Clinical Trials Targeting Secondary Damage after Traumatic Spinal Cord Injury.

Spinal cord injury (SCI) often causes loss of sensory and motor function resulting in a significant reduction in quality of life for patients. Currently, no therapies are available that can repair spinal cord tissue. After the primary SCI, an acute inflammatory response induces further tissue damage in a process known as secondary injury. Targeting secondary injury to prevent additional tissue damage during the acute and subacute phases of SCI represents a promising strategy to improve patient outcomes. Here, we review clinical trials of neuroprotective therapeutics expected to mitigate secondary injury, focusing primarily on those in the last decade. The strategies discussed are broadly categorized as acute-phase procedural/surgical interventions, systemically delivered pharmacological agents, and cell-based therapies. In addition, we summarize the potential for combinatorial therapies and considerations.

Inflammation Drives Retraction, Stiffening, and Nodule Formation via Cytoskeletal Machinery in a Three-Dimensional Culture Model of Aortic Stenosis.

In calcific aortic valve disease, the valve cusps undergo retraction, stiffening, and nodular calcification. The inflammatory cytokine, tumor necrosis factor (TNF)-α, contributes to valve disease progression; however, the mechanisms of its actions on cusp retraction and stiffening are unclear. We investigated effects of TNF-α on murine aortic valvular interstitial cells (VICs) within three-dimensional, free-floating, compliant, collagen hydrogels, simulating their natural substrate and biomechanics. TNF-α increased retraction (percentage of diameter), stiffness, and formation of macroscopic, nodular structures with calcification in the VIC-laden hydrogels. The effects of TNF-α were attenuated by blebbistatin inhibition of myosin II-mediated cytoskeletal contraction. Inhibition of actin polymerization with cytochalasin-D, but not inhibition of Rho kinase with Y27632, blocked TNF-α-induced retraction in three-dimensional VIC hydrogels, suggesting that actin stress fibers mediate TNF-α-induced effects. In the hydrogels, inhibitors of NF-κB blocked TNF-α-induced retraction, whereas simultaneous inhibition of c-Jun N-terminal kinase was required to block TNF-α-induced stiffness. TNF-α also significantly increased collagen deposition, as visualized by Masson's trichrome staining, and up-regulated mRNA expression of discoidin domain receptor tyrosine kinase 2, fibronectin, and α-smooth muscle actin. In human aortic valves, calcified cusps were stiffer and had more collagen deposition than noncalcified cusps. These findings suggest that inflammation, through stimulation of cytoskeletal contractile activity, may be responsible for valvular cusp retraction, stiffening, and formation of calcified nodules.

Injectable Hydrogels for Spinal Cord Repair: A Focus on Swelling and Intraspinal Pressure.

Spinal cord injury (SCI) is a devastating condition that leaves patients with limited motor and sensory function at and below the injury site, with little to no hope of a meaningful recovery. Because of their ability to mimic multiple features of central nervous system (CNS) tissues, injectable hydrogels are being developed that can participate as therapeutic agents in reducing secondary injury and in the regeneration of spinal cord tissue. Injectable biomaterials can provide a supportive substrate for tissue regeneration, deliver therapeutic factors, and regulate local tissue physiology. Recent reports of increasing intraspinal pressure after SCI suggest that this physiological change can contribute to injury expansion, also known as secondary injury. Hydrogels contain high water content similar to native tissue, and many hydrogels absorb water and swell after formation. In the case of injectable hydrogels for the spinal cord, this process often occurs in or around the spinal cord tissue, and thus may affect intraspinal pressure. In the future, predictable swelling properties of hydrogels may be leveraged to control intraspinal pressure after injury. Here, we review the physiology of SCI, with special attention to the current clinical and experimental literature, underscoring the importance of controlling intraspinal pressure after SCI. We then discuss how hydrogel fabrication, injection, and swelling can impact intraspinal pressure in the context of developing injectable biomaterials for SCI treatment.

Multichannel bridges and NSC synergize to enhance axon regeneration, myelination, synaptic reconnection, and recovery after SCI.

