Surface Modification Of PHB/PLLA Scaffolds For Anterior Cruciate Ligament Tissue Engineering

The anterior cruciate ligament(ACL) is an important structure for the knee stability.Because of the incapacity in self-healing of torn ACL, various reconstruction surgeries with various grafts under arthroscope have been performed to restore normal function of knee.Graft materials include autografts,allografts,synthetic grafts.However,each of these has their specific problems and limitations.Autografts are limited by a lack of an adequate supply of donors and by donor site morbidity.Allografts and xenografts own problems with the potential transfer of pathogens and immune rejection.Synthetic grafts exhibit uncertainly of long-term clinical outcome. Tissue engineering may potentially provide solution for these problems.Seeding cells,scaffold and tissue construction are three essential elements of tissue engineering.Scaffold material is nowadays one of difficult subjects for ligament tissue engineering.In the present study,we designed a novel kind of three-dimensional hybrid biomaterial consisting of PHB and PLLA.The scaffold was further hybridized with typeⅠcollagen to prepare the PHB/PLLA-collagen hybrid scaffold.Cellular responds of skin fibroblasts(SF) labeled by DiI to tile PLLA and PLLA-collagen hybrid braids were evaluated in vitro.Hybridized and original scaffolds combined with SF were implanted into the articular genu of rabbits.The aim was to investigate the degradation of the PHB/PLLA-collagen hybrid scaffold.1,Preparation of the PHB/PLLA scaffold and hybridized with typeⅠcollagenObjective The PHB/PLLA scaffold was constructed into a structure of sandwich fashion.The scaffold was further hybridized with typeⅠcollagen to prepare the PHB/PLLA-collagen hybrid scaffold.Methods A tile chaff made of PHB/PLLA(1:1) was clasped with fibre on its surface.The fibre was made of PHB and PLLA.The PHB/PLLA scaffold was synthesized into sandwich structure and was further hybridized with typeⅠcollagen to prepare the PHB/PLLA-collagen hybrid scaffold.The surface structure was observed under the scanning electronic microscope.The parameter of porosity of the scaffold was also measured.Re,nits The white,uniformity and tenacious PHB/PLLA scaffold composed of 1 mm chaff and 0.5 mm silk. Its porosity was 76.5%.The space among the PLLA fiber bundles in the PHB/PLLA-collagen hybrid scaffold was filled with collagen evenly.After hybridization,the collagen sponges intercrossed with the PHB/PLLA fiber and the braid become more compact.Condnsions We prepared a three-dimensional PHB/PLLA scaffold and was hybridized with collagen. The scaffold has favorable mechanics compatibility.2,Thestudy of skin fi broblasts labded by Dilin vitroObjective To offer a new feasible method to label S F through observing the change of SF labeled by DiI.Methods The third passage SF of rabbits digested were divided into two groups:the experiment group was labeled by DiI,and the normal group not.Then by the morphology under the microscope,trypanblau testing,drawing the growth curve,we surveyed the influence of DiI on the growth of SF.They were planted into cell culture plates with 12 holes at the same level,each group with 6 holes per plate. From then on,we took one plate each day digest,count cells for 7 days continuously and draw the growth curve.Results The growth curve of each group is not significant on the whole,and so are the cell numbers on different time(P＞0.05).Condusions Compared with the normal cells,the proliferation and appearance of SF labeled by DiI are not significant.The SF labeled by DiI can be observed continuously in living state.The novel method of labeling SF can be utilized extensively in the ACL tissue engineering.3,The study of combination of skin fibroblasts and PHB/PLLA -collagen hybrid scaffoldObjective The aim of this study was to examine the biological activity of the scaffold material,through observing the combination of SF labeled by DiI and the PHB/PLLA and hybridized scaffold.The hybridized and original scaffold were combined with self-SF and were implanted into the articular genu of rabbits to study the degradation rate of them.Methods The third passage SF of rabbits were labeled by DiI and were seeded into the hybridized and original scaffold.The experiment group was the hybridized scaffold and the control group was the original scaffold Digesting and counting each group cells on the day of 3,5,7,to observe the increasing state with the hybridized and original scaffold,and to investigate the growth state of the DiI labeled cells by fluorescent microscope.Cell growth and morphology were observed at 5d and 7d by scanning electron microscopy(SEM).The hybridized and original scaffold were combined with self-SF and were cultered for 7 days in vitro.Then they were implanted into the articular genu of rabbits.The test and control scaffolds were gathered in 4 week and 8 wek after operation and they were analyzed by SEM.Results The water absorption capability of the test groups is higher than the control groups(p＜0.05).The increasing rates of SF on the two groups were significant on the day of 3,5,7 (p＜0.05).More extensive cell proliferation and formation of large cellular networks which bridge the fibers were observed in the hybridized scaffold than in the PHB/PLLA scaffold.After these scaffolds were implanted for 4w and 8w, the degradation of the hybridized scaffold were more obviously than the original scaffold by SEM.Condusions The SF labeled by DiI can be well compatible with hy bridized scaffold and can be observed continuously in living state.The hybridization of the PHB/PLLA with collagen sponge increased hydrophilicity and facilitated cell seeding compared to pure PHB/PLLA scaffolds.And the degradation rate of the hybridized scaffold were better than the original scaffold.Our research has demonstrated that cell attachment,proliferation, differentiation on PHB/PLLA scaffold were greatly enhanced with collagen coating.The PHB/PLLA scaffold modified by collagen sponge is expected to be of useful value in ACL tissue engineering.