Assessment of mesh shrinkage using fibroblast-populated collagen matrices: a proof of concept for in vitro hernia mesh testing.

PURPOSE: This study uses free-floating contractile fibroblast-populated collagen matrices (FPCMs) to test the shrinkage of different hernia mesh products. We hope to present this model as a proof of concept for the development of in vitro hernia mesh testing-a novel technology with interesting potential. METHODS: FPCMs were formed by seeding Human Dermal Fibroblasts into collagen gels. FPCMs were seeded with three different cell densities and cast at a volume of 500 µl into 24-well plates. Five different mesh products were embedded within the collagen constructs. Gels were left to float freely within culture media and contract over 5 days. Photographs were taken daily and the area of the collagen gel and mesh were measured. Media samples were taken at days 2 and 4 for the purposes of measuring MMP-9 release. After 5 days, dehydrated FPCMs were also examined under light and fluorescence microscopy to assess cell morphology. RESULTS: Two mesh products-the mosquito net and large pore lightweight mesh were found to shrink notably more than others. This pattern persisted across all three cell densities. There were no appreciable differences observed in MMP-9 release between products. CONCLUSIONS: This study has successfully demonstrated that commercial mesh products can be successfully integrated into free-floating contractile FPCMs. Not only this, but FPCMs are capable of applying a contractile force upon those mesh products-eliciting different levels of contraction between mesh products. Such findings demonstrate this technique as a useful proof of concept for future development of in vitro hernia mesh testing.

The experimental methodology and comparators used for in vivo hernia mesh testing: a 10-year scoping review.

PURPOSE: Before being marketed, hernia mesh must undergo in vivo testing, which often includes biomechanical and histological assessment. Currently, there are no universal standards for this testing and methods vary greatly within the literature. A scoping review of relevant studies was undertaken to analyse the methodologies used for in vivo mesh testing. METHODS: Medline and Embase databases were searched for relevant studies. 513 articles were identified and 231 duplicates excluded. 126 papers were included after abstract and full text review. The data extraction was undertaken using standardised forms. RESULTS: Mesh is most commonly tested in rats (53%). 78% of studies involve the formation of a defect; in 52% of which the fascia is not opposed. The most common hernia models use mesh to bridge an acute defect (50%). Tensile strength testing is the commonest form of mechanical testing (63%). Testing strip widths and test speeds vary greatly (4-30 mm and 1.625-240 mm/min, respectively). There is little consensus on which units to use for tensile strength testing. Collagen is assessed for its abundance (54 studies) more than its alignment (18 studies). Alignment is not measured quantitatively. At least 21 histological scoring systems are used for in vivo mesh testing. CONCLUSIONS: The current practice of in vivo mesh testing lacks standardisation. There is significant inconsistency in every category of testing, both in methodology and comparators. We would call upon hernia organisations and materials testing institutions to discuss the need for a standardised approach to this field.

In vitro characterisation of low-cost synthetic meshes intended for hernia repair in the UK.

PURPOSE: Low-cost meshes (LCM) were repurposed for the repair of hernias in the developing world. In vivo studies have shown LCM to have comparable results to commercial meshes (CM) at a fraction of the cost. However, little has been done to characterise the mechanical and biocompatible properties of LCM, preventing its clinical use in the UK. The objectives of the research are to assess mechanical and ultrastructural properties of two UK-sourced low-cost meshes (LCM) and the characterisation of the LCMs in vitro biocompatibility. METHODS: Mechanical properties of the two LCM were measured through uniaxial tensile test and ultrastructure was evaluated with Scanning Electron Microscopy. LIVE/DEAD® Viability/Cytotoxicity Assay kit and alamarBlue were used to assess cellular viability and proliferation, respectively. Images were acquired with a fluorescence microscope and analysed using ImageJ (NIH, USA). RESULTS: LCM1 and LCM2 were both multifilament meshes, with the first having smaller pores than the latter. LCM1 exhibited significantly higher tensile strength (p < 0.05) than LCM2 but significantly lower extensibility (p < 0.0001), while Young's Modulus of the two samples was not significantly different. No significant difference was found in the cellular viability and morphology cultured in LCM1 and LCM2 conditioned media. Metabolic assay and fluorescence imaging showed cellular attachment and proliferation on both LCMs over 14 days. CONCLUSION: The characterisation of the two UK-sourced LCMs showed in vitro biocompatibility and mechanical and ultrastructural properties comparable to the equivalent CM. This in vitro data represents a step forward for the feasibility of adopting LCM for surgical repair of hernias in the UK.