Regeneration in the injured spinal cord is limited by physical and chemical barriers. Acute implantation of a multichannel poly(lactide-co-glycolide) (PLG) bridge mechanically stabilizes the injury, modulates inflammation, and provides a permissive environment for rapid cellularization and robust axonal regrowth through this otherwise inhibitory milieu. However, without additional intervention, regenerated axons remain largely unmyelinated (<10%), limiting functional repair. While transplanted human neural stem cells (hNSC) myelinate axons after spinal cord injury (SCI), hNSC fate is highly influenced by the SCI inflammatory microenvironment, also limiting functional repair. Accordingly, we investigated the combination of PLG scaffold bridges with hNSC to improve histological and functional outcome after SCI. In vitro, hNSC culture on a PLG scaffold increased oligodendroglial lineage selection after inflammatory challenge. In vivo, acute PLG bridge implantation followed by chronic hNSC transplantation demonstrated a robust capacity of donor human cells to migrate into PLG bridge channels along regenerating axons and integrate into the host spinal cord as myelinating oligodendrocytes and synaptically integrated neurons. Axons that regenerated through the PLG bridge formed synaptic circuits that connected the ipsilateral forelimb muscle to contralateral motor cortex. hNSC transplantation significantly enhanced the total number of regenerating and myelinated axons identified within the PLG bridge. Finally, the combination of acute bridge implantation and hNSC transplantation exhibited robust improvement in locomotor recovery. These data identify a successful strategy to enhance neurorepair through a temporally layered approach using acute bridge implantation and chronic cell transplantation to spare tissue, promote regeneration, and maximize the function of new axonal connections.

Molecular weight of hyaluronic acid crosslinked into biomaterial scaffolds affects angiogenic potential.

While hyaluronic acid (HA)-based hydrogels have been used clinically for decades, the mechanisms by which HA exerts molecular weight-dependent bioactivity and how chemical modification and crosslinking may affect molecular weight-dependent bioactivity remain poorly understood. This knowledge gap presents a significant barrier to designing HA hydrogels with predictable bioactivities. As HA has been widely reported to have molecular weight-dependent effects on endothelial cells (ECs), we investigated how the molecular weight of HA in either soluble or crosslinked forms affects angiogenesis and interrogated CD44 clustering on the surface of endothelial cells as a candidate mechanism for these affects. Using soluble HA, our results show high molecular weight (HMW) HA, but not low molecular weight (LMW) HA, increased viability and tube formation in cultured human cerebral microvascular ECs (HCMVECs). No size of HA affected proliferation. When HCMVECs were cultured with crosslinked HA of varying molecular weights in the form of HA-based microporous annealed particle scaffold (HMAPS), the cell response was comparable to when cultured with soluble HA. Similarly, when implanted subcutaneously, HMAPS with HMW HA were more vascularized than those with LMW HA. We also show that antibody-mediated CD44 clustering resulted in HCMVECs with increased viability and tube-like structure formation in a manner comparable to exposure to HMW HA, suggesting that HMW acts through CD44 clustering. STATEMENT OF SIGNIFICANCE: Biomaterials based on hyaluronic acid (HA), a bioactive extracellular matrix polysaccharide, have been used in clinical products for several years. Despite the knowledge that HA molecular weight heavily influences its bioactivity, molecular weight has been largely ignored in the development of HA-based biomaterials. Given the high viscosity of high molecular weight HA typically found in native tissues, lower molecular weight polysaccharides have been used most commonly for biomaterial fabrication. By comparing the ability of injectable, microporous annealed particle scaffolds (MAPS) fabricated from variably sized HA to promote angiogenesis, this study demonstrates that MAPS with high molecular weight HA better support vascularization, likely through an unique ability to induce clustering of CD44 receptors on endothelial cells.

Glioblastoma Spheroid Invasion through Soft, Brain-Like Matrices Depends on Hyaluronic Acid-CD44 Interactions.

