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.

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.

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.

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells.

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Brain-Mimetic 3D Culture Platforms Allow Investigation of Cooperative Effects of Extracellular Matrix Features on Therapeutic Resistance in Glioblastoma.

Glioblastoma (GBM) tumors exhibit potentially actionable genetic alterations against which targeted therapies have been effective in treatment of other cancers. However, these therapies have largely failed in GBM patients. A notable example is kinase inhibitors of EGFR, which display poor clinical efficacy despite overexpression and/or mutation of EGFR in >50% of GBM. In addressing this issue, preclinical models may be limited by the inability to accurately replicate pathophysiologic interactions of GBM cells with unique aspects of the brain extracellular matrix (ECM), which is relatively enriched in hyaluronic acid (HA) and flexible. In this study, we present a brain-mimetic biomaterial ECM platform for 3D culturing of patient-derived GBM cells, with improved pathophysiologic properties as an experimental model. Compared with orthotopic xenograft assays, the novel biomaterial cultures we developed better preserved the physiology and kinetics of acquired resistance to the EGFR inhibition than gliomasphere cultures. Orthogonal modulation of both HA content and mechanical properties of biomaterial scaffolds was required to achieve this result. Overall, our findings show how specific interactions between GBM cell receptors and scaffold components contribute significantly to resistance to the cytotoxic effects of EGFR inhibition.Significance: Three-dimensional culture scaffolds of glioblastoma provide a better physiological representation over current methods of patient-derived cell culture and xenograft models. Cancer Res; 78(5); 1358-70. ©2017 AACR.

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.

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.

Photodegradable hydrogels for capture, detection, and release of live cells.

Cells may be captured and released using a photodegradable hydrogel (photogel) functionalized with antibodies. Photogel substrates were used to first isolate human CD4 or CD8 T-cells from a heterogeneous cell suspension and then to release desired cells or groups of cells by UV-induced photodegradation. Flow cytometry analysis of the retrieved cells revealed approximately 95% purity of CD4 and CD8 T-cells, suggesting that this substrate had excellent specificity. To demonstrate the possibility of sorting cells according to their function, photogel substrates that were functionalized with anti-CD4 and anti-TNF-α antibodies were prepared. Single cells captured and stimulated on such substrates were identified by the fluorescence "halo" after immunofluorescent staining and could be retrieved by site-specific exposure to UV light through a microscope objective. Overall, it was demonstrated that functional photodegradable hydrogels enable the capture, analysis, and sorting of live cells.