The subcutaneous space is an attractive alternative site for engineering vascular networks, since it provides relatively easy accessibility, minimal invasiveness, and potentially high transplant capacity. However, it’s also a poorly vascularized site that often fails to achieve the level of vascularization necessary to maintain function of thick grafts.1 Here, we patterned our cell-laden hydrogels to form a 4 mm-thick constructs with gradient mechanical properties to create perfused vascular network within 7 days in vivo. Moreover, this construct was applied to encapsulate islets, and successfully supported and increased viability and function of transplanted islets at the subcutaneous site in diabetic mouse.
a. Creating thick functional vascular networks
Mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) were gently mixed into prepolymer solution of gelatin methacrylate (GelMA). After crosslinking under the designed hexagonal photomasks with suitable light exposure time, the cell-laden or acellular 4 mm-thick hydrogels with gradient mechanical properties were formed by layer-stacking technique. Then, 4 mm-thick cell-laden and acellular hydrogels constructs were implanted into subcutaneous space in nude mouse for 7 days.
b. Engineering vascularized islet tissues at subcutaneous site of mouse
900 islets encapsulated in 200 μL collagen were placed in the middle of 4 mm-thick HUVECs and MSCs-laden or acellular GelMA hydrogels constructs. Then, constructs were implanted into subcutaneous space in nude mouse for 3 months. Non-fasting blood glucose (BG) levels were monitored every 2 days, and intraperitoneal glucose tolerance tests (IPGTT) were done every 3 weeks after implantation. Implants were retrieved after 3 months, after that, BG levels were monitored every day, and explants were analyzed by immunohistochemistry staining.
Results and Conclusions
Results showed that thick functional vascular networks with uniformly-distributed vessels and few connective tissues were created in the cell-laden hydrogels in vivo within 7 days, which serves as the vascularized group. To demonstrate the importance of engineered vascular networks for transplanted islets, the acellular GelMA hydrogels was served as non-vascularized group. Meanwhile, 900 islets were encapsulated in collagen hydrogels to protect islets from hypoxia. In the vascularized group, having islets and collagen embedded in cell-laden GelMA hydrogels, BG levels of diabetic mouse (≧450 mg/dL) decreased to ＜250mg/dL, which is in the healthy normal region, one week after implantation and maintained for 3 months after implantation. Moreover, BG levels soared to ≧450 mg/dL after retrieving the implant. Immunostaining also confirmed that islets in the vascularized group were functional with insulin positive and highly vascularized both externally and internally. On the contrary, in the non-vascularized group, having islets and collagen embedded in acellular GelMA hydrogels, BG levels maintained at high level after implantation. In addition, there’re few micro-vessels and most islets were necrosis in the explants. Taken together, our engineered thick vascular networks are able to significantly support and increase viability and functionality of transplanted islets at the subcutaneous site in diabetic mouse, and successfully achieve correction of chemically induced diabetes in mouse for 3 months.
- Tatarkiewicz, K. et al., Transplantation. 67(5), 665–671 (1999).