Acute mechanical overload increases IGF-I and MMP-9 mRNA in 3D tissue-engineered skeletal muscle.

Skeletal muscle (SkM) is a tissue that responds to mechanical load following both physiological (exercise) or pathophysiological (bed rest) conditions. The heterogeneity of human samples and the experimental and ethical limitations of animal studies provide a rationale for the study of SkM plasticity in vitro. Many current in vitro approaches of mechanical loading of SkM disregard the three-dimensional (3D) structure in vivo. Tissue engineered 3D SkM, that displays highly aligned and differentiated myotubes, was used to investigate mechano-regulated gene transcription of genes implicated in hypertrophy/atrophy. Static loading (STL) and ramp loading (RPL) at 10 % strain for 60 min were used as mechano-stimulation with constructs sampled immediately for RNA extraction. STL increased IGF-I mRNA compared to both RPL and CON (control, p = 0.003 and 0.011 respectively) whilst MMP-9 mRNA increased in STL and RPL compared to CON (both p < 0.05). IGFBP-2 mRNA was differentially regulated in RPL and STL compared to CON (p = 0.057), whilst a reduction in IGFBP-5 mRNA was found for STL and RPL compared to CON (both p < 0.05). There was no effect in the expression of putative atrophic genes, myostatin, MuRF-1 and MAFBx (all p > 0.05). These data demonstrate a transcriptional signature associated with SkM hypertrophy within a tissue-engineered model that more greatly recapitulates the in vivo SkM structure compared previously published studies.

Characterization and optimization of a simple, repeatable system for the long term in vitro culture of aligned myotubes in 3D.

Increased recent research activity in exercise physiology has dramatically improved our understanding of skeletal muscle development and physiology in both health and disease. Advances in bioengineering have enabled the development of biomimetic 3D in vitro models of skeletal muscle which have the potential to further advance our understanding of the fundamental processes that underpin muscle physiology. As the principle structural protein of the extracellular matrix, collagen-based matrices are popular tools for the creation of such 3D models but the custom nature of many reported systems has precluded their more widespread adoption. Here we present a simple, reproducible iteration of an established 3D in vitro model of skeletal muscle, demonstrating both the high levels of reproducibility possible in this system and the improved cellular architecture of such constructs over standard 2D cell culture techniques. We have used primary rat muscle cells to validate this simple model and generate comparable data to conventional established cell culture techniques. We have optimized culture parameters for these cells which should provide a template in this 3D system for using muscle cells derived from other donor species and cell lines.

Mechanisms of structure generation during plastic compression of nanofibrillar collagen hydrogel scaffolds: towards engineering of collagen.

Operator control of cell/matrix density of plastically compressed collagen hydrogel scaffolds critically depends on reproducibly limiting the extent of scaffold compaction, as fluid expulsion. A functional model of the compression process is presented, based on the idea that the main fluid-leaving surface (FLS) behaves as an ultrafiltration membrane, allowing fluid (water) out but retaining collagen fibrils to form a cake. We hypothesize that accumulation of collagen at the FLS produces anisotropic structuring but also increases FLS hydraulic resistance (R(FLS) ), in turn limiting the flux. Our findings show that while compressive load is the primary determinant of flux at the beginning of compression (load-dependent phase), increasing FLS collagen density (measured by X-ray attenuation) and increasing R(FLS) become the key determinants of flux as the process proceeds (flow-dependent phase). The model integrates these two phases and can closely predict fluid loss over time for a range of compressive loads. This model provides a useful tool for engineering cell and matrix density to tissue-specific levels, as well as generating localized 3D nano micro-scale structures and zonal heterogeneity within scaffolds. Such structure generation is important for complex tissue engineering and forms the basis for process automation and up-scaling.