Increased secretion of hyaluronic acid (HA), a glycosaminoglycan abundant in the brain extracellular matrix (ECM), correlates with worse clinical outcomes for glioblastoma (GBM) patients. GBM cells aggressively invade the brain parenchyma while encountering spatiotemporal changes in their local ECM, including HA concentration. To investigate how varying HA concentrations affect GBM invasion, patient-derived GBM cells are cultured within a soft, 3D matrix in which HA concentration is precisely varied and cell migration observed. Data demonstrate that HA concentration can determine the invasive activity of patient-derived GBM cells in a biphasic and highly sensitive manner, where the absolute concentration of HA at which cell migration peaked is specific to each patient-derived line. Furthermore, evidence that this response relies on phosphorylated ezrin, which interacts with the intracellular domain of HA-engaged CD44 to effectively link the actin cytoskeleton to the local ECM is provided. Overall, this study highlights CD44-HA binding as a major mediator of GBM cell migration that acts independently of integrins and focal adhesion complexes and suggests that targeting HA-CD44-ezrin interactions represents a promising therapeutic strategy to prevent tumor cell invasion in the brain.

Microenvironmental stiffness induces metabolic reprogramming in glioblastoma.

The mechanical properties of solid tumors influence tumor cell phenotype and the ability to invade surrounding tissues. Using bioengineered scaffolds to provide a matrix microenvironment for patient-derived glioblastoma (GBM) spheroids, this study demonstrates that a soft, brain-like matrix induces GBM cells to shift to a glycolysis-weighted metabolic state, which supports invasive behavior. We first show that orthotopic murine GBM tumors are stiffer than peritumoral brain tissues, but tumor stiffness is heterogeneous where tumor edges are softer than the tumor core. We then developed 3D scaffolds with µ-compressive moduli resembling either stiffer tumor core or softer peritumoral brain tissue. We demonstrate that the softer matrix microenvironment induces a shift in GBM cell metabolism toward glycolysis, which manifests in lower proliferation rate and increased migration activities. Finally, we show that these mechanical cues are transduced from the matrix via CD44 and integrin receptors to induce metabolic and phenotypic changes in cancer cells.

Multichannel bridges and NSC synergize to enhance axon regeneration, myelination, synaptic reconnection, and recovery after SCI.

Regeneration in the injured spinal cord is limited by physical and chemical barriers. Acute implantation of a multichannel poly(lactide-co-glycolide) (PLG) bridge mechanically stabilizes the injury, modulates inflammation, and provides a permissive environment for rapid cellularization and robust axonal regrowth through this otherwise inhibitory milieu. However, without additional intervention, regenerated axons remain largely unmyelinated (<10%), limiting functional repair. While transplanted human neural stem cells (hNSC) myelinate axons after spinal cord injury (SCI), hNSC fate is highly influenced by the SCI inflammatory microenvironment, also limiting functional repair. Accordingly, we investigated the combination of PLG scaffold bridges with hNSC to improve histological and functional outcome after SCI. In vitro, hNSC culture on a PLG scaffold increased oligodendroglial lineage selection after inflammatory challenge. In vivo, acute PLG bridge implantation followed by chronic hNSC transplantation demonstrated a robust capacity of donor human cells to migrate into PLG bridge channels along regenerating axons and integrate into the host spinal cord as myelinating oligodendrocytes and synaptically integrated neurons. Axons that regenerated through the PLG bridge formed synaptic circuits that connected ipsilateral forelimb muscle to contralateral motor cortex. hNSC transplantation significantly enhanced the total number of regenerating and myelinated axons identified within the PLG bridge. Finally, the combination of acute bridge implantation and hNSC transplantation exhibited robust improvement in locomotor recovery vs. control and hNSC transplant alone. These data identify a successful novel strategy to enhance neurorepair through a temporally layered approach using acute bridge implantation and chronic cell transplantation to spare tissue, promote regeneration, and maximize the function of new axonal connections.

Engineering Tissues of the Central Nervous System: Interfacing Conductive Biomaterials with Neural Stem/Progenitor Cells.

