Development of a tissue engineered implantable device for the surgical repair of the peripheral nervous system

Peripheral nerve injury as a result of trauma affects approximately 1 million people in Europe and America annually. The current clinical gold standard treatment for repairing long gaps is the nerve autograft, in which only ~50% of cases result in satisfactory functional recovery. Tissue-engineered cellular bridging devices for surgical implantation into peripheral nerve injury sites could provide an attractive alternative to the autograft. This project reports the development of a robust, anisotropic biomaterial with highly aligned cells that can form the basis of a peripheral nerve repair device. Engineered neural tissue (EngNT), which is formed from columns of Schwann cells or stem cells within a 3D aligned collagen matrix, can promote directed neurite outgrowth in vitro. This study demonstrates that sheets of EngNT can be arranged to form the 'endoneurium' of a peripheral nerve repair device within a NeuraWrap™ outer tube, and can be used for the repair of critical sized defects in rat. Schwann cells are the preferred cell type for peripheral nerve repair because of their ability to enhance axon migration and secrete factors that further increase regeneration. However the use of autologous Schwann cells has a number of disadvantages, including the sacrifice of host nerve tissue for their extraction and slow expansion times in vitro. Various therapeutic cell types and a bovine collagen source that can potentially be used to make EngNT to form the device core were investigated. EngNT devices containing Schwann cell-like cells from adipose-derived stem cells (dADSC) or human neural progenitor cells differentiated to glial cells (dCX) were tested in a critical sized gap in the rat sciatic nerve model. The in vivo experiments demonstrated that there is potential, for the dADSCs to be used for peripheral nerve repair. The results from the dCX repairs were less clear. The technology reported here offers a simple, rapid and effective method for the manufacture of an aligned cellular biomaterial, and could be applied to a range of tissue engineering applications. This study demonstrates that there is potential for EngNT to be used in the construction of nerve repair conduits

Aligned cellular and acellular collagen guidance substrates for peripheral nerve repair

There is a clinical demand to shorten the delay of reinnervation and improve functional recovery after peripheral nerve injury. A peripheral nerve repair device with the ability to direct and promote axon growth across a lesion would be a promising alternative to nerve autograft repair, the current gold standard treatment. The growth of axons across a lesion is most effective when supported by columns of aligned Schwann cells, as found in an autograft. Here we report the development of a robust aligned cellular collagen biomaterial that supports and directs neuronal growth. We also investigate the potential of these aligned cells to precondition the collagen biomaterial, before they are freeze-killed, leaving an acellular guidance matrix

Investigating the requirement for dual cell co-culture platforms in creating regenerative cell therapies for CNS injury

Injuries to the central nervous system (CNS) can be devastating. CNS injuries include those to the spinal cord, where there can be a complete loss of function below the point of injury. Spinal cord injury impacts up to 500,000 people worldwide every year and where function is lost, quality of life can be severely limited. Olfactory ensheathing cells (OECs) are a candidate cell therapy for spinal cord injury as they can promote neuronal survival and facilitate regeneration of severed axons. Despite their unique properties, OECs are very challenging cells to work with because they are difficult to isolate, difficult to sustain in culture for prolonged periods and there is still controversy around how to characterize their identity and potency. The overall aim of this project is to identify methods to enhance the survival, growth and function of OECs. It has been reported that for OECs to be truly functional they require interaction with fibroblasts. Therefore, we sought to investigate whether it is necessary to use fibroblasts as a feeder layer to support OECs via physical cell-cell contact, or whether paracrine soluble factors in the conditioned media from fibroblasts would provide the trophic support necessary to enhance OEC survival and growth. A human mucosal fibroblast cell line was used as a feeder layer. Primary rat OECs were cultured for 14 days on the feeder layer or control substrate (laminin-coated dishes). After 14 days, the morphology of cells was assessed and an algorithm generated using ImageJ was used to ascribe a mathematical value to OEC morphology to determine whether a correlation of morphology to expression of markers could be made. Cells cultured on feeders adopted a more spindle-like appearance compared with cells cultured on laminin, which adopted an enlarged morphology. The algorithm was used to analyse the circularity of cells that labelled positive for candidate identity marker S100b. It was found that cells cultured on feeders had a lower circularity, and therefore more elongated shape compared to those cultured on laminin (p=0.037). Additionally, a significant increase in p75NTR expression (a second candidate OEC marker) was observed (p=0.01) on feeders. To further investigate the relationship between the OECs and the feeders, cells were cultured in the presence of conditioned media from the fibroblasts. When cells were cultured in conditioned media there was a significant (p=0.002) upregulation of Thy1, an undesirable marker, and the significance of this will be investigated further with work underway to compare different feeder types and their impact on marker expression

Fabrication of an endoneurium using engineered neural tissue within a peripheral nerve repair conduit