First implantable device for hypoxia-mediated angiogenic induction.

Delayed or inadequate vascularisation is one of the major factors leading to tissue infarction and poor graft survival. Current vascularisation strategies that rely on delivering single growth factors have proved ineffective or hard to control in practise. An alternative approach has been identified by this group that relies on stimulation of physiological angiogenic factor cascades by engineering local cell-hypoxia, within a nano-fibrillar collagen material. Here we report on a novel, practical and effective implantable device for delivering engineered angiogenic signalling, on demand. Human dermal fibroblast-seeded dense-collagen depots were pre-conditioned under physiological cell-generated hypoxia to up-regulate production of key angiogenic factors, including HIF1α and VEGF(165). The level of VEGF(165) protein retained within depots (indicating general angiogenic factor production) was directly correlated to the duration of pre-conditioning. Angiogenic factor delivery from pre-conditioned, non-viable depots rapidly induced an angiogenic response within endothelial cell-seeded constructs in vitro, while implanted acellular 3D constructs incorporating such angiogenic depots in their core were infiltrated with perfused vessels by 1 week in vivo, at which stage non-angiogenic implants were minimally perfused. Depot stability, tuneability of cell/matrix composition with long clinical experience of the collagen material, together with cost effectiveness, make this angiogenic therapy a promising addition to a clinician's tool kit for improving local tissue perfusion.

Shear-aggregated fibronectin with anti-adhesive properties.

Biomaterials based on proteins, such as fibronectin, have the potential to guide cell and tissue behaviour during healing as a function of their unique mechanical and bioactive properties. Fibronectin has been reported as a scaffold for attachment of fibroblasts and subsequent deposition of collagen. We have recently developed a derivative process of shear-aggregated fibronectin that prevents cell attachment without causing cell death. This has potential applications in clinical situations where adhesions form across gliding surfaces and cause loss of function, e.g. peritoneal or flexor tendon adhesions. This in vitro study tested this derivative fibronectin biomaterial and its effects on aggressive adhesion-forming cells, using rabbit flexor tendon synovial fibroblasts. We investigated degradation of the novel biomaterial, and attachment of fibroblasts to glass coated with the biomaterial, relative to fibroblast attachment to uncoated and fibronectin-coated glass. We assessed infiltration of the derivative fibronectin biomaterial by fibroblasts and cytotoxicity of the biomaterial to fibroblasts. The interaction between fibroblasts and the derivative fibronectin biomaterial was visualized using time-lapse photography. The derivative fibronectin biomaterial dissolved by 88% of its mass by 3 weeks. Fibroblast attachment to the novel biomaterial was significantly reduced at 6 h. After 24 h of exposure to the novel biomaterial, fibroblasts did not migrate into it, there was no cell death and no attachment was seen using time-lapse. This novel derivative fibronectin biomaterial combines inhibition of fibroblast attachment with barrier effects and has suitable mechanical properties for surgical use in preventing adhesions in vivo.

Controlling physiological angiogenesis by hypoxia-induced signaling.

The full sequence of signals leading to new blood vessel formation is a physiological response to tissue hypoxia through upregulation of angiogenic factor cascades. Controlled initiation of this mechanism for therapeutic/engineered angiogenesis must rely on precisely localized hypoxia. Here we have designed a 3D in vitro model able to test the effect and predictability of spatially positioned local hypoxic stimuli using defined cell depots within a 3D collagen matrix. Cell-mediated hypoxia was engineered using human dermal fibroblasts (HDFs), to generate a local population of Hypoxia-Induced Signaling (HIS) cells. HIS cell depots released angiogenic factors which induced directional endothelial cell (EC) migration and tubule formation in a spatially defined assay system. Non-hypoxic baseline control cultures induced minimal EC migration with little tubule formation. Furthermore, depots of HIS cells, positioned in the core of 3D collagen constructs directed host vessel in-growth deep into the implant by 1 week, which was at least 7 days earlier than in non-hypoxia pre-conditioned constructs. The functionality of in vivo vascularisation was verified by real-time monitoring of O2 levels in the core of implanted constructs. These findings establish the angiogenic potential of HIS cells applicable to in vitro tissue modeling, implant vascularization and engineering predictable angiogenic therapies.