Conductive biomaterials provide an important control for engineering neural tissues, where electrical stimulation can potentially direct neural stem/progenitor cell (NS/PC) maturation into functional neuronal networks. It is anticipated that stem cell-based therapies to repair damaged central nervous system (CNS) tissues and ex vivo, "tissue chip" models of the CNS and its pathologies will each benefit from the development of biocompatible, biodegradable, and conductive biomaterials. Here, technological advances in conductive biomaterials are reviewed over the past two decades that may facilitate the development of engineered tissues with integrated physiological and electrical functionalities. First, one briefly introduces NS/PCs of the CNS. Then, the significance of incorporating microenvironmental cues, to which NS/PCs are naturally programmed to respond, into biomaterial scaffolds is discussed with a focus on electrical cues. Next, practical design considerations for conductive biomaterials are discussed followed by a review of studies evaluating how conductive biomaterials can be engineered to control NS/PC behavior by mimicking specific functionalities in the CNS microenvironment. Finally, steps researchers can take to move NS/PC-interfacing, conductive materials closer to clinical translation are discussed.

Matrix stiffness mediates pancreatic cancer chemoresistance through induction of exosome hypersecretion in a cancer associated fibroblasts-tumor organoid biomimetic model.

In pancreatic ductal adenocarcinoma (PDAC), the abundant stromal cells which comprise the tumor microenvironment constitute more than 90% of the primary tumor bulk. Moreover, this desmoplastic environment has been found to be three times stiffer than normal pancreas tissue. Despite the importance of studying the desmoplastic environment of PDAC, there is still a lack of models designed to adequately recapitulate this complex stiff microenvironment, a critical hallmark of the disease that has been shown to induce chemoresistance. Here, we present a bio-mimetic, 3-dimensional co-culture system that integrates tumor organoids and host-matching stromal cancer associated-fibroblasts (CAFs) that recapitulates the complex, fibrotic matrix of PDAC using advanced biomaterials. With this model, we show that matrix-activated CAFs are able to "re-engineer" the fibrotic environment into a significantly stiffer environment through lysyl-oxidase dependent crosslinking. Moreover, we show that culture of CAFs in this model leads to an increase of exosomes; extracellular vesicles known to promote chemoresistance. Finally, using previously identified exosome inhibitors, climbazole and imipramine, we demonstrate how abrogation of exosome hypersecretion can reduce matrix stiffness-induced chemoresistance. These data highlight the importance of the development of new models that recapitulate not only the cellular composition found in PDAC tumors, but also the biophysical stresses, like stiffness, that the cells are exposed to in order to identify therapies that can overcome this critical feature which can contribute to the chemoresistance observed in patients. We believe that the 3D bio-mimetic model we have developed will be a valuable tool for the development, testing, and optimization of "mechano-medicines" designed to target the biophysical forces that lead to tumor growth and chemoresistance.

Brain-on-a-chip: Recent advances in design and techniques for microfluidic models of the brain in health and disease.

Recent advances in biomaterials, microfabrication, microfluidics, and cell biology have led to the development of organ-on-a-chip devices that can reproduce key functions of various organs. Such platforms promise to provide novel insights into various physiological events, including mechanisms of disease, and evaluate the effects of external interventions, such as drug administration. The neuroscience field is expected to benefit greatly from these innovative tools. Conventional ex vivo studies of the nervous system have been limited by the inability of cell culture to adequately mimic in vivo physiology. While animal models can be used, their relevance to human physiology is uncertain and their use is laborious and associated with ethical issues. To date, organ-on-a-chip systems have been developed to model different tissue components of the brain, including brain regions with specific functions and the blood brain barrier, both in normal and pathophysiological conditions. While the field is still in its infancy, it is expected to have major impact on studies of neurophysiology, pathology and neuropharmacology in future. Here, we review advances made and limitations faced in an effort to stimulate development of the next generation of brain-on-a-chip devices.

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models.

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

A molecular interactome of the glioblastoma perivascular niche reveals integrin binding sialoprotein as a mediator of tumor cell migration.

