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    ABSTRACT: Musculoskeletal reconstructive surgery often requires removal of significant quantities of bone tissue, such as the periosteum, causing critical problems following surgery like friction between different tissues and adhesion of soft tissues to the underlying bone. We studied the long-term host response and closure of large bone defects for periosteal reconstruction using Hyalonect, a novel membrane comprising knitted fibers of esterified hyaluronan, (HYAFF11). For biological characterization, 162 rats were used in a defect model in which a section of the dorsal muscular fascia was removed, and the membrane behavior observed over 540 d using conventional histology, with sham operated rats as controls. In addition, Hyalonect was used to cover defects made in the humeri of 7 dogs, filled with a variety of conventional bone filling compounds, and the regeneration process observed after 6 wks using histology. Low levels of inflammation were observed in the dorsal muscle fascia defect model, with cellular colonization of the mesh by 30 d, vascularization by 120 days, matrix fiber organization by 270 d, and the appearance of connective tissue identical to the surrounding tissue between 365 and 540 d, without the formation of fibrotic tissue. In addition, Hyalonect was shown to allow the regeneration of bone within the humeral defects whilst preventing fibrotic tissue in-growth, and allowing regeneration of tissue which, by 6 wk, had begun to resemble natural periosteal tissue. Hyalonect is suitable for improving the outcome of the final phases of orthopedic and trauma reconstructive surgical procedures, especially in the reconstruction of periosteal tissue.
    Journal of Surgical Research 10/2010; 168(1):e31-8. DOI:10.1016/j.jss.2010.09.015
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    ABSTRACT: Esterified hyaluronan scaffolds offer significant advantages for tissue engineering. They are recognized by cellular receptors, interact with many other extracellular matrix proteins and their metabolism is mediated by intrinsic cellular pathways. In this study differences in the viability and structural integrity of vascular tissue models cultured on hyaluronan scaffolds under laminar flow conditions highlighted potential differences in the biodegradation kinetics, processes and end-products, depending on the culture environment. Critical factors are likely to include seeding densities and the duration and magnitude of applied biomechanical stress. Proteomic evaluation of the timing and amount of remodelling protein expression, the resulting biomechanical changes arising from this response and metabolic cell viability assay, together with examination of tissue morphology, were conducted in vascular tissue models cultured on esterified hyaluronan felt and PTFE mesh scaffolds. The vascular tissue models were derived using complete cell sheets derived from harvested and expanded umbilical cord vein cells. This seeding method utilizes high-density cell populations from the outset, while the cells are already supported by their own abundant extracellular matrix. Type I and type IV collagen expression in parallel with MMP-1 and MMP-2 expression were monitored in the tissue models over a 10 day culture period under laminar flow regimes using protein immobilization technologies. Uniaxial tensile testing and scanning electron microscopy were used to compare the resulting effects of hydrodynamic stimulation upon structural integrity, while viability assays were conducted to evaluate the effects of shear on metabolic function. The proteomic results showed that the hyaluronan felt-supported tissues expressed higher levels of all remodelling proteins than those cultured on PTFE mesh. Overall, a 21% greater expression of type I collagen, 24% higher levels of type IV collagen, 24% higher levels of MMP-1 and 34% more MMP-2 were observed during hydrodynamic stress. This was coupled with a loss of structural integrity in these models after the introduction of laminar flow, as compared to the increases in all mechanical properties observed in the PTFE mesh-supported tissues. However, under flow conditions, the hyaluronan-supported tissues showed some recovery of the viability originally lost during static culture conditions, in contrast to PTFE mesh-based models, where initial gains were followed by a decline in metabolic viability after applied shear stress. Proteomic, cell viability and mechanical testing data emphasized the need for extended in vitro evaluations to enable better understanding of multi-stage remodelling and reparative processes in tissues cultured on biodegradable scaffolds. This study also highlighted the possibility that in high-density tissue culture with a biodegradable component, dynamic conditions may be more conducive to optimal tissue development than the static environment because they facilitate the efficient removal of high concentrations of degradation end-products accumulating in the pericellular space.
