Cell responses to biomimetic protein scaffolds used in tissue repair and engineering.

Basic science research in tissue engineering and regenerative medicine aims to investigate and understand the deposition, growth, and remodeling of tissues by drawing together approaches from a range of disciplines. This review discusses approaches that use biomimetic proteins and cellular therapies, both in the development of clinical products and of model platforms for scientific investigation. Current clinical approaches to repairing skin, bone, nerve, heart valves, blood vessels, ligaments, and tendons are described and their limitations identified. Opportunities and key questions for achieving clinical goals are discussed through commonly used examples of biomimetic scaffolds: collagen, fibrin, fibronectin, and silk. The key questions addressed by three-dimensional culture models, biomimetic materials, surface chemistry, topography, and their interaction with cells in terms of durotaxis, mechano-regulation, and complex spatial cueing are reviewed to give context to future strategies for biomimetic technology.

An efficient method and device for transfer of semisolid materials into solid-state NMR spectroscopy rotors.

The study of mass-limited biological samples by magic angle spinning (MAS) solid-state NMR spectroscopy critically relies upon the high-yield transfer of material from a biological preparation into the MAS rotor. This issue is particularly important for maintaining biological activity and hydration of semi-solid samples such as membrane proteins in lipid bilayers, pharmaceutical formulations, microcrystalline proteins and protein fibrils. Here we present protocols and designs for rotor-packing devices specifically suited for packing hydrated samples into Pencil-style 1.6 mm, 3.2 mm standard, and 3.2 mm limited speed MAS rotors. The devices are modular and therefore readily adaptable to other rotor and/or ultracentrifugation tube geometries.

Development of an RNA isolation procedure for the characterisation of human endothelial cell interactions with polyurethane cardiovascular bypass grafts.

To date no reliable method has been developed for the isolation of RNA from cells seeded onto cylindrical vascular grafts. This study was performed in order to develop a reliable methodology for isolating RNA from cylindrical conduits made from poly(carbonate-urea)urethane (PU). Human umbilical vein EC were seeded onto PU vascular grafts and an Alamar blue assay performed to assess cell viability. Cells were prepared for RNA extraction by trypsinisation, cell scraping and direct application of cell lysis buffer. In all cases RNA was extracted using a "Qiagen RNeasy" kit. Alamar blue showed viable cells were present on all of the seeded PU vascular grafts. Levels of RNA extracted from the cells removed from the graft by the trypsinisation yielded 0.130 microg/microl, by scraping 0.078 microg/microl and by direct lysing 0.093 microg/microl of RNA, respectively. RTPCR was conducted successfully for GAPDH and TGF-beta1. Trypsinisation prior to RNA extraction provided the highest RNA yield and attained near complete cell removal ensuring that gene expression obtained was representative.

Neural tissue engineering: a self-organizing collagen guidance conduit.

We report a novel implantable device that will deliver a tethered aligned collagen guidance conduit containing Schwann cells into a peripheral nerve injury site. Cells (Schwann cells and fibroblasts) incorporated into tethered rectangular collagen gels contracted and resulted in uniaxial alignment. This tissue-engineered construct was tested in three-dimensional culture and demonstrated the ability to guide neurite extension from dissociated dorsal root ganglia. A silicone tube was adapted to provide tethering sites for an implantable construct such that uniaxial cell-generated tension resulted in the formation of a bridge of aligned collagen fibrils, with a resident Schwann cell population. The potential of this device for surgical nerve regeneration was assessed in a 5-mm defect in a rat sciatic nerve model. Neural regeneration through this device was significantly greater than in controls, demonstrating that this system has potential both as a simple robust clinical implant and as a three-dimensional engineered tissue model.

Photographic-Based Optical Evaluation of Tissues and Biomaterials Used for Corneal Surface Repair: A New Easy-Applied Method.

