Direct-writing Process and in vivo Evaluation of Prevascularized Composite Constructs for Muscle Tissue Engineering Application

Tissue engineered skeletal muscle is expected to treat muscle defects caused by trauma and disease.However,designing and manufacturing thick and complex tissue engineered skeletal muscle requires vascularization to ensure its internal cell viability and nutrient supply in vitro.In this article,we developed a set of Direct-Writing(DW)bio-printing procedure to manufacture a prevascularized composite construct with Human Umbilical Vein Endothelial Cell(HUVEC)and C2C12 cells for muscle tissue engineering application.We put the cells into the construct during the DW process to obtain the prevascularization and intend to promote its vascularization in vivo later.The constructs with cells or without cells were implanted respectively into nude mice back for 3 weeks,after which the mice healthily live for all the time and all the implants are tightly bonded to the host.From immunohistochemical analysis,CD31-positive blood vessels existed in the implanted samples with cells are more substantial than those without cells,but the implanted samples with HUVEC and C2C12 cells have much more number of small blood vessels distributing evenly.Moreover,the implants with cells,especially that with HUVEC and C2C12 cells,are able to get better fusion with the host skin and subcutaneous tissues.Histological analysis demonstrates that our DW-based constructs have the potential to be getting to vascularize the tissue engineered muscle.

Biomimetic glycosaminoglycan-based scaffolds improve skeletal muscle regeneration in a Murine volumetric muscle loss model

Volumetric muscle loss(VML)injuries characterized by critical loss of skeletal muscle tissues result in severe functional impairment.Current treatments involving use of muscle grafts are limited by tissue availability and donor site morbidity.In this study,we designed and synthesized an implantable glycosaminoglycan-based hydrogel system consisting of thiolated hyaluronic acid(HA)and thiolated chondroitin sulfate(CS)cross-linked with poly(ethylene glycol)diacrylate to promote skeletal muscle regeneration of VML injuries in mice.The HA-CS hydrogels were optimized with suitable biophysical properties by fine-tuning degree of thiol group substitution to support C2C12 myoblast proliferation,myogenic differentiation and expression of myogenic markers MyoD,MyoG and MYH8.Furthermore,in vivo studies using a murine quadriceps VML model demonstrated that the HA-CS hydrogels supported integration of implants with the surrounding host tissue and facilitated migration of Pax7+satellite cells,de novo myofiber formation,angiogenesis,and innervation with minimized scar tissue formation during 4-week implantation.The hydrogel-treated and autograft-treated mice showed similar functional improvements in treadmill performance as early as 1-week post-implantation compared to the untreated groups.Taken together,our results demonstrate the promise of HA-CS hydrogels as regenerative engineering matrices to accelerate healing of skeletal muscle injuries.

Preparation and Endothelialization of Multi-level Vessel-like Network in Enzymated Gelatin Scaffolds

Loss of function of large tissues is an urgent clinical problem. Although the artificial microfluidic network fabricated in large tis- sue-engineered constructs has great promise, it is still difficult to develop an efficient vessel-like design to meet the requirements of the biomimetic vascular network for tissue engineering applications. In this study, we used a facile approach to fabricate a branched and multi-level vessel-like network in a large muscle scaffolds by combining stereolithography （SL） technology and enzymatic crosslinking mechanism. The morphology of microchannel cross-sections was characterized using micro-computed tomography. The square cross-sections were gradually changed to a seamless circular microfluidic network, which is similar to the natural blood vessel. In the different micro-channels, the velocity greatly affected the attachment and spread of Human Umbilical Vein Endothelial Cell （HUVEC）-Green Fluorescent Protein （GFP）. Our study demonstrated that the branched and multi-level microchannel network simulates biomimetic microenvironments to promote endothelialization. The gelatin scaffolds in the circular vessel-like networks will likely support myoblast and surrounding tissue for clinical use.