Glioblastoma (GBM) is characterized by extensive microvascular hyperproliferation. In addition to supplying blood to the tumor, GBM vessels also provide trophic support to glioma cells and serve as conduits for migration into the surrounding brain, promoting recurrence. Here, we enrich CD31-expressing glioma vascular cells (GVCs) and A2B5-expressing glioma tumor cells (GTCs) from primary GBM and use RNA sequencing to create a comprehensive molecular interaction map of the secreted and extracellular factors elaborated by GVCs that can interact with receptors and membrane molecules on GTCs. To validate our findings, we utilize functional assays, including a hydrogel-based migration assay and in vivo mouse models to demonstrate that one identified factor, the little-studied integrin binding sialoprotein (IBSP), enhances tumor growth and promotes the migration of GTCs along the vasculature. This perivascular niche interactome will serve as a resource to the research community in defining the potential functions of the GBM vasculature.

Extracellular Matrix Proteins Confer Cell Adhesion-Mediated Drug Resistance Through Integrin α v in Glioblastoma Cells.

Chemotherapy resistance to glioblastoma (GBM) remains an obstacle that is difficult to overcome, leading to poor prognosis of GBM patients. Many previous studies have focused on resistance mechanisms intrinsic to cancer cells; the microenvironment surrounding tumor cells has been found more recently to have significant impacts on the response to chemotherapeutic agents. Extracellular matrix (ECM) proteins may confer cell adhesion-mediated drug resistance (CAMDR). Here, expression of the ECM proteins laminin, vitronectin, and fibronectin was assessed in clinical GBM tumors using immunohistochemistry. Then, patient-derived GBM cells grown in monolayers on precoated laminin, vitronectin, or fibronectin substrates were treated with cilengitide, an integrin inhibitor, and/or carmustine, an alkylating chemotherapy. Cell adhesion and viability were quantified. Transcription factor (TF) activities were assessed over time using a bioluminescent assay in which GBM cells were transduced with lentiviruses containing consensus binding sites for specific TFs linked to expression a firefly luciferase reporter. Apoptosis, mediated by p53, was analyzed by Western blotting and immunocytofluorescence. Integrin α v activation of the FAK/paxillin/AKT signaling pathway and effects on expression of the proliferative marker Ki67 were investigated. To assess effects of integrin α v activation of AKT and ERK pathways, which are typically deregulated in GBM, and expression of epidermal growth factor receptor (EGFR), which is amplified and/or mutated in many GBM tumors, shRNA knockdown was used. Laminin, vitronectin, and fibronectin were abundant in clinical GBM tumors and promoted CAMDR in GBM cells cultured on precoated substrates. Cilengitide treatment induced cell detachment, which was most pronounced for cells cultured on vitronectin. Cilengitide treatment increased cytotoxicity of carmustine, reversing CAMDR. ECM adhesion increased activity of NFκB and decreased that of p53, leading to suppression of p53-mediated apoptosis and upregulation of multidrug resistance gene 1 (MDR1; also known as ABCB1 or P-glycoprotein). Expression of Ki67 was correlative with activation of the integrin α v -mediated FAK/paxillin/AKT signaling pathway. EGFR expression increased with integrin α v knockdown GBM cells and may represent a compensatory survival mechanism. These results indicate that ECM proteins confer CAMDR through integrin α v in GBM cells.

Injectable, macroporous scaffolds for delivery of therapeutic genes to the injured spinal cord.

