James B. Phillips

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.

Engineered neural tissue for peripheral nerve repair.

A new combination of tissue engineering techniques provides a simple and effective method for building aligned cellular biomaterials. Self-alignment of Schwann cells within a tethered type-1 collagen matrix, followed by removal of interstitial fluid produces a stable tissue-like biomaterial that recreates the aligned cellular and extracellular matrix architecture associated with nerve grafts. Sheets of this engineered neural tissue supported and directed neuronal growth in a co-culture model, and initial in vivo tests showed that a device containing rods of rolled-up sheets could support neuronal growth during rat sciatic nerve repair (5 mm gap). Further testing of this device for repair of a critical-sized 15 mm gap showed that, at 8 weeks, engineered neural tissue had supported robust neuronal regeneration across the gap. This is, therefore, a useful new approach for generating anisotropic engineered tissues, and it can be used with Schwann cells to fabricate artificial neural tissue for peripheral nerve repair.

Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite outgrowth in an aligned tissue-engineered collagen construct in vitro

In the present study, we evaluated the differentiation potential of human dental pulp stem cells (hDPSCs) toward Schwann cells, together with their functional capacity with regard to myelination and support of neurite outgrowth in vitro. Successful Schwann cell differentiation was confirmed at the morphological and ultrastructural level by transmission electron microscopy. Furthermore, compared to undifferentiated hDPSCs, immunocytochemistry and ELISA tests revealed increased glial marker expression and neurotrophic factor secretion of differentiated hDPSCs (d‐hDPSCs), which promoted survival and neurite outgrowth in 2‐dimensional dorsal root ganglia cultures. In addition, neurites were myelinated by d‐hDPSCs in a 3‐dimensional collagen type I hydrogel neural tissue construct. This engineered construct contained aligned columns of d‐hDPSCs that supported and guided neurite outgrowth. Taken together, these findings provide the first evidence that hDPSCs are able to undergo Schwann cell differentiation and support neural outgrowth in vitro, proposing them to be good candidates for cell‐based therapies as treatment for peripheral nerve injury.—Martens, W., Sanen, K., Georgiou, M., Struys, T., Bronckaers, A., Ameloot, M., Phillips, J., Lambrichts, I. Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite outgrowth in an aligned tissue‐engineered collagen construct in vitro. FASEB J. 28, 1634–1643 (2014). www.fasebj.org

Alignment of Astrocytes Increases Neuronal Growth in Three-Dimensional Collagen Gels and Is Maintained Following Plastic Compression to Form a Spinal Cord Repair Conduit

After injury to the spinal cord, reactive astrocytes form a glial scar consisting of highly ramified cell processes that constitute a major impediment to repair, partly due to their lack of orientation and guidance for regenerating axons. In some nonmammalian vertebrates, successful central nervous system regeneration is attributed to the alignment of reactive glia, which guide axons across the lesion site. Here, a three-dimensional mammalian cell-seeded collagen gel culture system was used to explore the effect of astrocyte alignment on neuronal growth. Astrocyte alignment was mapped within tethered rectangular gels and was significantly greater at the edge and middle of the gels compared to the control unaligned regions. When neurons were seeded on and within astrocyte gels, neurite length was greatest in the areas of astrocyte alignment. There was no difference in expression of astrocyte reactivity markers between aligned and control areas. Having established the potential utility of astrocyte alignment, the aligned gels were plastic compressed, transforming them into mechanically robust implantable devices. After compression, astrocytes remained viable and aligned and supported neurite outgrowth, yielding a novel method for assembling aligned cellular constructs suitable for tissue engineering and highlighting the importance of astrocyte alignment as a possible future therapeutic intervention for spinal cord repair.

Engineered neural tissue with aligned, differentiated adipose-derived stem cells promotes peripheral nerve regeneration across a critical sized defect in rat sciatic nerve.

Adipose-derived stem cells were isolated from rats and differentiated to a Schwann cell-like phenotype in vitro. The differentiated cells (dADSCs) underwent self-alignment in a tethered type-1 collagen gel, followed by stabilisation to generate engineered neural tissue (EngNT-dADSC). The pro-regenerative phenotype of dADSCs was enhanced by this process, and the columns of aligned dADSCs in the aligned collagen matrix supported and guided neurite extension in vitro. EngNT-dADSC sheets were rolled to form peripheral nerve repair constructs that were implanted within NeuraWrap conduits to bridge a 15 mm gap in rat sciatic nerve. After 8 weeks regeneration was assessed using immunofluorescence imaging and transmission electron microscopy and compared to empty conduit and nerve graft controls. The proportion of axons detected in the distal stump was 3.5 fold greater in constructs containing EngNT-dADSC than empty tube controls. Our novel combination of technologies that can organise autologous therapeutic cells within an artificial tissue construct provides a promising new cellular biomaterial for peripheral nerve repair.

A versatile 3D culture model facilitates monitoring of astrocytes undergoing reactive gliosis

A major impediment to CNS repair is the glial scar, which forms following damage and is composed mainly of ramified, ‘reactive’ astrocytes that inhibit neuronal regrowth. The transition of astrocytes into this reactive phenotype (reactive gliosis) is a potential therapeutic target, but glial scar formation has proved difficult to study in monolayer cultures because they induce constitutive astrocyte activation. Here we demonstrate a 3D collagen gel system in which primary rat astrocytes were maintained in a persistently less reactive state than comparable cells in monolayer, resembling their status in the undamaged CNS. Reactivity, proliferation and viability were monitored and quantified using confocal, fluorescence and time‐lapse microscopy, 3D image analysis, RT–PCR and ELISA. To assess the potential of this system as a model of reactive gliosis, astrocytes in 3D were activated with TGFβ1 to a ramified, reactive phenotype (elevated GFAP, Aquaporin 4, CSPG, Vimentin and IL‐6 secretion). This provides a versatile system in which astrocytes can be maintained in a resting state, then be triggered to undergo reactive gliosis, enabling real‐time monitoring and quantitative analysis throughout and providing a powerful new tool for research into CNS damage and repair. Copyright

