A Preparation Study Of Sodium Alginate-based Multi-layer Integrated Osteochondral Composite Tissue Engineering Scaffold

Articular cartilage lesion is a common clinical problem, which is accompanied by subchondral bone lesion most time. In the anatomic structure, the cartilage and subchondral bone are linked together closely so that they have a mutual influence on each other in the biological function. Their differences in composition and mechanical properties demonstrate the importance to construct the cartilage/bone biofunctional interface. This study was aimed at solving the problems of the integration between bone and cartilage in osteochondral tissue engineering scaffolds as well as the integration between these scaffolds and the surrounding host osteochondral tissues, which would carry out the study on integrated multi-layer scaffolds for osteochondral tissue engineering. This study used degradable PCL nanofibers and degradable nonstoichiometric micro or nano hydroxyapatite crystals to compose with RGD-grafted heparin sulfate-grafted sodium alginate to produce a composite, so as to improve the mechanical property of the material and cell compatibility, and meanwhile use the controlled release technology of gene to achieve combined and sequential delivery of growth factors for osteochondral tissue engineering, so as to effectively guide the formation and reconstruction of bone and cartilage via in vitro and in vivo tissue engineering. The effect of material factors on cell adhesion, growth, proliferation and differentiation would be determined; the mechanisms and laws in material degradation, growth factor release as well as the in vitro and in vivo formation of osteochondral tissue in scaffolds would be explored, based on the study on the preparation of materials, cell adhesion on modified materials, degradation of materials, growth factor release and the in vitro and in vivo study and so on. These will provide certain theory basis for the preparation of materials, which match osteochondral tissue better and are closer to the physiological metabolism, for osteochondral reconstruction and repair.In this study, micro and nano hydroxyapatite particles were synthesized via a hydrothermal method and used to form the hybrid hydrogel while electrostatic spinning technology was used to prepare PCL/PEG nanofiber membrane and coaxial short fiber. The bioactive OSA/NSC composite hydrogel and OSA/NSC/μmHA composite hydrogel were prepared by using the Schiff base reaction between oxidation sodium alginate and N-succinyl-chitosan. The SA/nmHA composite scaffolds with parallel pore structure were prepared by using the physical crosslinking method. Based on the design of "integrated multi-layer scaffold", we constructed the multi-layer scaffold via a reasonable in-situ assembling method. The physical and chemical properties of the as-prepared materials were characterized by using FT-IR, XRD, SEM, EDX, TEM, fluorescence microscope, optical microscope and mechanical testing machine. Additionally, cell culture was used to ecvluate the biocompatibility of the as-prepared scaffolds. The results showed that not only the multi-layer scaffolds had excellent structural integrity and obvious regional stratification but also the interfaces could be bonded together closely; the scaffolds also presented good biocompatibility.To evaluate the biofunction of of the intergrated multi-layer scaffolds under growth factors and the LIPUS effect, animal experimental study was conducted. The controlled release technology of gene was used to achieve combined and sequential delivery of growth factors for osteochondral tissue engineering and low intensity pulsed ultrasound (LIPUS) was applied as mechanical stimulation so as to effectively guide the formation and reconstruction of bone and cartilage via in vitro and in vivo tissue engineering. The scaffolds were implanted in rabbit’osteochondral defects via "an anteromedial parapatellar longitudinal and capsular incision" standard operation. These rabbits were sacrificed using an overdose ear vein injection method of pentobarbital sodium at 1 week,2 weeks,4 weeks, 8 weeks, and 12 weeks after operation. The specimens were harvested and then were fixed in 10% neutral buffered formalin, decalcified in a special type of decalcifying fluid, embedded in paraffin and sectioned into 5μm in thickness and stained for histological evaluation. The results showed that multi-layer integrated implants could achieve effective fixation and good intergration with host tissue in the early stage. The growth factor+LIPUS experiment group showed the best ability in repairing osteochondral defects among all experiment groups. The formation of vascularization was observed at 2 weeks postoperation, which helps to generate distinct metabolic microenvironment for angiogenesis and osteogenesis. Subchondral bone started to regenerate at 4 weeks. The number of chondrocyte-like cells increased more significantly than other groups. The reconstruction of subchondral bone was almost completed and the integration with the host bone was successfully achieved. These findings suggest that the hybrid use of growth factors and LIPUS might provide a optimal approach for a high level of angiogenesis and osteochondral-genesis at early stage and long-term osteochondral repair.