S. Downes

Growth of human osteoblast‐like cells on alkanethiol on gold self‐assembled monolayers: The effect of surface chemistry

Primary human osteoblasts were cultured on self-assembled monolayers (SAMs) of alkylthiols on gold with carboxylic acid and methyl termini, and the kinetics of cell attachment and proliferation were measured. Over 90 min approximately twice as many cells attached to carboxylic-acid-terminated monolayers as attached to methyl-terminated monolayers. After 24 h the number of cells attached to carboxylic-acid-terminated monolayers was ten times that attached to the methyl-terminated monolayers. Cell morphology and cytoskeletal actin organization also were found to be different. Osteoblasts were cultured on SAMs that were patterned by photolithographic techniques. Cells attached almost exclusively to carboxylic-acid-functionalized areas of the patterned surfaces, leaving methyl-functionalized regions bare. The patterns strongly influenced the morphology of the attached cells. After 24 h, cells were observed to bridge between carboxylic-acid-terminated regions separated by 75 microns, methyl-terminated regions but not those separated by 150 microns methyl-terminated regions. After 6 days in culture osteoblasts formed multilayers on the carboxylic-acid-terminated regions of the pattern.

Biocomposites of non-crosslinked natural and synthetic polymers

Biocomposite films comprising a non-crosslinked, natural polymer (collagen) and a synthetic polymer, poly(epsilon-caprolactone) (PCL), have been produced by impregnation of lyophilised collagen mats with a solution of PCL in dichloromethane followed by solvent evaporation. This approach avoids the toxicity problems associated with chemical crosslinking. Distinct changes in film morphology, from continuous surface coating to open porous format, were achieved by variation of processing parameters such as collagen:PCL ratio and the weight of the starting lyophilised collagen mat. Collagenase digestion indicated that the collagen content of 1:4 and 1:8 collagen:PCL biocomposites was almost totally accessible for enzymatic digestion indicating a high degree of collagen exposure for interaction with other ECM proteins or cells contacting the biomaterial surface. Much reduced collagen exposure (around 50%) was measured for the 1:20 collagen:PCL materials. These findings were consistent with the SEM examination of collagen:PCL biocomposites which revealed a highly porous morphology for the 1:4 and 1:8 blends but virtually complete coverage of the collagen component by PCL in the 1:20 samples. Investigations of the attachment and spreading characteristics of human osteoblast (HOB) cells on PCL films and collagen:PCL materials respectively, indicated that HOB cells poorly recognised PCL but attachment and spreading were much improved on the biocomposites. The non-chemically crosslinked, collagen:PCL biocomposites described are expected to provide a useful addition to the range of biomaterials and matrix systems for tissue engineering.

Regulation of cell surface tissue transglutaminase: effects on matrix storage of latent transforming growth factor-beta binding protein-1.

Using a cytochemical approach, we examined the role of tissue transglutaminase (tTgase, Type II) in the incorporation of latent TGF-β binding protein-1 (LTBP-1) in the extracellular matrix of Swiss 3T3 fibroblasts in which tTgase expression can be modulated through a tetracycline-controlled promoter. Increased tTgase expression led to an increased rate of LTBP-1 deposition in the matrix, which was accompanied by an increased pool of deoxycholate-insoluble fibronectin. Matrix deposition of LTBP-1 could also be reduced by the competitive amine substrate putrescine. Immunolocalization at the fluorescence and electron microscopic level showed that extracellular tTgase is located at the basal and apical surfaces of cells and at cell-cell contacts, and that LTBP-1 is co-distributed with cell surface tTgase, suggesting an early contribution of tTgase to the binding of LTBP-1 to matrix proteins. LTPB-1 was also found to co-localize with both intracellular and extracellular fibronectin, and increased immunoreactivity for LTBP-1 and fibronectin was found in large molecular weight polymers in the deoxycholate-insoluble matrix of fibroblasts overexpressing tTgase. We conclude that regulation of tTgase expression is important for controlling matrix storage of latent TGF-β1 complexes and that fibronectin may be one extracellular component to which LTBP-1 is crosslinked when LTBP-1 and tTgase interact at the cell surface.