Peripheral nerves in the rat exhibit localized heterogeneity of tensile properties during limb movement

Peripheral nerves in the limbs stretch to accommodate changes in length during normal movement. The aim of this study was to determine how stretch is distributed along the nerve relative to local variations in mechanical properties. Deformation (strain) in joint and non‐joint regions of rat median and sciatic nerves was measured in situ during limb movement using optical image analysis. In each nerve the strain was significantly greater in the joint rather than the non‐joint regions (2‐fold in the median nerve, 5‐ to 10‐fold in the sciatic). In addition, this difference in strain was conserved in the median nerve ex vivo, demonstrating an in‐built longitudinal heterogeneity of mechanical properties. Tensile testing of isolated samples of joint and non‐joint regions of both nerves showed that joint regions were less stiff (more compliant) than their non‐joint counterparts with joint: non‐joint stiffness ratios of 0.5 ± 0.07 in the median nerve, and 0.8 ± 0.02 in the sciatic. However, no structural differences identified at the light microscope level in fascicular/non‐fascicular tissue architecture between these two nerve regions could explain the observed tensile heterogeneity. This identification of localized functional heterogeneity in tensile properties is particularly important in understanding normal dynamic nerve physiology, provides clues to why peripheral nerve repair outcomes are variable, and suggests potential novel therapeutic targets.

Glucose-Coated Gold Nanoparticles Transfer across Human Brain Endothelium and Enter Astrocytes In Vitro

The blood-brain barrier prevents the entry of many therapeutic agents into the brain. Various nanocarriers have been developed to help agents to cross this barrier, but they all have limitations, with regard to tissue-selectivity and their ability to cross the endothelium. This study investigated the potential for 4 nm coated gold nanoparticles to act as selective carriers across human brain endothelium and subsequently to enter astrocytes. The transfer rate of glucose-coated gold nanoparticles across primary human brain endothelium was at least three times faster than across non-brain endothelia. Movement of these nanoparticles occurred across the apical and basal plasma membranes via the cytosol with relatively little vesicular or paracellular migration; antibiotics that interfere with vesicular transport did not block migration. The transfer rate was also dependent on the surface coating of the nanoparticle and incubation temperature. Using a novel 3-dimensional co-culture system, which includes primary human astrocytes and a brain endothelial cell line hCMEC/D3, we demonstrated that the glucose-coated nanoparticles traverse the endothelium, move through the extracellular matrix and localize in astrocytes. The movement of the nanoparticles through the matrix was >10 µm/hour and they appeared in the nuclei of the astrocytes in considerable numbers. These nanoparticles have the correct properties for efficient and selective carriers of therapeutic agents across the blood-brain barrier.

Investigating mechanical behaviour at a core–sheath interface in peripheral nerve

Abstract  As peripheral nerves bend and stretch, internal elements need to move in relation to each other. However, the way in which intraneural components interact is poorly understood. Previous work identified a distinct core and sheath in the rat sciatic nerve and provides a useful model with which to investigate this interaction. Here we have focused on identifying the mechanical and anatomical characteristics of the interface between core and sheath. Nerve samples, 15 and 20 mm long, of rat sciatic nerves were harvested and placed in a purpose‐built jig, and a tensile testing machine was used to pull core from sheath. Mechanical tests of specimens in which core had been previously pulled from sheath by 25% of its initial length achieved a mean pull‐out force approximately six times smaller than that achieved using intact controls. These results are consistent with the proposal that core–sheath interactions involve physical connections rather than a viscous fluid interface. Anatomical features of this interface were characterised using transmission electron microscopy. It appeared that sheath was derived from epineurium and most of the perineurium, whilst core consisted of endoneurium and a small proportion of the perineurium: the plane of cleavage appeared to involve the innermost perineurial cell layer.

THE NEUROPROTECTIVE EFFECTS OF FIBRONECTIN MATS AND FIBRONECTIN PEPTIDES FOLLOWING SPINAL CORD INJURY IN THE RAT

We have shown previously that mats made from the glycoprotein fibronectin are permissive for axonal growth when implanted into the injured spinal cord. Recent evidence has indicated that fibronectin and its peptides also have neuroprotective effects in the CNS. We have therefore examined the neuroprotective effects of fibronectin applied to a spinal cord injury site. Adult rats with fibronectin mats implanted into a spinal cord lesion cavity had decreased apoptosis in the intact adjoining spinal cord tissue at 1 and 3 days post-injury compared to rats that had gelfoam implanted into the lesion cavity. Rats with fibronectin mat implants also showed enhanced hindlimb locomotor performance for the first 3 weeks post-surgery compared to control animals. To further examine the neuroprotective potential of fibronectin following spinal cord injury, we examined the effects of placing fibronectin mats over the site of a spinal cord hemisection or of delivering a solution derived from a dissolved fibronectin mat. The effects of these treatments were compared with control animals and animals that were treated with a fibronectin peptide (PRARIY) that has been shown to decrease secondary damage in a rodent model of cerebral ischemia. Results showed that both types of fibronectin mat treatment resulted in decreased lesion size, apoptosis, and axonal damage within the first week post-injury compared to control animals and were comparable in their neuroprotective efficacy to treatment with the fibronectin peptide. The results of the current study indicate that fibronectin based biomaterials have neuroprotective effects following spinal cord injury, in addition to their previously reported ability to promote axonal regeneration.