the cartilage implant site. When seeded with mesenchymal stem cells (MSCs), the cells adhered on the surface of the Coll-20 mesh and they remained viable over a period of 28 days; however, they were unable to infiltrate through the dense meshwork. Cell compatibility was also noted in the chondrogenic environment as the MSCs differentiated into chondrocytes with the expression of Sox9, aggrecan and collagen II. More importantly, the mesh did not induce a hypertrophic response from the cells. The current findings support the use of Coll-20 as a cartilage patch, and future implantation studies are anticipated.

Response of cells on surface-induced nanopatterns: Fibroblasts and mesenchymal progenitor cells

Ultrathin films of a poly(styrene)-block-poly(2-vinylpyrindine) diblock copolymer (PS-b-P2VP) and poly(styrene)-block-poly(4-vinylpyrindine) diblock copolymer (PS-b-P4VP) were used to form surface-induced nanopattern (SINPAT) on mica. Surface interaction controlled microphase separation led to the formation of chemically heterogeneous surface nanopatterns on dry ultrathin films. Two distinct nanopatterned surfaces, namely, wormlike and dotlike patterns, were used to investigate the influence of topography in the nanometer range on cell adhesion, proliferation, and migration. Atomic force microscopy was used to confirm that SINPAT was stable under cell culture conditions. Fibroblasts and mesenchymal progenitor cells were cultured on the nanopatterned surfaces. Phase contrast and confocal laser microscopy showed that fibroblasts and mesenchymal progenitor cells preferred the densely spaced wormlike patterns. Atomic force microscopy showed that the cells remodelled the extracellular matrix differently as they migrate over the two distinctly different nanopatterns