Enhancing the Repair of Substantial Volumetric Muscle Loss by Creating Different Levels of Blood Vessel Networks Using Pre-Vascularized Nerve Hydrogel Implants.

Volumetric muscle loss (VML), a severe muscle tissue loss from trauma or surgery, results in scarring, limited regeneration, and significant fibrosis, leading to lasting reductions in muscle mass and function. A promising approach for VML recovery involves restoring vascular and neural networks at the injury site, a process not extensively studied yet. Collagen hydrogels have been investigated as scaffolds for blood vessel formation due to their biocompatibility, but reconstructing blood vessels and guiding innervation at the injury site is still difficult. In this study, collagen hydrogels with varied densities of vessel-forming cells are implanted subcutaneously in mice, generating pre-vascularized hydrogels with diverse vessel densities (0-145 numbers/mm2) within a week. These hydrogels, after being transplanted into muscle injury sites, are assessed for muscle repair capabilities. Results showed that hydrogels with high microvessel densities, filling the wound area, effectively reconnected with host vasculature and neural networks, promoting neovascularization and muscle integration, and addressing about 63% of the VML.

Engineering large and geometrically controlled vascularized nerve tissue in collagen hydrogels to restore large-sized volumetric muscle loss.

Developing scalable vascularized and innervated tissue is a critical challenge for the successful clinical application of tissue-engineered constructs. Collagen hydrogels are extensively utilized in cell-mediated vascular network formation because of their naturally excellent biological properties. However, the substantial increase in hydrogel contraction induced by populated cells limits their long-term use. Previous studies attempted to mitigate this issue by concentrating collagen pre-polymer solutions or synthesizing covalently crosslinked collagen hydrogels. However, these methods only partially reduce hydrogel contraction while hindering blood vessel formation within the hydrogels. To address this challenge, we introduced additional support in the form of a supportive spacer to counteract the contraction forces of populated cells and prevent hydrogel contraction. This approach was found to promote cell spreading, resist hydrogel contraction, control hydrogel/tissue geometry, and even facilitate the engineering of functional blood vessels and host nerve growth in just one week. Subsequently, implanting these engineered tissues into muscle defect sites resulted in timely anastomosis with the host vasculature, leading to enhanced myogenesis, increased muscle innervation, and the restoration of injured muscle functionality. Overall, this innovative strategy expands the applicability of collagen hydrogels in fabricating large vascularized nerve tissue constructs for repairing volumetric muscle loss (∼63 %) and restoring muscle function.

Reconstructing vascular networks promotes the repair of skeletal muscle following volumetric muscle loss by pre-vascularized tissue constructs.

Current treatment for complex and large-scale volumetric muscle loss (VML) injuries remains a limited success and have substantial disadvantages, due to the irreversible loss of muscle mass, slow muscle regeneration, and rapid formation of non-functional fibrosis scars. These VML injuries are accompanied by denervation and the destruction of native vasculature which increases difficulties in the functional restoration of muscle. Here, reconstruction of the vascular network at the injury site was offered as a possible solution for improving the repair of muscle defects through the timely supply of nutrients and oxygen to surrounding cells. A hydrogel-based tissue construct containing various densities of the vascular network was successfully created in the subcutaneous space of mice by manipulating hydrogel properties, and then implanted into the VML injury site. One month after implantation, the mouse treated with the highly vascularized tissue had extensive muscle repair at the injury site and only spent a shorter time completing the inclined plane tests. These findings suggest that the reconstruction of the functional vascular network at the VML injury site accelerated muscle fiber repair through a timely supply of sufficient blood and avoided invasion by host fibroblasts.

Strategy for improving cell-mediated vascularized soft tissue formation in a hydrogen peroxide-triggered chemically-crosslinked hydrogel.

The physically-crosslinked collagen hydrogels can provide suitable microenvironments for cell-based functional vascular network formation due to their biodegradability, biocompatibility, and good diffusion properties. However, encapsulation of cells into collagen hydrogels results in extensive contraction and rapid degradation of hydrogels, an effect known from their utilization as a pre-vascularized graft in vivo. Various types of chemically-crosslinked collagen-based hydrogels have been successfully synthesized to decrease volume contraction, retard the degradation rate, and increase mechanical tunability. However, these hydrogels failed to form vascularized tissues with uniformly distributed microvessels in vivo. Here, the enzymatically chemically-crosslinked collagen-Phenolic hydrogel was used as a model to determine and overcome the difficulties in engineering vascular networks. Results showed that a longer duration of inflammation and excessive levels of hydrogen peroxide limited the capability for blood vessel forming cells-mediated vasculature formation in vivo. Lowering the unreacted amount of crosslinkers reduced the densities of infiltrating host myeloid cells by half on days 2-4 after implantation, but blood vessels remained at low density and were mainly located on the edge of the implanted constructs. Co-implantation of a designed spacer with cell-laden hydrogel maintained the structural integrity of the hydrogel and increased the degree of hypoxia in embedded cells. These effects resulted in a two-fold increase in the density of perfused blood vessels in the hydrogel. Results agreed with computer-based simulations. Collectively, our findings suggest that simultaneous reduction of the crosslinker-induced host immune response and increase in hypoxia in hydrogen peroxide-triggered chemically-crosslinked hydrogels can effectively improve the formation of cell-mediated functional vascular networks.

Viscous Fingering as a Rapid 3D Pattering Technique for Engineering Cell-Laden Vascular-Like Constructs.