Physical and biocompatibility properties of poly-ε-caprolactone produced using in situ polymerisation: a novel manufacturing technique for long-fibre composite materials

Preliminary investigations into a novel process for the production of poly-epsilon-caprolactone (PCL) to be used as a matrix material in a bioabsorbable composite material are detailed. This material is primarily being developed as a bone substitute for use in maxillofacial reconstructive surgery, however, the technique described could be adapted to other areas where bioabsorbable composite materials may be used. The development of a totally bioabsorbable long-fibre composite material would allow a two-stage degradation to occur with the matrix material degrading first leaving a scaffold structure of degradable fibres which would be absorbed at a later stage. Caprolactone monomer was polymerised in situ within a tool cavity to produce a net shape moulding. Inclusion of a fibre preform within the tool cavity which was impregnated by the liquid monomer produces a long-fibre composite material. PCL with a range of molecular weights has been produced using this liquid moulding technique to assess the physical and biocompatibility properties compared to commercially available PCL. Osteoblast-like cells derived from human craniofacial bone (CFC) have been used to assess the in vitro biocompatibility of the PCL. The results show that high-quality PCL with a narrow molecular weight distribution and properties similar to commercially available PCL can be produced using this technique. Polymerisation of the monomer around a woven fibre preform made of a poly(lactic acid) (PLA)/poly(glycolic acid) (PGA) copolymer (vicryl mesh) produced a bioabsorbable long-fibre composite material. Further work is ongoing to develop this system towards a method for improving craniofacial bone reconstruction.

Influence of packaging conditions on the properties of gamma-irradiated UHMWPE following accelerated ageing and shelf ageing

The ageing behaviour of ultra-high molecular weight polyethylene (UHMWPE) has been studied following gamma irradiation (25 or 40 kGy) in air or in inert atmosphere (vacuum packed). Accelerated ageing procedures used elevated temperature (70 degrees C) and/or pressurised oxygen (5 bar). Shelf ageing for up to six months was also performed. The variation in polymer properties with depth into the polymer was determined using density measurements, infra-red spectroscopy and differential scanning calorimetry. Tensile properties of the irradiated polymer after accelerated ageing were also determined. Accelerated ageing in air for 20 days or pressurised oxygen for 4 days resulted in peaks in polymer density, crystallinity and degree of oxidation at the polymer surface. Accelerated ageing in pressurised oxygen for 8 days resulted in peaks in these properties 500 microns below the polymer surface. Where gamma irradiation was performed in inert atmosphere the amount of polymer degradation following accelerated ageing (in oxidising conditions) was generally lower but still significant. Differences were also observed in tensile properties between material gamma-irradiated in air and in vacuum. This study indicated that performing gamma sterilisation procedures under inert conditions can reduce the level of UHMWPE degradation when exposed to an oxidising environment.

The influence of gamma irradiation and aging on degradation mechanisms of ultra-high molecular weight polyethylene.

The aging behavior of ultra-high molecular weight polyethylene (UHMWPE) has been studied following gamma irradiation in air. Accelerated aging procedures used elevated temperature (70 °C), pressurized oxygen (5 bar) and applied stress. Shelf and in vivo aged components have also been investigated. The variation in polymer properties with depth into the polymer was determined using density measurements, infra-red spectroscopy and differential scanning calorimetry. Accelerated aging in pressurized oxygen resulted in peaks in polymer density and degree of oxidation up to 500 μm below the polymer surface. Shelf and in vivo aging was also found to result in increased density at or below the component surfaces. Changes in density were mainly due to changes in crystallinity within the UHMWPE and, to a smaller extent, due to oxygen incorporation within the polymer. The application of stress did not appear to influence the accelerated aging of UHMWPE. A method for estimating the residual stress distribution in the UHMWPE using the measured changes in density is proposed. This study has indicated that oxidation of UHMWPE may lead to the development of tensile residual stresses, near the component surface, in the region of 1.7 MPa. These stresses may contribute to the failure mechanism of UHMWPE acetabular cups or knee tibial trays during service.

The role of protein adsorption on chondrocyte adhesion to a heterocyclic methacrylate polymer system

Chondrocyte adhesion to a polymer system consisting of poly(ethyl methacrylate) and tetrahydrofurfuryl methacrylate (PEMA/THFMA) has been investigated in vitro. The adhesive glycoproteins, fibronectin and vitronectin were studied for their role in promoting cell attachment. Fibronectin was the best substrate for chondrocyte attachment, if it was pre-adsorbed and did not have to compete with other proteins for attachment sites. Chondrocytes began to spread on fibronectin coated glass although they remained rounded on the libronectin coated PEMA/THFMA system. Vitronectin was better at competing with the other proteins in serum and was the main adhesive protein for chondrocyte attachment to TCP and the PEMA/THFMA system in normal serum medium. Serum contains non-adhesive proteins that compete for binding sites and hence reduce cell attachment. The alpha5beta1 and alpha(v)beta3/beta5 integrins were detected on the chondrocytes although there may be a difference in expression between different material surfaces.

