Combinatorial strategies for cell transplantation in traumatic spinal cord injury.

Spinal cord injury (SCI) substantially reduces the quality of life of affected individuals. Recovery of function is therefore a primary concern of the patient population and a primary goal for therapeutic interventions. Currently, even with growing numbers of clinical trials, there are still no effective treatments that can improve neurological outcomes after SCI. A large body of work has demonstrated that transplantation of neural stem/progenitor cells (NSPCs) can promote regeneration of the injured spinal cord by providing new neurons that can integrate into injured host neural circuitry. Despite these promising findings, the degree of functional recovery observed after NSPC transplantation remains modest. It is evident that treatment of such a complex injury cannot be addressed with a single therapeutic approach. In this mini-review, we discuss combinatorial strategies that can be used along with NSPC transplantation to promote spinal cord regeneration. We begin by introducing bioengineering and neuromodulatory approaches, and highlight promising work using these strategies in integration with NSPCs transplantation. The future of NSPC transplantation will likely include a multi-factorial approach, combining stem cells with biomaterials and/or neuromodulation as a promising treatment for SCI.

Electroconductive PEDOT nanoparticle integrated scaffolds for spinal cord tissue repair.

BACKGROUND: Hostile environment around the lesion site following spinal cord injury (SCI) prevents the re-establishment of neuronal tracks, thus significantly limiting the regenerative capability. Electroconductive scaffolds are emerging as a promising option for SCI repair, though currently available conductive polymers such as polymer poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) present poor biofunctionality and biocompatibility, thus limiting their effective use in SCI tissue engineering (TE) treatment strategies. METHODS: PEDOT NPs were synthesized via chemical oxidation polymerization in miniemulsion. The conductive PEDOT NPs were incorporated with gelatin and hyaluronic acid (HA) to create gel:HA:PEDOT-NPs scaffolds. Morphological analysis of both PEDOT NPs and scaffolds was conducted via SEM. Further characterisation included dielectric constant and permittivity variances mapped against morphological changes after crosslinking, Young's modulus, FTIR, DLS, swelling studies, rheology, in-vitro, and in-vivo biocompatibility studies were also conducted. RESULTS: Incorporation of PEDOT NPs increased the conductivity of scaffolds to 8.3 × 10-4 ± 8.1 × 10-5 S/cm. The compressive modulus of the scaffold was tailored to match the native spinal cord at 1.2 ± 0.2 MPa, along with controlled porosity. Rheological studies of the hydrogel showed excellent 3D shear-thinning printing capabilities and shape fidelity post-printing. In-vitro studies showed the scaffolds are cytocompatible and an in-vivo assessment in a rat SCI lesion model shows glial fibrillary acidic protein (GFAP) upregulation not directly in contact with the lesion/implantation site, with diminished astrocyte reactivity. Decreased levels of macrophage and microglia reactivity at the implant site is also observed. This positively influences the re-establishment of signals and initiation of healing mechanisms. Observation of axon migration towards the scaffold can be attributed to immunomodulatory properties of HA in the scaffold caused by a controlled inflammatory response. HA limits astrocyte activation through its CD44 receptors and therefore limits scar formation. This allows for a superior axonal migration and growth towards the targeted implantation site through the provision of a stimulating microenvironment for regeneration. CONCLUSIONS: Based on these results, the incorporation of PEDOT NPs into Gel:HA biomaterial scaffolds enhances not only the conductive capabilities of the material, but also the provision of a healing environment around lesions in SCI. Hence, gel:HA:PEDOT-NPs scaffolds are a promising TE option for stimulating regeneration for SCI.

Biomimetic 3D-printed scaffolds for spinal cord injury repair.

Current methods for bioprinting functional tissue lack appropriate biofabrication techniques to build complex 3D microarchitectures essential for guiding cell growth and promoting tissue maturation1. 3D printing of central nervous system (CNS) structures has not been accomplished, possibly owing to the complexity of CNS architecture. Here, we report the use of a microscale continuous projection printing method (µCPP) to create a complex CNS structure for regenerative medicine applications in the spinal cord. µCPP can print 3D biomimetic hydrogel scaffolds tailored to the dimensions of the rodent spinal cord in 1.6 s and is scalable to human spinal cord sizes and lesion geometries. We tested the ability of µCPP 3D-printed scaffolds loaded with neural progenitor cells (NPCs) to support axon regeneration and form new 'neural relays' across sites of complete spinal cord injury in vivo in rodents1,2. We find that injured host axons regenerate into 3D biomimetic scaffolds and synapse onto NPCs implanted into the device and that implanted NPCs in turn extend axons out of the scaffold and into the host spinal cord below the injury to restore synaptic transmission and significantly improve functional outcomes. Thus, 3D biomimetic scaffolds offer a means of enhancing CNS regeneration through precision medicine.