PURPOSE: Tissues and biomaterials used for corneal surface repair require fulfilling specific optical standards prior to implantation in the patient. However, there is not a feasible evaluation method to be applied in clinical or Good Manufacturing Practice settings. In this study, we describe and assess an innovative easy-applied photographic-based method (PBM) for measuring functional optical blurring and transparency in corneal surface grafts. METHODS: Plastic compressed collagen scaffolds (PCCS) and multilayered amniotic membranes (AM) samples were optically and histologically evaluated. Transparency and image blurring measures were obtained by PBM, analyzing photographic images of a standardized band pattern taken through the samples. These measures were compared and correlated to those obtained applying the Inverse Adding-Doubling (IAD) technique, which is the gold standard method. RESULTS: All the samples used for optical evaluation by PBM or IAD were histological suitable. PCCS samples presented transmittance values higher than 60%, values that increased with increasing wavelength as determined by IAD. The PBM indicated that PCCS had a transparency ratio (TR) value of 80.3 ± 2.8%, with a blurring index (BI) of 50.6 ± 4.2%. TR and BI obtained from the PBM showed a high correlation (ρ>|0.6|) with the diffuse transmittance and the diffuse reflectance, both determined using the IAD (p<0.005). The AM optical properties showed that there was a largely linear relationship between the blurring and the number of amnion layers, with more layers producing greater blurring. CONCLUSIONS: This innovative proposed method represents an easy-applied technique for evaluating transparency and blurriness of tissues and biomaterials used for corneal surface repair.

Stabilisation of cables of fibronectin with micromolar concentrations of copper: in vitro cell substrate properties.

We have previously described the production of large cables of fibronectin, a large extracellular matrix cell adhesion glycoprotein, which has a potential application in tissue engineering. Here we have stabilised these cables for longer survival and looked at their ultrastructural cell-substrate behaviour in vitro. Dissolution experiments showed that low concentrations of copper not only caused significant material stabilisation but left pores which could promote cell ingrowth, as we have previously reported with Fn-mats. Indeed, the greatest amount of cell ingrowth was observed for copper treated cables. Immunostaining showed S-100(+) multi-layers of cells around the edge of cables while ultrastructural analysis confirmed the presence of a mixture of fibroblasts and bipolar cells associated with fragments of basal lamina, which is a Schwann cell phenotype. Interestingly, the outermost layers of cells consisted of S-100(-) cells, presumed fibroblasts, apparently 'capping' the Schwann cells. Toxicity tests revealed that Schwann cells were only able to grow at the lowest concentration of copper used (1microM) while fibroblasts grew at all concentrations tested. These results could be used to design biomaterials with optimum properties for promoting cellular ingrowth and survival in tissue engineered grafts which may be used to improve peripheral nerve repair.

Fluid shear in viscous fibronectin gels allows aggregation of fibrous materials for CNS tissue engineering.

Fibronectin (Fn) materials prepared from human plasma have been used in various forms as substrates for tissue engineering. Such purposes require that the soluble protein aggregates into insoluble fibrous structures which encourage the attachment and migration of cells. The method of aggregation due to mechanical shear was investigated by applying fluid shear forces directly to a viscous solution of Fn. Structural analysis revealed that mechanical shear resulted in the formation of an orientated fibrous protein material that was less soluble than its non-sheared counterpart. The suitability of this shear aggregated Fn material for CNS repair purposes was assessed in vitro where it supported the growth of fibroblasts, S100 immunoreactive Schwann cells and GFAP immunoreactive astrocytes. Implantation of the shear aggregated Fn material into a rat model of spinal cord injury provided a permissive environment for axonal growth. This was extended using an impermeable coating to improve orientation and straightness of axonal growth.

Roofed grooves: rapid layer engineering of perfusion channels in collagen tissue models.

Surface patterning (micro-moulding) of dense, biomimetic collagen is a simple tool to produce complex tissues using layer-by-layer assembly. The aim here was to channelise three-dimensional constructs for improved perfusion. Firstly, collagen fibril accumulation was measured by comparative image analysis to understand the mechanisms of structure formation in plastically compressed collagen during µ-moulding. This showed that shape (circular or rectangular) and dimensions of the template affected collagen distribution around moulded grooves and consequently their stability. In the second part, this was used for effective fabrication of multi-layered plastically compressed collagen constructs with internal channels by roofing the grooves with a second layer. Using rectangular templates of 25/50/100 µm widths and 75 µm depth, grooves were µ-moulded into the fluid-leaving surface of collagen layers with predictable width/depth fidelities. These grooves were then roofed by addition of a second plastically compressed collagen layer on top to produce µ-channels. Resulting µ-channels retained their dimensions and were stable over time in culture with fibroblasts and could be cell seeded with a lining layer by simple transfer of epithelial cells. The results of this study provide a valuable platform for rapid fabrication of complex collagen-based tissues in particular for provision of perfusing microchannels through the bulk material for improved core nutrient supply.