The effect of cell density on the maturation and contractile ability of muscle derived cells in a 3D tissue-engineered skeletal muscle model and determination of the cellular and mechanical stimuli required for the synthesis of a postural phenotype.

The successful engineering of a truly biomimetic model of skeletal muscle could have a significant impact on a number of biomedical disciplines. Although a variety of techniques are currently being developed, there is, as of yet, no widely available and easily reproducible culture system for the synthesis of 3D artificial muscle tissues. In attempting to generate such a model it is essential to optimise any protocol in order to generate a tissue that best represents the in vivo environment. Since the maturation of muscle derived cells in culture is critically dependent on density, a major factor to be addressed in the development of these models is the ideal concentration at which to seed cells in order to generate an optimal response. In studying the effect of cell density on the performance of cells in an established 3D collagen based model of skeletal muscle, we demonstrate that an optimum density does exist in terms of peak force generation and myogenic gene expression data. Greater densities however, lead to the formation of a more physiologically relevant tissue with a phenotype characteristic of slow, postural muscle.

Interface integration of layered collagen scaffolds with defined matrix stiffness: implications for sheet-based tissue engineering.

Successful application of sheet-based engineering for complex tissue reconstruction requires optimal integration of construct components. An important regulator of cellular responses (such as migration and collagen deposition) mediating interface integration is matrix stiffness. In this study we developed a sheet-based 3D model of interface integration that allows control of interface matrix stiffness. Fluid was removed from acellular or fibroblast-seeded bilayer collagen hydrogel constructs, using plastic compression to increase collagen density and matrix stiffness. Cell-seeded constructs were either compressed at day 0 and cultured for 7 days (compressed culture, high stiffness) or left uncompressed during culture and compressed on day 7 (compliant-compressed culture, low stiffness). Constructs were fitted onto a mechanical testing system to measure interface adhesive strength. Analysis of stresses by finite element modelling predicted a sharp rise of stress and rapid failure at the interface. While cell-seeded constructs showed a six-fold increase in interface adhesive strength compared to acellular control constructs (p < 0.05), there was no significant difference between low- and high-stiffness cultures after 1 week. Cell migration across the interface was greater in low- compared to high-stiffness constructs at 24 h (p < 0.05); however, no significant difference was observed after 1 week. Visualization of interfaces showed fusion of the two layers in low- but not in high-stiffness constructs after 1 week of culture. The ability to regulate cellular behaviour at an interface by controlling matrix stiffness could provide an important tool for modelling the integration of sheet-based bioengineered tissues in bioreactor culture or post-implantation.

Close dependence of fibroblast proliferation on collagen scaffold matrix stiffness.

Human dermal fibroblasts (HDFs) in free-floating collagen matrices show minimal proliferation, although this may increase when the matrix is 'under tension'. We have investigated the detailed mechanics underlying one of the possible controls of this important cell behaviour, in particular the hypothesis that this is a response to substrate stiffness. Hyperhydrated collagen gels were plastic-compressed (PC) to give a predetermined collagen density and stiffness. Mechanical properties were tested using a dynamic mechanical analyser; cell number by Alamar blue assay. In the stiffest PC matrices, cell proliferation was rapid and seeding density-dependent, with a population doubling time of 2 days. In contrast, compliant attached matrices showed a 4 day lag period and a doubling time of 6 days. HDF growth was directly related to matrix stiffness, such that increasing stiffness using a range of compression levels (0-75% fluid removal) supported increasing proliferation rate, doubling times and matrix elastic modulus. HDF quiescence in compliant matrices was reversible, such that increasing stiffness in situ by compression at 1 and 5 days initiated proliferation. We conclude that collagen matrix stiffness regulates proliferation of fibroblasts (a duro-response), with important implications for understanding fibroblast-matrix feedback controls during wound healing and the design and regulation of engineered connective tissues based on collagen and other hydrogel-based scaffolds.