Peripheral nerve growth within a hydrogel microchannel scaffold supported by a kink-resistant conduit.

Nerve repair in several mm-long nerve gaps often requires an interventional technology. Microchannel scaffolds have proven effective for bridging nerve gaps and guiding axons in the peripheral nervous system (PNS). Nonetheless, fabricating microchannel scaffolds at this length scale remains a challenge and/or is time consuming and cumbersome. In this work, a simple computer-aided microdrilling technique was used to fabricate 10 mm-long agarose scaffolds consisting of 300 µm-microchannels and 85 µm-thick walls in less than an hour. The agarose scaffolds alone, however, did not exhibit adequate stiffness and integrity to withstand the mechanical stresses during implantation and suturing. To provide mechanical support and enable suturing, poly caprolactone (PCL) conduits were fabricated and agarose scaffolds were placed inside. A modified salt-leaching technique was developed to introduce interconnected porosity in PCL conduits to allow for tuning of the mechanical properties such as elastic modulus and strain to failure. It was shown that the PCL conduits were effective in stabilizing the agarose scaffolds in 10 mm-long sciatic nerve gaps of rats for at least 8 weeks. Robust axon ingress and Schwann cell penetration were observed within the microchannel scaffolds without using growth factors. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3392-3399, 2017.

Hierarchically Ordered Porous and High-Volume Polycaprolactone Microchannel Scaffolds Enhanced Axon Growth in Transected Spinal Cords.

The goal of this work was to design nerve guidance scaffolds with a unique architecture to maximize the open volume available for nerve growth. Polycaprolactone (PCL) was selected as the scaffold material based on its biocompatibility and month-long degradation. Yet, dense PCL does not exhibit suitable properties such as porosity, stiffness, strength, and cell adhesion to function as an effective nerve guidance scaffold. To address these shortcomings, PCL was processed using a modified salt-leaching technique to create uniquely controlled interconnected porosity. By controlling porosity, we demonstrated that the elastic modulus could be controlled between 2.09 and 182.1 MPa. In addition, introducing porosity and/or coating with fibronectin enhanced the PCL cell attachment properties. To produce PCL scaffolds with maximized open volume, porous PCL microtubes were fabricated and translated into scaffolds with 60 volume percent open volume. The scaffolds were tested in transected rat spinal cords. Linear axon growth within both the microtubes as well as the interstitial space between the tubes was observed, demonstrating that the entire open volume of the scaffold was available for nerve growth. Overall, a novel scaffold architecture and fabrication technique are presented. The scaffolds exhibit significantly higher volume than state-of-the-art scaffolds for promising spinal cord nerve repair.

Bone Marrow Stromal Cell Intraspinal Transplants Fail to Improve Motor Outcomes in a Severe Model of Spinal Cord Injury.

Bone marrow stromal cells (BMSCs) have been reported to exert potential neuroprotective properties in models of neurotrauma, although precise mechanisms underlying their benefits are poorly understood. Despite this lack of knowledge, several clinical trials have been initiated using these cells. To determine whether local mechanisms mediate BMSC neuroprotective actions, we grafted allogeneic BMSCs to sites of severe, compressive spinal cord injury (SCI) in Sprague-Dawley rats. Cells were administered 48 h after the original injury. Additional animals received allogeneic MSCs that were genetically modified to secrete brain-derived neurotrophic factor (BDNF) to further determine whether a locally administered neurotrophic factor provides or extends neuroprotection. When assessed 2 months post-injury in a clinically relevant model of severe SCI, BMSC grafts with or without BDNF secretion failed to improve motor outcomes. Thus, allogeneic grafts of BMSCs do not appear to act through local mechanisms, and future clinical trials that acutely deliver BMSCs to actual sites of injury within days are unlikely to be beneficial. Additional studies should address whether systemic administration of BMSCs alter outcomes from neurotrauma.

Characterizing the degradation of alginate hydrogel for use in multilumen scaffolds for spinal cord repair.