Pre-crosslinked polymeric collagen in 3-D models of mechanically stiff tissues: blended collagen polymer hydrogels for rapid layer fabrication.

Currently one factor hindering the development of collagen hydrogel constructs for tissue engineering is the mismatch between initial cellularity and mechanical strength. The main advantage of collagen hydrogel tissue constructs is their ability to support interstitially seeded cells. However, cells are sensitive to their environment, in particular, substrate stiffness, which cannot easily be replicated within hydrogels without cytotoxic cross-linking treatment. In this study, pre-crosslinked polymeric collagen fibrils are introduced as a starting material, thereby avoiding artificial cross-linking. Shear aggregation of this material in solution results in fibril alignment, but cell addition is only possible when polymeric collagen is blended with its monomeric counterparts to slow the aggregation of collagen fibrils. The hydrogel can then be brought to physiological collagen density by plastic compression. Interstitially seeded fibroblasts were supported for 14days. Although compression of blended gels resulted in some cell death due to increased rate of fluid expulsion, not normally seen in conventional collagen hydrogels, the surviving cell population recovers during subsequent culture. Importantly, the compression process can be controlled and customized to limit cell damage. This is the first report of native polymeric collagen used in a tissue engineering context, for the rapid production of a stiff collagen-cell constructs.

Less is more: new biomimetic approach to control spatial and temporal cell loading for tissue engineering.

It is increasingly recognized that use of stiff biodegradable polymers in connective tissue engineering has an inherent flaw. Although polymer stiffness has early benefit for mechanical strength of implants, such pseudoprosthetic material function inevitably stress shields embedded cells, switching off their synthetic/remodeling functions. This core conundrum represents a tension between early mechanical benefits of polymer stiffness against blocking of cell load-dependent matrix production. In effect, an ideal system would produce a gradual, transfer of load onto resident cells and their matrix. Toward this target, our "less is more" (LiM) hypothesis proposes that less stress shielding (polymer stiffness) will lead to more cell-dependent tissue formation. To test this we have designed a hybrid segregation solution in which the cells are segregated into a native (but weak) collagen-gel matrix while the external mechanical loading is taken by temporary, reinforcing polyglycolic acid (PGA) fibers, with gradual, load transfer as the polymer µ-fibers fracture. Dermal fibroblasts grew predictably in the hybrid construct and the fine, parallel PGA fibers fractured and fragmented due to hydrolysis, giving a fall of construct stiffness to near collagen-only levels, over 14 days. The same fiber fracture and fall in stiffness occurred over 14 days in constructs implanted in vivo. In this case a cell dependent, net enhancement of connective tissue stiffness could be identified in hybrid constructs, supporting the LiM hypothesis for cytomechanical control of matrix. This is the first demonstration of spatiotemporal load transfer as a customizable tool for improved, biomimetic connective tissue engineering.

Direct collagen-material engineering for tissue fabrication.

So what is the "big deal" about engineering of collagen materials? It is certainly not enough to produce the familiar line about it being "more physiological." This phrase explains very little of the substance and gets nowhere near to the base of the question. We need to be clear about why this is important enough to justify a major new research investment.

Development of conical soluble phosphate glass fibers for directional tissue growth.

One of the challenges of tissue engineering is the regulation of vascularization and innervations of the implant by the host. Here, we propose that using soluble phosphate glass (SPG) fibers, incorporated in dense collagen constructs will allow us to control the rate and direction of tissue ingrowth. The idea here was to generate channels with tailored direction using conical phosphate glass fibers. The changing surface area-to-mass ratio of conical fibers will make them to dissolve faster from their narrow ends opening up channels in that direction ahead of any ingrowing cells. In this study, we show that SPG fibers can be manipulated to produce conical shape fibers using graded dissolution. Our result shows that 40 µm fibers of composition ratio 0.5 (P(2)O(5)):0.25 (CaO):0.25 (Na(2)O) and dissolution time of 8-10 h have a mean reduction in fiber diameter of 8.85 ± 2.8 µm over 19.5 mm fiber length, i.e., a mean rate of 0.5 µm/mm (n=20) change. These conically shaped fibers can also be manipulated and potentially used to promote uniaxial cell-tissue ingrowth for improved innervations and vascularization of tissue engineered constructs.