Tissues are much larger than the diffusion limit distance, so rapidly providing blood vessels to supply embedded cells inside tissues with sufficient nutrients and oxygen is regarded as a major strategy for the success of bioengineered large and thick tissue constructs. Here, a patterning technique, viscous fingering, is developed to bioengineer vascularized-like tissues within a few minutes. By controlling viscosity, flow rate, and the volume of photo-cross-linkable prepolymer, macro- and microscale vascular network structures can be precisely engineered using the Hele-Shaw cell that is designed in this study. After cross-linking, a vascular-like gel with fingering structures is formed between the bottom and top base gels, creating a sandwich-like structure. Cells can be incorporated into the fingers, bases, or both gels. The spreading and growth direction of the embedded cells are successfully controlled and guided by manipulating the physical properties of the fingering and base gels individually. Moreover, fingering is generated, connected, and surrounded prepared cell-laden microgels in base prepolymers to form prevascularized tissue-like constructs. Taken together, the 3D cell patterning technique extends the potential for modeling and fabricating large and stackable vascularized tissue-like constructs for both ex vivo and in vivo applications.

A strategy to engineer vascularized tissue constructs by optimizing and maintaining the geometry.

The success of engineered tissues is limited by the need for rapid perfusion of a functional vascular network that can control tissue engraftment and promote survival after implantation. Diabetic conditions pose an additional challenge, because high glucose and lipid concentrations cause an aggressive oxidative environment that impairs vessel remodeling and stabilization and impedes integration of engineered constructs into surrounding tissues. Thus, to achieve rapid vasculogenesis, angiogenesis, and anastomosis, hydrogels incorporating cells in their structure have been developed to facilitate formation of functional vascular networks within implants. However, their transport diffusivity decreases with increasing thickness, preventing the formation of a thick vascularized tissue. To address this, we used diffusion-based computational simulations to optimize the geometry of hydrogel structures. The results show that the micro-patterned constructs improved diffusion, thus supporting cell viability, and spreading while retaining their mechanical properties. Thick cell-laden bulk, linear, or hexagonal infill patterned hydrogels were implanted; and structural stability due to skin mobility was improved by the protective spacer. Larger and thicker perfused vascular networks formed in the hexagonal structures (∼17 mm diameter, ∼1.5 mm thickness) in both normal and diabetic mice on day 3, and they became functional and uniformly distributed on day 7. Moreover, transplanted islets were rapidly integrated subcutaneously in this engineered functional vascular bed, which significantly enhanced islet viability and insulin secretion. In summary, we developed a promising strategy for generating large, thick vascularized tissue constructs, which may support transplanted islet cells. These constructs showed potential for engineering other vascularized tissues in regenerative therapy. STATEMENT OF SIGNIFICANCE: Diffusion-based computational simulations were used to optimize the geometry of hydrogel structures, i.e., hexagonal cell-laden hydrogels. To maintain the hydrogel's structural integrity, a spacer was designed and co-implanted subcutaneously to increase the permeability of explants. The spacer provides the structural integrity to improve the permeability of the implanted hydrogel. Otherwise, the implanted hydrogel may be easily squeezed and deformed by compression from the skin mobility of mice. Here, we successfully developed a cell-based strategy for rapidly generating large, functional vasculature (diameter ∼17 mm and thickness ∼1.5 mm) in both normal and diabetic mice and demonstrated its advantages over currently available methods in a clinically-relevant animal model. This concept could serve as a basis for engineering and repairing other tissues in animals.

Dynamically and Spatially Controllable Albumin-Based Hydrogels for the Prevention of Postoperative Adhesion.

Since the degree of severity and the geometry of wounds vary, it is necessary to prepare an antiadhesive hydrogel that possesses dynamically controllable material properties, exhibits biodegradability, and possesses drug-releasing properties. Injectable, oxygen peroxide-sensitive, and photo-cross-linkable hydrogels that permit in situ dynamic and spatial control of their physicochemical properties were synthesized for the prevention of postoperative adhesion. Albumin is the most abundant protein in blood serum and serves as a carrier for several molecules that exhibit poor water solubility. It is therefore a suitable biomaterial for the fabrication of hydrogels since it presents a low risk of life-threatening complications and does not require immunosuppressive therapy for preventing graft rejection. The physicochemical properties of this hydrogel can then be spatially postadjusted via transdermal exposure to light to release drugs, depending on what is required for the injury. A significant reduction in postoperative peritoneal adhesion was observed in an animal model involving severe sidewall and bowel abrasions. This study demonstrated that the fabricated dually cross-linked, albumin-based hydrogels have great potential in such applications because they showed a low immune response, easy handling, full wound coverage, and tunable biodegradability. Precise spatial and controllable drug-release profiles may also be achieved via in situ transdermal post-tuning of the biomaterials, depending on the injury.

Two-Stage Patterned Cell-Based Treatments for Skin Regeneration.

During the process of wound healing, avoiding the formation of aligned collagen fibrils and subsequent scarring has become the focus of numerous research efforts. However, the goal of regeneration of native or scar-free skin remains a challenge. The complex and equivocal connection between inflammation and regeneration within the process of healing contributes to unsatisfactory treatment outcomes. Inspired by the scarless repair observed in fetal wound healing, we create a two-stage treatment combining the hydrocolloid dressing to attenuate the immune response in the initial three days, and the biomimetic cell-laden hydrogel to improve skin regeneration, which meet the specific needs of each stage in the healing process. To further accelerate the skin regeneration, the patterned cell-laden hydrogels were fabricated by photo-mask based photolithography technique. The efficacy and possible mechanisms of skin regeneration using this patterned cell-laden hydrogel therapy was investigated. Results show that these two-stage patterned cell-laden treatments were able to promote vascular network formation, accelerate wound closure, decrease scar formation, increase tissue regeneration and restore structure and mechanical properties of the skin in a full-thickness murine wound model. These data suggest that our patterned cell-based two-stage treatments can be used as a promising therapeutic option for wound healing by accelerating skin tissue regeneration.