Biomaterials are being developed as therapeutics for spinal cord injury (SCI) that can stabilize and bridge acute lesions and mediate the delivery of transgenes, providing a localized and sustained reservoir of regenerative factors. For clinical use, direct injection of biomaterial scaffolds is preferred to enable conformation to unique lesions and minimize tissue damage. While an interconnected network of cell-sized macropores is necessary for rapid host cell infiltration into-and thus integration of host tissue with-implanted scaffolds, injectable biomaterials have generally suffered from a lack of control over the macrostructure. As genetic vectors have short lifetimes in vivo, rapid host cell infiltration into scaffolds is a prerequisite for efficient biomaterial-mediated delivery of transgenes. We present scaffolds that can be injected and assembled in situ from hyaluronic acid (HA)-based, spherical microparticles to form scaffolds with a network of macropores (∼10 µm). The results demonstrate that addition of regularly sized macropores to traditional hydrogel scaffolds, which have nanopores (∼10 nm), significantly increases the expression of locally delivered transgene to the spinal cord after a thoracic injury. Maximal cell and axon infiltration into scaffolds was observed in scaffolds with more regularly sized macropores. The delivery of lentiviral vectors encoding the brain-derived neurotrophic factor (BDNF), but not neurotrophin-3, from these scaffolds further increased total numbers and myelination of infiltrating axons. Modest improvements to the hindlimb function were observed with BDNF delivery. The results demonstrate the utility of macroporous and injectable HA scaffolds as a platform for localized gene therapies after SCI.

A highly selective low-background fluorescent imaging agent for nitric oxide.

We introduce a novel sensing mechanism for nitric oxide (NO) detection with a particular easily synthesized embodiment (NO(550)), which displays a rapid and linear response to NO with a red-shifted 1500-fold turn-on signal from a dark background. Excellent selectivity was observed against other reactive oxygen/nitrogen species, pH, and various substances that interfere with existing probes. NO(550) crosses cell membranes but not nuclear membranes and is suitable for both intra- and extracellular NO quantifications. Good cytocompatibility was found during in vitro studies with two different cell lines. The high specificity, dark background, facile synthesis, and low pH dependence make NO(550) a superior probe for NO detection when used as an imaging agent.

Bioengineered scaffolds for 3D culture demonstrate extracellular matrix-mediated mechanisms of chemotherapy resistance in glioblastoma.

Originating in the brain, glioblastoma (GBM) is a highly lethal and virtually incurable cancer, in large part because it readily develops resistance to treatments. While numerous studies have investigated mechanisms enabling GBM cells to evade chemotherapy-induced apoptosis, few have addressed how their surrounding extracellular matrix (ECM) acts to promote their survival. Here, we employed a biomaterial-based, 3D culture platform to investigate systematically how interactions between patient-derived GBM cells and the brain ECM promote resistance to alkylating chemotherapies - including temozolomide, which is used routinely in clinical practice. Scaffolds for 3D culture were fabricated from hyaluronic acid (HA) - a major structural and bioactive component of the brain ECM - and functionalized with the RGD (arginine-glycine-aspartic acid) tripeptide to provide sites for integrin engagement. Data demonstrate that cooperative engagement of CD44, through HA, and integrin αV, through RGD, facilitates resistance to alkylating chemotherapies through co-activation of Src, which inhibited downstream expression of BCL-2 family pro-apoptotic factors. In sum, a bioengineered, 3D culture platform was used to gain new mechanistic insights into how ECM in the brain tumor microenvironment promotes resistance to chemotherapy and suggests potential avenues for the development of novel, matrix-targeted combination therapies designed to suppress chemotherapy resistance in GBM.

Defining the molecular target of an antibody derived from nuclear extract of Jurkat cells using protein arrays.

Many diagnostic antibodies are generated by immunization with whole cells or cell extracts and are shown by screening on tissue sections to label specific cell populations. However, their target molecule then needs to be identified, and this can be technically demanding. Here we describe the use of protein arrays to define the targets of new or uncharacterized monoclonal antibodies. The technique involves screening protein arrays containing thousands of recombinant human proteins. An initial experiment showed that a well-characterized monoclonal antibody against nucleophosmin identified 22 clones on the array encoding this protein. Next, the antibody JJ166, for which the antigen had not yet been identified, was screened. This antibody was generated by immunizing with a nuclear extract of Jurkat cells and was detected in immunohistochemistry due to its distinctive nuclear staining of lymphoid tissue cells. However, its molecular target had remained unidentified using traditional approaches. A protein array screen rapidly identified the mitotic spindle-associated molecule NUMA1 (nuclear mitotic apparatus protein 1). To confirm this putative specificity, JJ166 was shown to react with COS-1 cells transiently transfected with the complementary DNA for NUMA1. Furthermore, a commercially available antibody against NUMA1 showed nearly identical staining in immunohistochemistry on human tissue and cells. Overall, this method represents an effective and quick strategy for defining the protein targets of new or uncharacterized monoclonal antibodies identified as having diagnostic or other potential value on the basis of their immunostaining patterns.

