Experimental Study on Construction of Intervertebral Disc Adduius Fibrosus Scaffolds for Tissue Engineering
|School||Second Military Medical University|
|Keywords||annulus fibrosus intervertebral disc tissue engineering acellular scaffold construction|
Objective：Degeneration of intervertebral discs (IVDs) is a common cause ofcervical-shoulder and lumbodorsal pain, which is a significant health problem in today’ssociety. Current treatments are to relieve patients’ symptoms, but that cannot solve thepotential problems of the biological structure of intervertebral disc degeneration. Tissueengineering is becoming the ideal method of treatment of disc degeneration because it canrestore function of natural tissues. Although a large number of studies on nucleus pulposustissue engineering has achieved much success in the laboratory, without annulus fibrosusintervertebral disc tissue engineering is difficult to make a breakthrough. The purpose ofthis study is to take advantage of New Zealand white rabbits to prepare natural anddecellularized intervertebral disc scaffolds. Decellularized scaffolds have natural structureof annulus fibrosus extracellular matrix, anatomic morphology, mechanical and biologicalfunctions. Culturing and evaluating the rabbit bone marrow mesenchymal stem cells、fibrous ring cells. Both can grow well and secrete collagen matrix on the scaffold. All canillustrate that acellular annulus fibrosus have good histocompatibility without obviouscytotoxicity. So it is feasible for acellular annulus fibrosus to become intervertebral disctissue engineering annulus fibrosus scaffolds.Methods: Take1month old New Zealand white rabbits for cell isolating and culturing.After anesthesia do extract about3-5ml bone marrow from the iliac or femoral with bonemarrow puncture needle, putting bone marrow into culture flasks with plentiful culturemedium. Isolate and culture the BMSCs in terms of whole bone marrow culture. Get thelumbar spine, remove the periphery soft tissues and vertebral bone, rinse the intervertebraldisc with PBS solution, and get rid of cartilage endplate on both sides of the disc. Alsoreject nucleus pulposus、the outermost and innermost layer of AF. Ophthalmic scissors canbe used to trim annulus fibrosus tissue small pieces around1mm×1mm×1mm size, whichare putting into cell culture flasks to isolate and culture the annulus fibrosus cells. After3-month-old New Zealand white rabbits death from overdosage of anaesthesia, get thelumbar spine, remove the periphery soft tissues and vertebral bone, rinse the intervertebraldisc with PBS solution, and get rid of cartilage endplate on both sides of the disc. Thediameter about12mm, sagittal diameter about7mm and height about3mm. Prepare theacellular scaffolds in the process of decellularizing the annulus fibrosus. Detect theacellular scaffolds on histology, light microscopy, electron microscopy, and measure the porosity, biomechanical parameters about annulus fibrosus. Combine BMSCs and AFCswith the acellular scaffolds respectively, implant the acellular scaffolds into the rabbit andalso analyse cytotoxicity and histocompatibility.Results：Annulus fibrosus cells fusiform or polygonal, bone mesenchymal stem cellsfusiform，both show a spiral-shaped growth of integration about70%-80%. MTT assayshows both BMSCs and AFCs express strong proliferative capacity, and reach a plateau inthe seventh day, subsequent proliferation minimal,with a declining trend. Annulus fibrosuscells have declined more obvious than bone marrow mesenchymal stem cells. Theannulus fibrosus decellularized scaffolds have milky, hollow oval-shaped, soft,three-dimensional porous structure. HE staining shows that annulus fibrosus scaffolds getno significant residual cellular components. The frozen section Hoechst33258colorprompts that normal scaffolds have obvious fluorescent reaction, no significantfluorescence reaction in the decellularized scaffolds, indicating that the the annulusfibrosus acellular scaffold have been decellularized thoroughly. Scanning electronmicroscopy and transmission electron microscopy illustrate the microstructure of theannulus fibrosus, the structural arrangement of the typical lamellar structure, the collagenfiber bundle and the elastic fibers between plates layers. The acellular collagen structureare more smooth and sloppy, full of collagen fibers, no significant changes in the scaffolds.Scaffolds owns uniform porosity, pores interlinked to each other. The porosity is42.86±3.41%. Vitro composite culture proves that active proliferation bone mesenchymal stemcells proliferate actively on the scaffolds and generate amount ofⅠ,Ⅱcollagen. Implantedafter8weeks, histological observation tells cell ingrowth the annulus fibrosus outer layer,inflammatory cells immersed between the inner and outer layers of the annulus fibrosus.The annulus fibrosus acellular scaffolds show the maximum tensile、elasticity modulusdiffer subtle between the inner and outer layers, and can meet the mechanicalrequirements.Conclusions:Aellular method can effectively remove cellular components from the rabbit fibrousannulus. Proper pore size、porosity and biomechanical strength belong to the rabbitintervertebral disc fibrous annulus acellular scaffolds. The acellular method does no harmto collagen fibers and biomechanics of the acellular scaffolds. Annulus fibrosus cells cangrow well on the scaffolds by negative pressure adsorption method. Acellular scaffolds have good biocompatibility without cytotoxicity. Annulus fibrosus can serve asintervertebral disc tissue engineering scaffold materials.