RESUMEN
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.
RESUMEN
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 (â¼17 mm diameter, â¼1.5 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 â¼17 mm and thickness â¼1.5 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.
Asunto(s)
Diabetes Mellitus Experimental , Animales , Diabetes Mellitus Experimental/terapia , Hidrogeles , Ratones , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
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.