Spatial differences of cellular origins and in vivo hypoxia modify contractile properties of pulmonary artery smooth muscle cells: lessons for arterial tissue engineering.

Tissue engineering of functional arteries is challenging. Within the pulmonary artery wall, smooth muscle cells (PASMCs) have site-specific developmental and functional phenotypes, reflecting differing contractile roles. The force generated by PASMCs isolated from the inner 25% and outer 50% of the media of intrapulmonary elastic arteries from five normal and eight chronically hypoxic (hypertensive) 14 day-old piglets was quantified in a three-dimensional (3D) collagen construct, using a culture force monitor. Outer medial PASMCs from normal piglets exerted more force (528 +/- 50 dynes) than those of hypoxic piglets (177 +/- 42 dynes; p < 0.01). Force generation by inner medial PASMCs from normal and hypoxic piglets was similar (349 +/- 35 and 239 +/- 60 dynes). In response to agonist (thromboxane) stimulation, all PASMCs from normal and hypoxic piglets contracted, but the increase in force generated by outer and inner hypoxic PASMCs (ranges 13-72 and 14-56 dynes) was less than by normal PASMCs (ranges 27-154 and 34-159 dynes, respectively; p < 0.05 for both). All hypoxic PASMCs were unresponsive to antagonist (sodium nitroprusside) stimulation, all normal PASMCs relaxed (range - 87 to - 494 dynes). Myosin heavy chain expression by both hypoxic PASMC phenotypes was less than normal (p < 0.05 for both), as was the activity of focal adhesion kinase, regulating contraction, in hypoxic inner PASMCs (p < 0.01). Chronic hypoxia resulted in the development of abnormal PASMC phenotypes, which in collagen constructs exhibited a reduction in contractile force and reactivity to agonists. Characterization of the mechanical response of spatially distinct cells and modification of their behaviour by hypoxia is critical for successful tissue engineering of major blood vessels.

Collagen stiffness regulates cellular contraction and matrix remodeling gene expression.

Cell-level mechanical and 3D spatial cues are essential to the organization and architecture of new tissues that form during growth, repair or in bioreactors. Fibroblast-seeded 3D collagen constructs have been used as bioartifical extracellular matrix (ECM) providing a 3D environment to embedded resident cells. As cells attach to scaffold fibrils, they generate quantifiable contractile forces which depend on cell type, cell attachment, cell density, growth factors, and matrix stiffness. The aim of this study was to quantify the cytomechanical and molecular responses of human dermal (HDF) and neonatal foreskin fibroblasts (HNFF) seeded in constructs of increased stiffness. We also tested the effect of blocking early attachment using serum starvation on these outputs. Constructs were placed under uniaxial strains of 0-10% to increase scaffold stiffness, prior to gel contraction, and force generation was monitored using a tensional culture force monitor (t-CFM). Increased matrix stiffness reduced generation of quantifiable cellular force (up to 70%) over 24 h in both cell types and delayed the onset of measurable contraction (upto sevenfold). The delay of measurable force generation was cell lineage dependent but not FCS dependent. Gene expression of MMP-2, TIMP-2, and collagen type III expression in HDFs were significantly upregulated in constructs of increased stiffness. HNFFs did not show any significant changes in these gene expressions indicating a lineage specific response.

Feedback inhibition of high TGF-beta1 concentrations on myofibroblast induction and contraction by Dupuytren's fibroblasts.

