Decellularization techniques and their applications for the repair and regeneration of the nervous system.

A variety of surgical and non-surgical approaches have been used to address the impacts of nervous system injuries, which can lead to either impairment or a complete loss of function for affected patients. The inherent ability of nervous tissues to repair and/or regenerate is dampened due to irreversible changes that occur within the tissue remodeling microenvironment following injury. Specifically, dysregulation of the extracellular matrix (i.e., scarring) has been suggested as one of the major factors that can directly impair normal cell function and could significantly alter the regenerative potential of these tissues. A number of tissue engineering and regenerative medicine-based approaches have been suggested to intervene in the process of remodeling which occurs following injury. Decellularization has become an increasingly popular technique used to obtain acellular scaffolds, and their derivatives (hydrogels, etc.), which retain tissue-specific components, including critical structural and functional proteins. These advantageous characteristics make this approach an intriguing option for creating materials capable of stimulating the sensitive repair mechanisms associated with nervous system injuries. Over the past decade, several diverse decellularization methods have been implemented specifically for nervous system applications in an attempt to carefully remove cellular content while preserving tissue morphology and composition. Each application-based decellularized ECM product requires carefully designed treatments that preserve the unique biochemical signatures associated within each tissue type to stimulate the repair of brain, spinal cord, and peripheral nerve tissues. Herein, we review the decellularization techniques that have been applied to create biomaterials with the potential to promote the repair and regeneration of tissues within the central and peripheral nervous system.

Engineering Tumor Stroma Morphogenesis Using Dynamic Cell-Matrix Spheroid Assembly.

The tumor microenvironment consists of resident tumor cells organized within a compositionally diverse, three-dimensional (3D) extracellular matrix (ECM) network that cannot be replicated in vitro using bottom-up synthesis. We report a new self-assembly system to engineer ECM-rich 3D MatriSpheres wherein tumor cells actively organize and concentrate microgram quantities of decellularized ECM dispersions which modulate cell phenotype. 3D colorectal cancer (CRC) MatriSpheres were created using decellularized small intestine submucosa (SIS) as an orthotopic ECM source that had greater proteomic homology to CRC tumor ECM than traditional ECM formulations such as Matrigel. SIS ECM was rapidly concentrated from its environment and assembled into ECM-rich 3D stroma-like regions by mouse and human CRC cell lines within 4-5 days via a mechanism that was rheologically distinct from bulk hydrogel formation. Both ECM organization and transcriptional regulation by 3D ECM cues affected programs of malignancy, lipid metabolism, and immunoregulation that corresponded with an in vivo MC38 tumor cell subpopulation identified via single cell RNA sequencing. This 3D modeling approach stimulates tumor specific tissue morphogenesis that incorporates the complexities of both cancer cell and ECM compartments in a scalable, spontaneous assembly process that may further facilitate precision medicine.

A bioengineered in situ ovary (ISO) supports follicle engraftment and live-births post-chemotherapy.

Female cancer patients who have undergone chemotherapy have an elevated risk of developing ovarian dysfunction and failure. Experimental approaches to treat iatrogenic infertility are evolving rapidly; however, challenges and risks remain that hinder clinical translation. Biomaterials have improved in vitro follicle maturation and in vivo transplantation in mice, but there has only been marginal success for early-stage human follicles. Here, we developed methods to obtain an ovarian-specific extracellular matrix hydrogel to facilitate follicle delivery and establish an in situ ovary (ISO), which offers a permissive environment to enhance follicle survival. We demonstrate sustainable follicle engraftment, natural pregnancy, and the birth of healthy pups after intraovarian microinjection of isolated exogenous follicles into chemotherapy-treated (CTx) mice. Our results confirm that hydrogel-based follicle microinjection could offer a minimally invasive delivery platform to enhance follicle integration for patients post-chemotherapy.

Microstructural evolution and resulting properties of differently sintered and heat-treated binder-jet 3D-printed Stellite 6.

Stellite 6 components are manufactured from gas-atomized powder using binder-jet 3D-printing (BJ3DP) followed by curing and sintering steps for densification. Green parts are sintered at temperatures ranging from 1260 °C to 1310 °C for 1 h. Microstructural evolution and phase formation during sintering and aging are studied by optical and scanning electron microscopy, elemental analysis and X-ray diffraction. It was found that solid-state sintering was present at temperatures below 1280 °C with Cr-rich carbides present within grains; while supersolidus liquid phase sintering was the dominant sintering mechanism during sintering at 1290 °C and higher in which the Co-rich solid solution regions are surrounded by eutectic carbides. Sintering at 1300 °C resulted in the maximum density of ~99.8%, mean grain size of ~98 ±â€¯6 µm with an average hardness of 307 ±â€¯15 HV0.1 and 484 ±â€¯30 HV0.1 within grain and at the boundaries, respectively. Aging was performed at 900 °C for 10 h leading to the martensitic transformation (fcc → hcp) as well as an increase in eutectic carbides at boundaries and nano-sized carbides within grains where the average hardness within grains and boundaries was enhanced to 322 ±â€¯29 HV0.1 and 491 ±â€¯58 HV0.1, respectively. Fibroblasts seeded on top of 3D-printed Stellite 6 discs displayed a cell viability of 98.8% ±â€¯0.2% after 48 h, which confirmed that these materials are non-cytotoxic. Presented results demonstrate that binder jetting can produce mechanically sound complex-shaped structures as shown here on a denture metal framework and small-scale knee model.

Preparation of Decellularized Biological Scaffolds for 3D Cell Culture.

The biggest challenge of designing and implementing an in vitro study is developing a microenvironment that most closely represents the interactions observed in vivo. Decellularization of tissues and organs has been shown to be an effective method for the removal of potentially immunogenic constituents while preserving essential growth factors and extracellular matrix (ECM ) proteins necessary for proper cell function. Enzymatic digestion of decellularized tissues allows these tissue-specific components to be reconstituted into bioactive hydrogels through a physical crosslinking of collagen. In the following protocol, we describe unique decellularization methods for both dermis and urinary bladder matrix (UBM) derived from porcine tissues. We then provide details for hydrogel formation and subsequent three-dimensional (3D) culture of two cell types: NIH 3T3 fibroblasts and C2C12 myoblasts .