    Journal of Tissue Engineering and Regenerative Medicine 02/2010; 4(2):83-95. DOI:10.1002/term.208
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    ABSTRACT: A range of poly epsilon-caprolactone (PCL) films mixed/doped with poly(lactide-co-glycolide) (PLGA) (65:35) in 0, 10, 20, and 30 wt % were produced, sterilized using ethylene oxide, and analyzed using FTIR. Characterized human mesenchymal stem cells (hMSCs) were cultured in contact with the materials in basal, chondrogenic, and osteogenic medium for time periods up to 28 days, to determine if the materials could induce differentiation of MSC both in the presence and absence of biological stimuli. Viable cell adhesion was analyzed under all conditions. Collagen I, collagen II, sox-9, osteocalcin, osteopontin, osteonectin, and CBFA1 were evaluated at both the mRNA (real-time PCR) and protein production levels (fluorescent immunohistochemistry) and used to identify cell differentiation. Pure PCL and PCL mixed with PLGA demonstrated a chondrogenic potential. Only PCL 8 (80 wt % PCL, 20 wt % PLGA) facilitated osteogenic differentiation of MSCs under osteogenic conditions. This was attributed to the increased hydrophilic nature of the surface allowing sufficient homogeneous cell attachment and the formation of filamentous F-actin in the cells, allowing osteogenic differentiation. Of all materials tested, PCL 7 (70 wt % PCL, 30 wt % PLGA) demonstrated the greatest chondrogenic differentiation potential under basal and stimulated conditions at both the mRNA and protein production level.
    Journal of Biomedical Materials Research Part A 04/2009; 89(1):1-12. DOI:10.1002/jbm.a.31966
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    ABSTRACT: The aim of the study was to assess the hard tissue response of a composite hydroxyapatite/poly L-lactic acid (HA/PLLA) interference screw for anterior cruciate ligament (ACL) reconstruction compared to a standard PLLA screw. Twelve skeletally mature rams underwent unilateral ACL reconstruction using an autologous bone-patellar tendon graft. Each animal received either two test HA/PLLA interference screws or two control PLLA interference screws. Animals were sacrificed at 6 and 12 months post-implantation and the operated knees excised. Undecalcified sections of the screw and surrounding tissues were cut from resin embedded samples and stained; sections were approximately parallel to the longitudinal axis of the screws. A quantitative assessment of bone formation between each screw type (PLLA vs. HA/PLLA) and adjacent tissue in both the tibia and femur was undertaken using automated image analysis (KS400, Zeiss, UK). The inflammatory response of each screw type was assessed by histological evaluation. New bone formation along the perimeter of the screw threads was statistically significantly higher with the HA/PLLA than the PLLA alone. The inflammatory response as assessed semi-quantitatively by histologically determining the number of inflammatory cells present in the tissue adjacent to the implant, was higher for PLLA than HA/PLLA. Significantly increased new bone formation and decreased inflammatory cells were observed in vivo with the composite screw in comparison with the standard polymer. A novel HA/PLLA composite biomaterial in the form of an interference screw demonstrated an improved hard-tissue response compared to PLLA in a large animal ACL reconstruction. This study determined the differences in the tissue response between PLLA and a composite material of HA/PLLA. The improved tissue related outcomes observed in vivo, may be of benefit clinically in ACL reconstruction.
    Knee Surgery Sports Traumatology Arthroscopy 08/2008; 16(7):655-60. DOI:10.1007/s00167-008-0528-8
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    ABSTRACT: Electrostatic spinning is receiving increasing attention in the field of tissue engineering, due to its ability to produce 3-dimensional, multidirectional, microfibrous scaffolds. These structures are capable of supporting a wide range of cell growth; however, there is little knowledge relating material substrates with specific cellular interactions and responses. The aim of this research was to investigate if electrostatically spun scaffolds, with controlled topographical features, would affect the adhesion mechanisms of contacting cells. A range of electrostatically spun Tecoflex SG-80A polyurethane scaffolds was characterized in terms of inter-fibre separation, fibre diameter, surface roughness, void fraction and fibre orientation. Human embryonic lung fibroblasts and human vein endothelial cells were cultured on these scaffolds for 7, 14, 28 days, and analysed for their expression of extracellular matrix and adhesion molecules using image analysis and laser scanning confocal microscopy. There were significant differences in adhesion mechanisms between scaffolds, cell types and culture periods. Fibroblast-scaffolds were stimulated and oriented to a greater degree, and at earlier cultures, by the controlled topographical features than the endothelial cells. These conclusions confirm that cellular behaviour can be influenced by the induced scaffold topography at both molecular and cellular levels, with implications for optimum application specific tissue engineering constructs.