Alginate was studied as a degradable nerve guidance scaffold material in vitro and in vivo. In vitro degradation rates were determined using rheology to measure the change in shear modulus vs time. The shear modulus decreased from 155 kPa to 5 kPa within 2 days; however, alginate samples maintained their superficial geometry for over 28 days. The degradation behavior was supported by materials characterization data showing alginate consisted of high internal surface area (400 m2 /g), which likely facilitated the release of cross-linking cations resulting in the rapid decrease in shear modulus. To assess the degradation rate in vivo, multilumen scaffolds were fabricated using a fiber templating technique. The scaffolds were implanted in a 2-mm-long T3 full transection rodent spinal cord lesion model for 14 days. Although there was some evidence of axon guidance, in general, alginate scaffolds degraded before axons could grow over the 2-mm-long lesion. Enabling alginate-based scaffolds for nerve repair will likely require approaches to slow its degradation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 611-619, 2016.

Brain derived neurotrophic factor release from layer-by-layer coated agarose nerve guidance scaffolds.

Agarose nerve guidance scaffolds (NGS) seeded with cells expressing brain derived neurotrophic factor (BDNF) have demonstrated robust nerve regeneration in the rat central nervous system. The purpose of this work was to explore whether agarose NGS coated with hydrogen-bonded layer-by-layer (HLbL) could provide an acellular method of delivering prolonged and consistent dosages of active BDNF. Our results show that HLbL-coated agarose NGS could release BDNF over 10days in consistent dosages averaging 80.5±12.5(SD)ng/mL. Moreover, the BDNF released from HLbL was confirmed active by in vitro cell proliferation assays. To our knowledge, this is the first report demonstrating that HLbL assembled onto a hydrogel can provide consistent, prolonged release of active BDNF in clinically relevant dosages.

Using templated agarose scaffolds to promote axon regeneration through sites of spinal cord injury.

The past 30 years of research in spinal cord injury (SCI) have revealed that, under certain conditions, some types of axons are able to regenerate. To aid these axons in bridging the lesion site, many experimenters place cellular grafts at the lesion. However, to increase the potential for functional recovery, it is likely advantageous to maximize the number of axons that reach the intact spinal cord on the other side of the lesion. Implanting linear-channeled scaffolds at the lesion site provides growing axons with linear growth paths, which minimizes the distance they must travel to reach healthy tissue. Moreover, the linear channels help the regenerating axons maintain the correct mediolateral and dorsoventral position in the spinal cord, which may also improve functional recovery by keeping the axons nearer to their correct targets. Here, we provide a protocol to perform a full spinal cord transection in rats that accommodates an implanted scaffold.

An engineered muscle flap for reconstruction of large soft tissue defects.

Large soft tissue defects involve significant tissue loss, requiring surgical reconstruction. Autologous flaps are occasionally scant, demand prolonged transfer surgery, and induce donor site morbidity. The present work set out to fabricate an engineered muscle flap bearing its own functional vascular pedicle for repair of a large soft tissue defect in mice. Full-thickness abdominal wall defect was reconstructed using this engineered vascular muscle flap. A 3D engineered tissue constructed of a porous, biodegradable polymer scaffold embedded with endothelial cells, fibroblasts, and/or myoblasts was cultured in vitro and then implanted around the femoral artery and veins before being transferred, as an axial flap, with its vascular pedicle to reconstruct a full-thickness abdominal wall defect in the same mouse. Within 1 wk of implantation, scaffolds showed extensive functional vascular density and perfusion and anastomosis with host vessels. At 1 wk posttransfer, the engineered muscle flaps were highly vascularized, were well-integrated within the surrounding tissue, and featured sufficient mechanical strength to support the abdominal viscera. Thus, the described engineered muscle flap, equipped with an autologous vascular pedicle, constitutes an effective tool for reconstruction of large defects, thereby circumventing the need for both harvesting autologous flaps and postoperative scarification.

Engineered vascular beds provide key signals to pancreatic hormone-producing cells.