Peripheral nerve injury as a result of trauma affects approximately 1 million people in Europe and America annually. The current clinical gold standard treatment for repairing long gaps is the nerve autograft, in which only ~50% of cases result in satisfactory functional recovery. Tissue-engineered cellular bridging devices for peripheral nerve repair could provide an attractive alternative to autografts. Sheets of engineered neural tissue (EngNT), which is formed from columns of Schwann cells within a 3D aligned collagen matrix, can promote directed neurite outgrowth in vitro. These sheets of EngNT can be arranged to form the ‘endoneurium’ of a peripheral nerve repair device. Two different arrangements, rod-based and sheet-based designs, were tested within a clinically approved tube, NeuraWrap™, in a 5mm gap in the rat sciatic nerve. Cross sections were stained to detect neurofilament after 4 weeks in vivo and revealed where the axons were growing in relation to the EngNT structures (this was divided into three zones for the analysis). The axon density was significantly greater in zone 1 than in zone 3 in the devices (P2 (mean ± SEM), compared to the sheet-based arrangement (B) (2920 ± 587 axons/mm2). The rod-based arrangement was more stable; there were no observed changes to its structure or orientation as a result of surgical handling or limb movement post-implantation. The designs are modular and can be adapted for the repair of bigger nerves by, for example, having multiple rod structures in the core of outer tubes or sheath wraps. Aligned cellular EngNT rods can form the basis of a functional conduit for peripheral nerve repair

A 3D <i>in vitro</i> model reveals differences in the astrocyte response elicited by potential stem cell therapies for CNS injury.

Aim: This study aimed to develop a 3D culture model to test the extent to which transplanted stem cells modulate astrocyte reactivity, where exacerbated glial cell activation could be detrimental to CNS repair success. Materials & methods: The reactivity of rat astrocytes to bone marrow mesenchymal stem cells, neural crest stem cells (NCSCs) and differentiated adipose-derived stem cells was assessed after 5 days. Schwann cells were used as a positive control. Results: NCSCs and differentiated Schwann cell-like adipose-derived stem cells did not increase astrocyte reactivity. Highly reactive responses to bone marrow mesenchymal stem cells and Schwann cells were equivalent. Conclusion: This approach can screen therapeutic cells prior to in vivo testing, allowing cells likely to trigger a substantial astrocyte response to be identified at an early stage. NCSCs and differentiated Schwann cell-like adipose-derived stem cells may be useful in treating CNS damage without increasing astrogliosis

Concrush: Understanding fugitive dust production and potential emission at a recycled concrete manufacturing facility

The production and emission of fugitive dust is a topic ofconcern that Concrush brought to the MISG, 2020. Concrushis recycled concrete manufacturing company in the Hunterregion of New South Wales. Concrush's operations producefugitive dust, fine particles that can escape the site. Fugitive dust can travel long distances from the site ofemission, and can have negative health impacts includingrespiratory illnesses. Presently, concrete recyclingfacilities are managed by the Environmental ProtectionAgency using guidelines initially developed for the coalindustry. Concrush seeks to understand the appropriatenessof these guidelines, and how they can reduce and managefugitive dust on their Teralba site. Mathematical modellingof dust emission and transport, together with a review ofsimilar processes in the literature, identified a number ofpractical options for Concrush to reduce their dustemissions. In addition, opportunities for improved datacollection are identified

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

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

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

Generation of c-MycERTAM-transduced human late-adherent olfactory mucosa cells for potential regenerative applications

Human olfactory mucosa cells (hOMCs) have been transplanted to the damaged spinal cord both pre-clinically and clinically. To date mainly autologous cells have been tested. However, inter-patient variability in cell recovery and quality, and the fact that the neuroprotective olfactory ensheathing cell (OEC) subset is difficult to isolate, means an allogeneic hOMC therapy would be an attractive “off-the-shelf” alternative. The aim of this study was to generate a candidate cell line from late-adherent hOMCs, thought to contain the OEC subset. Primary late-adherent hOMCs were transduced with a c-MycERTAM gene that enables cell proliferation in the presence of 4-hydroxytamoxifen (4-OHT). Two c-MycERTAM-derived polyclonal populations, PA5 and PA7, were generated and expanded. PA5 cells had a normal human karyotype (46, XY) and exhibited faster growth kinetics than PA7, and were therefore selected for further characterisation. PA5 hOMCs express glial markers (p75NTR, S100ß, GFAP and oligodendrocyte marker O4), neuronal markers (nestin and ß-III-tubulin) and fibroblast-associated markers (CD90/Thy1 and fibronectin). Co-culture of PA5 cells with a neuronal cell line (NG108-15) and with primary dorsal root ganglion (DRG) neurons resulted in significant neurite outgrowth after 5 days. Therefore, c-MycERTAM-derived PA5 hOMCs have potential as a regenerative therapy for neural cells

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