    Journal of Materials Science Materials in Medicine 05/2008; 19(4):1601-8. DOI:10.1007/s10856-008-3377-6
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    ABSTRACT: Scaffolds with 400 microm pores constructed from hyaluronan modified by benzyl esterification of the carboxylic acid groups (HYAFF-11) and viscous gels created from dodecyl-amidation of hyaluronan (HYADD-3) were implanted subcutaneously into rats for periods of up to 26 and 12 weeks, respectively. Tissue explants were infiltrated with methacrylate resin, sectioned and stained with a broad panel of inflammatory markers in addition to conventional histological stains. Both gels and sponges became rapidly infiltrated by cells that, in the case of HYAFF sponges, did not differentiate, whilst mature adipocytes were only observed at the margins of the sponges. This was combined with sustained inflammatory antigen expression. Conversely, in the HYADD gels, only moderate inflammatory staining was observed at 4 weeks which had diminished completely by 8 weeks. Mature and maturing adipocytes were observed deep within the gels. It is hypothesised that the gels present an excellent inflammatory cytokine profile which induces macrophage infiltration, proliferation then differentiation into adipocytes and is responsible for the generation of neoadipogenesis.
    Biomaterials 01/2008; 28(34):5131-6. DOI:10.1016/j.biomaterials.2007.08.004
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    ABSTRACT: Integration and regeneration are currently the fundamental objectives for medical interventions that are utilising materials and or cells to provide therapeutic benefit. These terms can form the basis of exciting research paradigms through logical progression, as both will derive control through understanding cognitive principles and theories. This potential for the subject to move from research into clinical applications may be expedited by a strictly analytical quantitative approach to provide controlled and successful long term interventions that are additionally safe and definable for regulation. There have been significant developments in the approach to interrogating the biological response to implanted materials, which have made full use of the timely and rapid evolution of the microprocessor. The breakdown and quantitative measurement and description of biological processes facilitates the provision of the detail required to unravel the key mechanisms and processes which will be required to be harnessed to integrate with human tissue to provide regeneration. In the future, a systems biology approach may provide significant potential as a research tool for the further development of regenerative medicine theories into clinic.
    Biomaterials 01/2008; 28(34):5128-30. DOI:10.1016/j.biomaterials.2007.07.016
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    ABSTRACT: This work concerned the endothelialization of vascular prostheses and subsequent improvement of functionality with respect to tissue engineering. The aim of the study was to investigate the initial, pre-shear stress cellular behavior with respect to three vascular biomaterials to explain subsequent cellular responses to physiological shear stresses. Expanded polytetrafluoroethylene (ePTFE), polyethyleneterephthalate (polyester; Dacron; PET), and electrostatically spun polyurethane (PU) (all pre-impregnated with collagen I/III) were cell-seeded with L929 immortalized murine fibroblasts or human umbilical vein endothelial cells (HUVECs). Cytoskeletal involvement, cell height profiles, and immunohistochemistry were examined after 7 d static culture. All three vascular biomaterials demonstrated different structures. Cell behavior varied both between the materials and the two cell types: cytoskeletal involvement was greater for the HUVECs and the more fibrous surfaces; height profiles were greater for the L929 and PET, and lowest on PU. Immunohistochemistry of HUVEC samples also showed differences: PU revealed the greatest expression of intercellular adhesion molecule-1 and E-selectin (PET and ePTFE the lowest, respectively); ePTFE produced the greatest for vascular cell adhesion molecule-1 (PET the lowest). Material substrate influenced the cellular response. Cells demonstrating firm adhesion increased their cytoskeletal processes and expression of cell-substratum and inter-cellular adhesion markers, which may explain their ability to adapt more readily to shear stress. The fibrous PU structure appeared to be most suited to further shear stress exposure. This study demonstrated the potential of the underlying vascular material to affect the long-term cellular functionality of the prosthesis.