Role of the cross-linking enzyme tissue transglutaminase in the biological recognition of synthetic biodegradable polymers

The calcium-dependent cross-linking enzyme tissue transglutaminase (tTgase, type II) is a potential novel player at the cell surface, where its contribution to cell adhesion and stabilization of the extracellular matrix is becoming increasingly recognized. We investigated whether tTgase enhances the biological recognition of poly (DL lactide co-glycolide) (PLG), poly (epsilon-caprolactone) (PCL), and poly (L lactide) (PLA), biomaterials widely used in medical implants. Three cell-model systems consisting of human osteoblasts, endothelial cells (ECV-304), and Swiss 3T3 fibroblasts were utilized, in which tTgase expression was modulated by gene transfer, and the ability of cells to spread on these polymers was quantified in relation to the altered level of expressed tTGase. Results show that over-expression of tTgase in human osteoblasts positively correlated with cell spreading on PLG, while no attachment and spreading was found on PCL and PLA. Antisense silencing of tTgase in the endothelial cells led to a marked reduction of cell spreading on all polymers. The hydrophobic nature of PLC also appeared to favor endothelial cell attachment. Spreading of Swiss 3T3 fibroblasts on these biomaterials was only slightly affected by increased expression of tTgase, although cell spreading on control glass was increased. We propose that the consideration of tTgase-mediated bioactivity in novel biomaterials may improve cell attachment and promote biocompatibility.

Release of bioactive human growth hormone from a biodegradable material : Poly(ε-caprolactone)

We have characterized the biodegradable material poly(ϵ-caprolactone) (PCL) as a delivery system for recombinant human growth hormone (hGH). Two contrasting methods for the manufacture of the biomaterial were investigated: namely, solvent casting and solvent casting particulate leaching; the latter yielded porous PCL discs. The degree of porosity, which was assessed by scanning electron microscopy, could be controlled by incorporating selected concentrations of particulate sodium chloride during the manufacturing process. Bioactive hGH released from the PCL preparations was quantified with a highly sensitive and precise bioassay which was based upon hGH activation of rat lymphoma Nb2 cells. Eluates obtained from control discs of PCL which had not been loaded with hGH proved to be nontoxic when tested on these cells. The release of bioactive hGH from hormone-loaded nonporous discs of PCL was found to be a direct function of the initial hormone loading dose. Increased porosity of the discs manufactured by solvent casting particulate leaching increased the delivery of hGH from discs which had been immersion loaded. However, hGH release after surface loading was independent of porosity. Hormone concentrations were also assessed by immunoassay so that the ratios of bio- to immunoactivity (B:I ratio) of the hormone release could be determined. We found that the B:I ratio of the hormone after release from unstored discs was identical to that of the hormone prior to its incorporation into the PCL, demonstrating that the mild incorporation procedures utilized had not adversely affected the structural integrity of the hormone. However, if the hormone-loaded discs were stored at 37°C prior to elution, the B:I ratios of the hGH released decreased indicating that this compromised the bioactive site.

Investigation into the release of bioactive recombinant human growth hormone from normal and low-viscosity poly(methylmethacrylate) bone cements

Previous studies showed that recombinant human growth hormone (hGH) released from hormone-loaded poly(methylmethacrylate) (PMMA) cement stimulated osteoid formation in a rabbit model. Local delivery of hGH from cemented hip arthroplasties may thereby provide a means of reducing the problem of aseptic loosening. We have investigated two different formulations of PMMA as delivery systems for bioactive hGH. The bioactivity of the hormone release in vitro was monitored with an eluted stain assay (ESTA). The hGH was also measured by an immunoassay, which provides an alternative assessment of structural integrity of the hormone released. In addition, we adapted the ESTA bioassay to assess the in vitro cytotoxicity of the cements. Using unloaded cements, the undiluted eluates from both types of PMMA proved cytotoxic. This cytotoxicity could be diluted out, and the procedure allowed us to measure the bioactivity of hGH in the eluates from hormone-loaded cements independent of their cytotoxicity. The major fraction of the bioactivity was released from both of the PMMA cements during the first 24 h, but the hormone remained detectable in eluates collected after 36 days of elution. Comparison of the bio- and immunoactivity of the hGH released showed that the ratio of these two activities (i.e., the B:I ratio) was constant over this time period. However in parallel studies in which hormone-loaded discs were stored under dry conditions prior to elution, we found that the B:I ratio then declined markedly. This suggests that fully hydrated conditions, such as when the discs are bathed in assay medium, are necessary to maintain the bioactivity of the hGH. Both cements released only approximately 1% of the hormone originally incorporated, but the hGH concentration which accumulated in the eluates were high in physiologic terms (approximately 1000 mU/L).