Myofibroblasts and TGF-beta1 are implicated in Dupuytren's contracture. Transforming growth fact- or beta1(TGF-beta1) (1-10ng/ml) increases myofibroblast induction in Dupuytren's fibroblasts and contraction in a collagen model. However, higher doses (20-30ng/ml) inhibit contraction in dermal fibroblasts. We hypothesized higher doses of TGF-beta1 would inhibit induction of myofibroblasts and contraction by Dupuytren's fibroblasts. Increasing doses of TGF-beta1 (0-30ng/ml) were tested on Dupuytren's fibroblasts using immunofluorescence to determine myofibroblast upregulation and a 3D collagen model used to determine contractile forces. Flexor retinaculum fibroblasts were used as controls. TGF-beta1 induced myofibroblasts in Dupuytren's fibroblasts (n=3) from 12% (0ng/ml) to 23% (12.5ng/ml) at 24 hours but dropped to 13% at 30ng/ml (P<0.05). This response was mirrored in the contraction profiles. These trends were similar for flexor retinaculum fibroblasts (n=3), but contractile forces and myofibroblast induction were significantly less (P<0.001). This is the first report of negative feedback inhibition of TGF-beta1 at higher concentrations in Dupuytren's fibroblasts.

Complex dependence of substrate stiffness and serum concentration on cell-force generation.

Collagen is a widely used biomaterial in tissue engineering. Mechanical stimulation of cell-seeded collagen constructs and its effects on cell orientation, intracellular signaling, and molecular responses have been reported. Our aim was to study the transfer of applied mechanical load to resident cells in 3D collagen constructs. Stainless steel markers were embedded in constructs as reporters of micromovement and uniaxial (0-15%) strain was applied. Cell-seeded collagen constructs were also subjected to (0-15%) uniaxial strain and material responses recorded. The viscoelastic properties of collagen resulted in comparatively small movement of the marker bars relative to gel deformation. Cell seeding density of 1 million/mL had no significant effect on the viscoelastic properties of collagen for the range of strain tested. Our findings indicate that viscoelastic properties of collagen result in minimal force transfer of applied loads as recorded by movement of stainless steel markers. At higher strain rates as collagen got stiffer the movement decreased. These findings indicate that as cell-seeded collagen constructs mature in a bioreactor and become stiffer due to ECM production/deposition, mechanical stimulation will have to be tailored over time to account for increased stiffness of constructs in vitro to elicit predictable and consistent cellular responses.

The different characteristics of Dupuytren's disease fibroblasts derived from either nodule or cord: expression of alpha-smooth muscle actin and the response to stimulation by TGF-beta1.

Mechanisms behind the onset and progression of Dupuytren's disease are poorly understood. Both myofibroblasts and transforming growth factor beta 1 (TGF-beta(1)) have been implicated. We studied fibroblast cultures derived from nodules or cords of Dupuytren's contracture tissue to determine the proportion of myofibroblasts present in comparison with flexor retinaculum fibroblast cultures. We identified myofibroblasts by immunohistochemical staining for alpha-SMA. We then investigated the effects of TGF-beta(1) stimulation on these fibroblasts. Basal myofibroblast/fibroblast proportions were 9.7% in nodule cell cultures, 2.7% in cord cell cultures and only 1.3% in flexor retinaculum cell cultures. Nodule and cord myofibroblast proportions increased to 25.4% and 24.2%, respectively, in response to TGF-beta(1) treatment. Flexor retinaculum cell cultures showed no response to TGF-beta(1) stimulation. Fibroblasts cultured from specific regions of Dupuytren's tissue retain myofibroblast features in culture. TGF-beta(1) stimulation causes an increased myofibroblast phenotype to similar levels in both nodule and cord, suggesting that previously quiescent cord fibroblasts can be reactivated to become myofibroblasts by TGF-beta(1). This could be an underlying reason for high recurrence rates seen after surgery or progression following injury.

3-D in vitro model of early skeletal muscle development.

