Geometric Control Of Vascular Network Is Regulated By Tensile Force In 3D Bioprinted Tissue

Objective: At present,skin wounds are usually treated with autologous tissue,allograft tissue,xenograft tissue and tissue engineered skin.However,the harvest of autologous tissues,such as skin graft and flap,may result in non-healing wound or scar formation in the donor site.In addition,if the wounded area is too big,it is difficult to get enough and high quality skin tissue from the patient.Because of immunological rejection after transplantation,the allograft tissue(such as cadaver skin)and xenograft tissue(such as pig skin)can only be used to cover the wound temporarily.Tissue engineered skin,which can be manufactured and has low immunogenicity,is one of the ideal materials for treating skin wound and is a hot topic in the research field of wound healing.Currently,commercialized tissue engineered skins are mainly classified as epidermal substitutes(such as Epidex and epidermal),dermal substitutes(such as Integra and Dermagraft)and epidermal-dermal substitutes(such as Apligraf).Due to the lack of vascular system,these skin substitutes are mainly used to treat burn wounds which have good vascular bed and are not used for the wounds with poor blood supply.Vascularized skin substitute will promote wound healing by establishing blood circulation with recipient tissue after transplantation.Therefore,developing vascularized skin substitute is one of the main subjects of skin tissue engineering.The objective of this study is to investigate the effect of tensile force on vessel formation in 3D bioprinted tissue and explore the underlying mechanisms.Methods and Results: In the present study,tissues with human umbilical vein endothelial cells(HUVECs)and fibroblasts was printed by using fibrin and gelatin as scaffolds.We examined the expression actin,collagen and CD31 in printed tissues by immunofluorescence during vessel formation,the effect of tensile force on vessel formation and explored mechanisms involved.The present study consists of three parts: In the first part,we established a 3D bioprinting model for studying the effect of tensile force on vessel formation in printed tissue.Polycaprolactone(PCL)was used to print the outer frame which fixed the printed tissue containing fibrin,and gelatin was printed between fibrin tissues.We found that the printed fibrin tissue was stable in culture when PCL was printed in 2 layers and there were 3 gelatin lines between fibrin tissues.In the second part,we studied the effect of the tensile force on vessel formation in 3D bioprinted tissue.Unprinted or printed tissues containing HUVECs and different concentrations of fibrin were cultured and examined for vessel formation by immunofluorescence staining with antibody against CD31.The results showed that unprinted tissues containing 1 mg/ml or 3 mg/ml fibrin were contracted in culture,but this phenomenon didn't occur when fibrin was higher than 7 mg/ml.Vessel-like structures grew in random direction were observed in unprinted tissue at 7 days after culture,and its length is longest when fibrin concentration is 5-7 mg/ml.In printed tissues containing different concentrations of fibrin,most of the vessel-like structures grew in parallel with the direction of tensile force,and formed lumen.Printed tissues with 5 mg/ml fibrin had longer vessels than tissues with other fibrin concentrations.These results indicate that tensile force plays important role in vessel formation.In the third part,we investigated the mechanism underlying tensile force regulated vessel formation in 3D bioprinted tissue.By immunofluorescence staining,we examined collagen,actin and CD31 in printed tissues during vessel formation,and explored the mechanisms involved by using Rho/ROCK inhibitor Y27632 and Myosin-II inhibitor Blebbistatin.Our results showed that in 3D bioprinted tissues,HUVECs,actin and collagen located randomly at one day after culture,began to distribute along the direction of tensile force at 3 days after culture,and mostly distributed along the direction of tensile force at 7 days after culture.However,the HUVECs,actin and collagen in unprinted tissues were distributed randomly all the time.These results indicate that cytoskeleton and collagen contribute to vessel formation regulation by tensile force.Further studies showed that printed tissues treated with Y27632 or Blebbistatin for 14 days had shorter vessel-like structure than untreated tissues.All together,our results demonstrated that actin and collagen were involved in the regulation of vessel formation by tensile force through Rho/ROCK signaling pathway.Conclusions: In summary,our results showed that fibrin at 5 mg/ml is the best concentration for vessel formation in 3D bioprinted tissues.Mechanical studies demonstrated that actin and collagen took part in the regulation of vascular formation by tensile force through Rho/ROCK related signaling pathway.