Hyaluronan hydration generates three-dimensional meso-scale structure in engineered collagen tissues.

Here, we show that the local incorporation of osmotically active hyaluronan into previously compressed collagen constructs results in further rapid dehydration/compression of collagen layers, channel formation and generation of new interfaces; these novel structures, at the nano-micro (i.e. meso-scale) were formed within native collagen gels, in a highly predictable spatial manner and offer important new methods of fabricating scaffolds (e.g. tubes and open-spirals) with potential for use in tissue regeneration such as in peripheral nerves and small vessels. This paper tests the possibility that the local fluid content of a dense collagen network can be controlled by incorporation of an osmotically active (native) macromolecule--hyluronan. This is an exemplar physiological, osmotic swelling agent. Hyaluronan is commonly secreted by cells deep in connective tissues, so is a good candidate for this role in a cell-driven system balancing mechanical compaction of bulk tissue collagen. These constructs may have potential as functional in vitro models representing developmental and pathological processes.

Plastic compressed collagen as a biomimetic substrate for human limbal epithelial cell culture.

We describe, for the first time, the use of cellular plastic compressed collagen as a substrate for human limbal epithelial cell expansion and stratification. The characteristics of expanded limbal epithelial cells on either acellular collagen constructs or those containing human limbal fibroblasts were compared to a human central cornea control. After compression, human fibroblasts in collagen constructs remained viable and limbal epithelial cells were successfully expanded on the surface. After airlifting, a multilayered epithelium formed with epithelial cell morphology very similar to that of cells in the central cornea. Immunochemical staining revealed expression of basement membrane proteins and differentiated epithelial cell markers found in native central cornea. Ultrastructural analysis revealed cells on collagen constructs had many features similar to central cornea, including polygonal, tightly opposed surface epithelial cells with microvilli and numerous desmosomes at cell-cell junctions. Taken together, these data demonstrate that plastic compressed collagen constructs can form the basis of a biomimetic tissue model for in vitro testing and could potentially provide a suitable alternative to amniotic membrane as a substrate for limbal epithelial cell transplantation.

The use of injectable forms of fibrin and fibronectin to support axonal ingrowth after spinal cord injury.

Many studies have described biomaterial devices (conduits and scaffolds) that can be implanted into experimental lesions and which support axonal growth. However, a disadvantage of such pre-formed devices is that tissue needs to be excised to allow their insertion. In this study we have therefore examined four biomaterials that can be injected into an injury site and which gel in situ; namely collagen, viscous fibronectin, fibrin, and fibrin + fibronectin (FB/FN). The materials were tested in an experimental knife-cut cavity in the rat spinal cord, and evaluated at 1 week and 4 weeks survival for their biocompatibility, neuroprotective efficacy, and permissiveness for axonal growth. At one week, all four materials showed good integration with the host spinal cord and supported some degree of axonal ingrowth, which was associated with infiltration of Schwann cells and deposition of laminin. However axon growth in the collagen implants was uneven because implants contained dense inclusions which were not penetrated by axons. At 4 weeks, axon growth was greatest in the fibronectin and FB/FN implants, however the fibronectin implants had large cavities at the interface between the implant and host spinal cord. The fibronectin implants also had fewer surviving neurons in the intact spinal cord adjoining the implant site. The FB/FN mixture thus had the best combination of properties in that it was easy to handle, integrated with the host spinal cord tissue, and supported robust growth of axons. It therefore has promise as an injectable biomaterial for filling cavities at spinal cord injury sites.

A poly(lactic acid-co-caprolactone)-collagen hybrid for tissue engineering applications.

A biodegradable hybrid scaffold consisting of a synthetic polymer, poly(lactic acid-co-caprolactone) (PLACL), and a naturally derived polymer, collagen, was constructed by plastic compressing hyperhydrated collagen gels onto a flat warp-knitted PLACL mesh. The collagen compaction process was characterized, and it was found that the duration, rather than the applied load under the test conditions in the plastic compression, was the determining factor of the collagen and cell density in the cell-carrying component. Cells were spatially distributed in three different setups and statically cultured for a period of 7 days. Short-term biocompatibility of the hybrid construct was quantitatively assessed with AlamarBlue and qualitatively with fluorescence staining and confocal microscopy. No significant cell death was observed after the plastic compression of the interstitial equivalents, confirming previous reports of good cell viability retention. The interstitial, epithelial, and composite tissue equivalents showed no macroscopic signs of contraction and good cell proliferation with a two- to threefold increase in cell number over 7 days. Quantitative analysis showed a homogenous cell distribution and good biocompatibility. The results indicate that viable and proliferating multilayered tissue equivalents can be engineered using the PLACL-collagen hybrid construct in the space of several hours.

