Study Of Hyaluronic Acid Oligosaccharides Modified Collagen For Tissue Engineered Vascular Scaffolds

Cardiovascular disease remain the first killer of human life.Nowadays,the vascular grafts used to replace the small diameter blood vessels?inner diameter<5mm?face the following difficulties:?1?Difficult to mimic the structure and components of nature blood,especially the difficulties in the endothelialization and anticoagulation of the lumen part;?2?Vascular activity lose caused by the abnormal phenotype of the smooth muscle cell,which will lead to the mechanical mismatch with the nature blood vessel and tend to cause thrombosis and hemadostenosis of the artificial blood vessel;?3?Long preparation time.Ideal artificial blood vessels should be able to form a new blood vessel with the same mechanical and biological properties comparing with the host artery during the healing process.Therefore,it has become a hot topic in the world to study the composition and structure of natural blood vessels,and to develop the small blood vessels based on biomimicking.This study focus on the dual function of hyaluronic oligosaccharides?oH A?,which is both anti-coagulation and able to promote endothelialization.Based on the whole principle of biomimicking,we aimed to mimic the components and structure of the natural small vascular endothelial layer matrix,and finally construct the scaffold with the functions of able to promote vascularization and anti-coagulation for vascular repair and regeneration research.Contents in this paper includes:firstly,modify the collagen which is the major components of the extracellular matrix with oHA,to construct the glycosylated collagen and to equip the scaffold with the dual function of angiogenesis and anti-coagulation.Then,assemble the oHA modified collagen into vascular scaffolds with the technology of electrospinning.Finally,inoculate the porcine iliac artery endothelial cells?PIECs?into the scaffold.With the dual function of angiogenic and anti-coagulation inspired by the oHA,a successful vascular endothelial layer with similar components and structure of the natural blood vessel will be constructed.The conclusions of this study are:I Enzymatic hydrolysis and gel chromatography methods optimized by this paper are effective in the preparation of oHA4,oHA6,oHA8 and oHA10;optimized reaction methods are:get type Bio-Gel P6,mobile phase:0.111mol/L NH4HCO₃,loading quantity:100mg,UV detection wave length:232nm.II By the method of EDC/NHS crosslinking or the reductive amination,HA?or oHA?can be successfully connected with collagen constructing the glycosylated collagen composite;optimized reaction methods are:activation time 30min,mass ratio of collagen to HA=8:2,reaction time 4h,molar ratio of EDC:oHA-COOH=4:1,resulted HA content ranges from 1.2%to 4.1%.For the reductive amination,optimized condition are:mass ratio of collagen to oHA=8:2,reagent:hexafluoro isopropanol-O.lmol/L NaHCO₃.When oHA reacts with the collagen,3 times ultrafiltration centrifugation of the products can remove the unlinked oHA totally,the resulted HA content is?1.44±0.32?%.When HA of 5k molecular weight reacts with the collagen,6 times ultrafiltration centrifugation can remove the unlinked oHA totally,the resulted HA content is?2.65±0.30?%.III HFP or HFP/trifluoro acetic acid?TFA?system can successfully dissolve the oHA-collagen linked products and be used for electrospinning.The obtained electrospun fiber membranes have certain porosity and can be used in the construction of vascular tissue engineered scaffold.25%glutaraldehyde vapor can be used to crosslink the COL,oHA-COL and HA5k-COL successfully;EDC/ethyl alcohol can be used to crosslink the HA870k-COL;the resulted crosslinked products can maintain nanofiber state with the structure resembles the natural extracellular matrix?ECM?very well thus can be used to construct the biommetic ECM.IV The PIECs can grow,proliferate and migrate very well on the oHA-collagen electrospun fiber membrane scaffold.The proliferation behavior of the PIECs on the COL,oHAs-COL,HA5k-COL and HA870k-COL indicate that HA of low molecular weight can promote the growth of PIECs whereas the HA of high molecular weight can inhibit the growth of PIECs.After 3days culturing on the above mentioned materials,mRNA expression of ICAM-1,PCNA,P53 and CD31 can be detected.The expression of ICAM-1 and PCNA indicated favorable proliferation and migration ability;the expression of P53 proved that these cells can still inhibit the formation of tumor;the expression of CD31 showed that after 3 days culturing,the cells still maintained the phenotype and angiogenic ability of endothelial cells.Thus the oHA-collagen scaffold have good biological compatibility,no obvious toxicity,does not significantly affect the normal function of the PIECs,thus is expected to become good scaffolds for vascular tissue engineering.