An understanding of the mechanical and mechano-molecular responses that occur during the differentiation of mouse C2C12 [corrected] myoblasts in 3-D culture is critical for understanding growth, which is important for progress towards producing a tissue-engineered muscle construct. We have established the main differences in force generation between skeletal myoblasts, dermal fibroblasts, and smooth muscle cells in a 3-D culture model in which cells contract a collagen gel construct. This model was developed to provide a reproducible 3-D muscle organoid in which differences in force generation could be measured, as the skeletal myoblasts fused to form myotubes within a collagen gel. Maintenance of the 3-D culture under sustained uni-axial tension, was found to promote fusion of myoblasts to form aligned multi-nucleate myotubes. Gene expression of both Insulin Like Growth Factor (IGF-1 Ea) and an isoform of IGF-1 Ea, Mechano-growth factor (IGF-1 Eb, also termed MGF), was monitored in this differentiating collagen construct over the time course of fusion and maturation (0-7 days). This identified a transient surge in both IGF-1 and MGF expression on day 3 of the developing construct. This peak of IGF-1 and MGF expression, just prior to differentiation, was consistent with the idea that IGF-1 stimulates differentiation through a Myogenin pathway [Florini et al., 1991: Mol. Endocrinol. 5:718-724]. MGF gene expression was increased 77-fold on day 3, compared to a 36-fold increase with IGF-1 on day 3. This indicates an important role for MGF in either differentiation or, more likely, a response to mechanical or tensional cues.

Contraction-mediated pinocytosis of RGD-peptide by dermal fibroblasts: inhibition of matrix attachment blocks contraction and disrupts microfilament organisation.

Force generation in collagen and matrix contraction are basic functions of fibroblasts and important elements of tissue repair. Cell-matrix attachment is critical to this contraction, involving RGD-binding integrins. We have investigated how this process operates, in terms of force generation (in the Culture Force Monitor) and cytoskeletal structure, using a synthetic RGD-decapeptide. The RGD-peptide blocked force generation over the first 6 h, followed by near complete recovery by 20 h. However, dose response was complex indicating multiple processes were operating. Analysis of cytoskeletal structure after treatment with RGD-peptide indicated major disruption with condensed aggregates of actin and microtubular fragmentation. Fluorescent labeling and tracking of the RGD-peptide demonstrated intracellular uptake into discrete cytoplasmic aggregates. Critically, these RGD-peptide pools co-localised with the condensed actin microfilament aggregates. It is concluded that RGD-peptide uptake was by a form of contraction-mediated pinocytosis, resulting from mechanical tension applied to the untethered RGD-peptide-integrin, as contractile microfilament were assembled. These findings emphasize the importance of sound mechanical attachment of ligand-occupied integrins (e.g., to extracellular matrix) for normal cytoskeletal function. Conversely, this aspect of unrestrained cytoskeletal contraction may have important pathogenic and therapeutic applications.

A study of the cellular response to orientated fibronectin material in healing extensor rat tendon.

3D orientated fibronectin (Fn) mats have been used as biocompatible and biodegradeable scaffolds to provide orientated cues using contact guidance for cell migration/adhesion and deposition of extracellular matrix. We have implanted Fn scaffolds in an established rat tendon(partial tenotomy) injury model to test its efficacy and monitor the early cellular and inflammatory response. Tendons were harvested at 0, 6 h, 1, 3, 5, 7 and 14 days for H&E, immunohistochemistry and TEM. Total cell counts within the window increased progressively with time with no significant differences between the Fn scaffolds and controls. CD45 (pan leukocyte) positive cell numbers peaked at 6 h and when expressed as a percentage of total cell counts as determined by H&E staining constituted 20% of the total cell number at 6 h but decreased to 5% of total number by 72 h. There were no significant differences in the inflammatory response between the control and implanted groups. Few CD44 (mesenchymal stem cell) positive cells identified had a surface location. A novel cell with long exaggerated cytoplasmic processes was identified by TEM. Our results show that the Fn scaffold did not degrade or elicit any untoward inflammatory response at the time points tested and has potential use in guiding the repair process.