The mechanisms underlying early islet graft failure are not entirely clear, but are thought to involve ischemic injury due to delayed vascularization. We hypothesize that blood vessels play an active role in cell-cell communications supporting islet survival and engraftment. To test this hypothesis and to uncouple endothelial cell (EC)-generated signaling stimuli from their nutritional and gas exchange functions, we developed three dimensional (3D) endothelial vessel networks in engineered pancreatic tissues prepared from islets, fibroblasts and ECs. The tri-culture setup, seeded on highly porous biocompatible polymeric scaffolds closely mimics the natural anatomical context of pancreatic vasculature. Enhanced islet survival correlating with formation of functional tube-like endothelial vessels was demonstrated. Addition of foreskin fibroblasts to islet-endothelial cultures promoted tube-like structure formation, which further supported islet survival as well as insulin secretion. Gene expression profiles of EC growth factors, extracellular matrix (ECM), morphogenes and differentiation markers were significantly different in 2D versus 3D culture systems and were further modified upon addition of fibroblasts. Implantation of prevascularized islets into diabetic mice promoted survival, integration and function of the engrafted engineered tissue, supporting the suggested role of ECs in islet survival. These findings present potential strategies for preparation of transplantable islets with increased survival prospects.

Improved vascular organization enhances functional integration of engineered skeletal muscle grafts.

Severe traumatic events such as burns, and cancer therapy, often involve a significant loss of tissue, requiring surgical reconstruction by means of autologous muscle flaps. The scant availability of quality vascularized flaps and donor site morbidity often limit their use. Engineered vascularized grafts provide an alternative for this need. This work describes a first-time analysis, of the degree of in vitro vascularization and tissue organization, required to enhance the pace and efficacy of vascularized muscle graft integration in vivo. While one-day in vitro was sufficient for graft integration, a three-week culturing period, yielding semiorganized vessel structures and muscle fibers, significantly improved grafting efficacy. Implanted vessel networks were gradually replaced by host vessels, coupled with enhanced perfusion and capillary density. Upregulation of key graft angiogenic factors suggest its active role in promoting the angiogenic response. Transition from satellite cells to mature fibers was indicated by increased gene expression, increased capillary to fiber ratio, and similar morphology to normal muscle. We suggest a "relay" approach in which extended in vitro incubation, enabling the formation of a more structured vascular bed, allows for graft-host angiogenic collaboration that promotes anastomosis and vascular integration. The enhanced angiogenic response supports enhanced muscle regeneration, maturation, and integration.

Engineering vessel-like networks within multicellular fibrin-based constructs.

Sufficient vascularization in engineered tissues can be achieved through coordinated application of improved biomaterial systems with proper cell types. In this study, we employed 3D fibrin gels alone or in combination with the synthetic poly(l-lactic acid) (PLLA)/polylactic-glycolic acid (PLGA) sponges to support in-vitro construct vascularization and to enhance neovascularization upon implantation. Two multicellular assays were embedded in these constructs: (a) co-culture of endothelial (EC) and fibroblast cells, and (b) a tri-culture combination of ECs, fibroblasts and tissue specific skeletal myoblast cells. In-vitro vessel network formation was examined under advanced confocal microscopy in various time points from cell seeding. Vessel network maturity levels and morphology were found to be highly regulated by fibrinogen concentrations in-vitro. Combination of PLLA/PLGA sponges with fibrin matrices provided added mechanical strength and featured highly mature vessels-like networks. Implantation studies revealed that the implanted ECs developed into 3D interconnected vessel-like networks in-vivo. The PLLA/PLGA scaffold proved to be a key stimulator of neovascularization and perfusion of implanted grafts. Our findings demonstrate that complex biomaterial platform involving fibrin and PLLA/PLGA synthetic scaffold provide a way to enhancing vascularization in-vitro and in-vivo.

Paclitaxel-clusters coated with hyaluronan as selective tumor-targeted nanovectors.

Paclitaxel (PTX) is a widely used anti-tumor agent in the treatment of solid tumors. Lack of selective strategies to target PTX into tumor cells together with poor solubility necessitating detergent, are severe clinical limitations. To address these hurdles, we devised a strategy that utilized PTX insolubility, mixing it with lipids that self-assemble into nanoparticle-like clusters. These clusters were then coated with hyaluronan, a glycosaminoglycan (GAG), and termed PTX-GAGs. These particles, delivered PTX selectively into tumor cells in a CD44-dependent manner. Injected systemically to mice bearing solid tumors, the PTX-GAGs showed high safety profile and tumor accumulation. Tumor progression was exponential upon treatment with free PTX or PTX in albumin nanoparticles (the FDA-approved Taxol and Abraxane, respectively). Under the same conditions, PTX-GAGs induced tumor arrest and were as potent as a 4-fold higher Taxol dose. Our findings suggest GAGs merit further investigation as vehicles for taxanes, and may be applicable as carriers in other therapeutic settings.