Bulk collagen incorporation rates into knitted stiff fibre polymer in tissue-engineered scaffolds: the rate-limiting step.

Fabrication of tissue-engineered constructs in vitro relies on sufficient synthesis of extracellular matrix (ECM) by cells to form a material suitable for normal function in vivo. Collagen synthesis by human dermal fibroblasts grown in vitro on two polymers, polyethylene terephthalate (PET) and polyglycolic acid (PGA), was measured by high-performance liquid chromatography (HPLC). Cells were either cultured in a dynamic environment, where meshes were loaded onto a pulsing tube in a bioreactor, or in a static environment without pulsing. Collagen synthesis by cells cultured on a static mesh increased by six-fold compared to monolayer culture, and increased by up to a further 5.4-fold in a pulsed bioreactor. However, little of the collagen synthesized was deposited onto the meshes, almost all being lost to the medium. The amount of collagen deposited onto meshes was highest when cells were cultured dynamically on PET meshes (17.6 microg), but deposition still represented only 1.4% of the total synthesized. Although total collagen synthesis was increased by the use of 3D culture and the introduction of pulsing, the results suggest that the limiting factor for fabrication of a tissue-engineered construct within practical timescales is not the amount of collagen synthesized but the quantity retained (i.e. deposited) within the construct during culture. This may be enhanced by systems which promote or assemble true 3D multi-layers of cells.

Effect of multiple unconfined compression on cellular dense collagen scaffolds for bone tissue engineering.

Plastic compression of hydrated collagen gels rapidly produces biomimetic scaffolds of improved mechanical properties. These scaffolds can potentially be utilised as cell seeded systems for bone tissue engineering. This work investigated the influence of multiple unconfined compression on the biocompatibility and mechanical properties of such systems. Single and double compressed dense collagen matrices were produced and characterised for protein dry weight, morphology and mechanical strength. Compression related maintenance of the seeded HOS TE85 cell line viability in relation to the extent of compression was evaluated up to 10 days in culture using the TUNEL assay. Fluorescence Live/Dead assay was conducted to examine overall cell survival and morphology. Cell induced structural changes in the dense collagenous scaffolds were assessed by routine histology. The mechanical properties of the cellular scaffolds were also evaluated as a function of time in culture. It is clear that a single plastic compression step produced dense collagenous scaffolds capable of maintaining considerable cell viability and function as signs of matrix remodeling, and maintenance of mechanical properties were evident. Such scaffolds should therefore be further developed as systems for bone tissue regeneration.

Evidence for sequential utilization of fibronectin, vitronectin, and collagen during fibroblast-mediated collagen contraction.

Contraction plays a major role in wound healing and is inevitably mediated through the mechanical interaction of fibroblast cytoskeleton and integrins with their extracellular matrix ligands. Cell-matrix attachment is critical for such events. In human dermal fibroblasts most such interactions are mediated by the beta1-type integrins. This study investigated the role played by key components in this system, notably fibronectin, vitronectin, and integrin subcomponents alpha2 and alpha5, which recognize collagen and fibronectin. Inhibition of adhesion through these ligands was studied either by antibody blocking or with fibronectin and/or vitronectin depletion. Functional effects of inhibition were monitored as force generation in collagen-glycosaminoglycan (IntegraTM) sponges, over 20 hours using a culture force monitor. Dose and time-course inhibition studies indicated that initial attachment and force generation (approx. 0-5 hours postseeding) was through fibronectin receptors and this was followed by vitronectin ligand and receptor utilization (4 hours onward). Utilization of the collagen integrin subcomponent alpha2 appeared to be increasingly important between 6 and 16 hours and dominant thereafter. Additionally, there was evidence for functional interdependence between the three ligand systems fibronectin, vitronectin, and collagen. We propose that there is a short cascade of sequential integrin-ligand interactions as cells attach to, extend through, and eventually contract their matrix. (WOUND REP REG 2002;10:-408)