Injectable, Hyaluronic Acid-Based Scaffolds with Macroporous Architecture for Gene Delivery.

INTRODUCTION: Biomaterials can provide localized reservoirs for controlled release of therapeutic biomolecules and drugs for applications in tissue engineering and regenerative medicine. As carriers of gene-based therapies, biomaterial scaffolds can improve efficiency and delivery-site localization of transgene expression. Controlled delivery of gene therapy vectors from scaffolds requires cell-scale macropores to facilitate rapid host cell infiltration. Recently, advanced methods have been developed to form injectable scaffolds containing cell-scale macropores. However, relative efficacy of in vivo gene delivery from scaffolds formulated using these general approaches has not been previously investigated. Using two of these methods, we fabricated scaffolds based on hyaluronic acid (HA) and compared how their unique, macroporous architectures affected their respective abilities to deliver transgenes via lentiviral vectors in vivo. METHODS: Three types of scaffolds-nanoporous HA hydrogels (NP-HA), annealed HA microparticles (HA-MP) and nanoporous HA hydrogels containing protease-degradable poly(ethylene glycol) (PEG) microparticles as sacrificial porogens (PEG-MP)-were loaded with lentiviral particles encoding reporter transgenes and injected into mouse mammary fat. Scaffolds were evaluated for their ability to induce rapid infiltration of host cells and subsequent transgene expression. RESULTS: Cell densities in scaffolds, distances into which cells penetrated scaffolds, and transgene expression levels significantly increased with delivery from HA-MP, compared to NP-HA and PEG-MP, scaffolds. Nearly 8-fold greater cell densities and up to 16-fold greater transgene expression levels were found in HA-MP, over NP-HA, scaffolds. Cell profiling revealed that within HA-MP scaffolds, macrophages (F4/80+), fibroblasts (ERTR7+) and endothelial cells (CD31+) were each present and expressed delivered transgene. CONCLUSIONS: Results demonstrate that injectable scaffolds containing cell-scale macropores in an open, interconnected architecture support rapid host cell infiltration to improve efficiency of biomaterial-mediated gene delivery.

Regenerative Therapies for Spinal Cord Injury.

Spinal cord injury (SCI) is a serious problem that primarily affects younger and middle-aged adults at its onset. To date, no effective regenerative treatment has been developed. Over the last decade, researchers have made significant advances in stem cell technology, biomaterials, nanotechnology, and immune engineering, which may be applied as regenerative therapies for the spinal cord. Although the results of clinical trials using specific cell-based therapies have proven safe, their efficacy has not yet been demonstrated. The pathophysiology of SCI is multifaceted, complex and yet to be fully understood. Thus, combinatorial therapies that simultaneously leverage multiple approaches will likely be required to achieve satisfactory outcomes. Although combinations of biomaterials with pharmacologic agents or cells have been explored, few studies have combined these modalities in a systematic way. For most strategies, clinical translation will be facilitated by the use of minimally invasive therapies, which are the focus of this review. In addition, this review discusses previously explored therapies designed to promote neuroregeneration and neuroprotection after SCI, while highlighting present challenges and future directions. Impact Statement To date there are no effective treatments that can regenerate the spinal cord after injury. Although there have been significant preclinical advances in bioengineering and regenerative medicine over the last decade, these have not translated into effective clinical therapies for spinal cord injury. This review focuses on minimally invasive therapies, providing extensive background as well as updates on recent technological developments and current clinical trials. This review is a comprehensive resource for researchers working towards regenerative therapies for spinal cord injury that will help guide future innovation.