    Journal of Surgical Research 10/2007; 149(1):39-46. DOI:10.1016/j.jss.2007.08.030
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    ABSTRACT: Unseeded sponges of benzyl-esterified hyaluronan (HYAFF11) and HYAFF11 coated with unmodified hyaluronan were implanted subcutaneously and intramuscularly in adult rats for 1, 2, 4, 8, 12, and 26 weeks. Explanted samples were stained tincturally using Van Geison, von Kossa, and hematoxylin and eosin, enzyme histochemically by chloroacetate esterase, and by immunohistochemistry for the specific identification of cell types and subpopulations, targeting immature (ED1) and mature macrophages (ED2), MHC-I subset, MHC-II subset, CD54, T-cell alpha-beta receptor, T-cell gamma-delta receptor, CD2, CD4, CD8, natural killer cells, B-cells, vimentin, and TGFbeta. Little or no fibrous tissue formation was observed in any sample in either sponge type at any implantation site. Little degradation was observed in either location until 26 weeks. Little neovascularization occurred at early time periods but was in evidence at 26 weeks. Complete cellular infiltration was observed after 4 weeks, with some mature adipocytes observed within the center of the subcutaneous implants, but these cells were mainly observed around the periphery of the sponges. At 26 weeks, cells were mostly macrophages, with small numbers of T-lymphocytes present. No natural killer cells, B-cells, helper/inducer, or cytotoxic/suppressor T-cells were observed in any sample. Most infiltrating cells were MHC-II positive, and discrete pockets of TGFbeta protein were observed within the sponges. While a sustained inflammatory response was observed within both sponge types at 26 weeks, it was relatively benign and nonspecific immunologically, and inflammatory markers such as MHC-II were declining after 12 weeks. No fibrous capsule was observed, and sponge degradation was only observed at 26 weeks, an event essential for induction of neovasculargenesis. At 26 weeks, there was significant staining for vimentin and ED2 on macrophages. Taken with the pattern of other macrophage activation markers, angiogenic environment and absence of inhibitory matrix proteins, the conditions were consistent with the onset of neoadipogenesis, although this would need to be confirmed by longer term studies. For the generation of neoadipose tissue for clinical therapy, we hypothesize that macrophages require an inflammatory stimulus for infiltration, then a reduction in proinflammatory cytokine secretion simultaneous with angiogenic conditions allowing macrophage differentiation into adipocytes.
    Biomacromolecules 10/2007; 8(9):2733-8. DOI:10.1021/bm070368p
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    ABSTRACT: Electrostatic spinning is a potentially significant technique for scaffold production within the field of tissue engineering; however, the effect of sterilisation upon these structures is not known. This research investigated the extent of any topographical alteration to electrostatically spun scaffolds post-production through sterilisation, and examined any subsequent effect on contacting cells. Scaffolds made from Tecoflex SG-80A polyurethane were sterilised using ethylene oxide and UV-ozone. Scaffold topography was characterized in terms of inter-fibre separation (ifs), fibre diameter (f.dia) and surface roughness. Cell culture was performed over 7 days with both mouse L929 and human embryonic lung fibroblasts, the results of which were assessed using SEM, image analysis and confocal microscopy. Sterilisation by UV-ozone and ethylene oxide decreased ifs and increased f.dia; surface roughness was decreased by UV-ozone but increased by ethylene oxide. Possible mechanisms to explain these observations are discussed, namely photo-oxidative degradation in the case of UV-ozone and process-induced changes in surface roughness. UV-ozone sterilised scaffolds showed greater cell coverage than those treated with ethylene oxide, but lower coverage than all the controls. Changes in cell attachment and morphology were thought to be due to the changes in topography brought about by the sterilisation process. We conclude that surface modification by sterilisation could prove to be a useful tool at the final stage of scaffold production to enhance cell contact, phenotype or function.
    Biomaterials 03/2007; 28(6):1014-26. DOI:10.1016/j.biomaterials.2006.10.014
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