Bionic Design And Initial Preparation Of Tissue Engineered Osteochondral Composites

The articular osteochondral defects caused by trauma or bone disease are common and become one of the main reasons of physically handicap in clinic. Those patients with osteochondral defects will frequently be with intractable pain, restrict joint activities and seriously impact on their quality of life. The incidence rate of osteochondral defects in the United States is 1.5‰to 3‰. In our country, it is about 5 to 6 times of that in the United States. With the increase in the proportion of elderly population, the incidence is increasing. Because cartilage have not nerves, blood vessels, lymphatic system and a enough self-repair capacity, in almost cartilage defects whose diameters is large than 2mm can not be completely restored, and the treatment of cartilage defects merging subchondral bone defect is more difficult. The current clinical treatment measures have obvious flaws, conservative treatment and joint clean-up operation could only temporarily alleviate the pain, can prevent the development of disease; autogenous bone graft is difficult to repair large defect for its limited source and may cause human-induced; osteochondral allograft transplantation may transmitted diseases; arthrodesis is possible to change joint anatomy of the original structure and loss of joint function and is difficult to accept by patients; joint replacement is costly, complications leaded to a high rate of revision and especially have a great physically and mentally impact to the younger patients. The rapid development of tissue engineering technology, especially single bone tissue engineering and tissue-engineered cartilage construction technology gradually get success, it provide new ideas and new technologies for the regeneration and repair of the osteochondral defects.The tissue engineering of osteochondral composite tissue has been deeply studied both in China and aboard. Preliminary results have been achieved from the feasibility of "construction by layers" to the experimental study of "integration construction". But the poor quality of the repair tissue in the area of osteochondral defects, poor integration with the host interface and the lack of corresponding mechanical functions are the major problems. On the base of correct understanding of normal and pathological state of the structure-function relationship ,tissue engineering apply life sciences and engineering principles and technology to research and develop biological substitutes for repairing, maintaining and promoting the human body in various injury or morphology state. Osteochondral composition is a more complex organ than a single cartilage and bone tissue. Therefore, we speculate the main problems of the current bone and cartilage tissue engineering are: (1) the lack of basic knowledge about the components, organizational structure, physiological functions of human osteochondral composite, and in-depth understanding of its micro-environment; disregard the important functional structure between the articular cartilage and subchondral bone - calcified cartilage zone; (2) there is a big difference between the biological materials and body composition, it is easily lead to immune rejection reaction in vivo, induce an acid environment during the degradation process (PLGA) to the detriment of cell proliferation; (3) the ideal strategy of construction will be build and host similar structure and function of the integration of osteochondral tissue engineering composite tissue in its normal structure, composition and foundation, rather than simply paste cartilage and bone tissue together.For the above reasons, this study first use the technology and methods related to tissue morphology, to study the normal human osteochondral composition, in particular its interface structure - calcified layer structure and the major component; and then based on the results of the study, with the use of collagen, and chitosan bionic raw materials, such as hydroxyapatite, using the technology of the mineralization of collagen and the methods such as spray-drying techniques to modify the regulation and make it with good plasticity and test the compatibility of their cell, tissue compatibility and biological safety; finally with the construction of the integration strategy, a preliminary tissue engineered osteochondral composite and animal experimental study was carried out with bionic structure and composition.PRIMARY PART: Normal Human Osteochondral Morphology and Component AnalysisUsing tissue morphology related technology and methods, to explore the morphology and component of osteochondral tissue and interface between bone and cartilage in normal human knee. Provide relevant theory for building bionic osteochondral tissue engineering composite. Materials and methods1. Normal human articular osteochondral morphology(1) To observe osteochondral composite tissue morphology in Fan-O / solid green staining and the Feng Savimbi staining.(2) To observe interface connections by scanning electron microscope and three-dimensional reconstruction technology.2. Normal human articular cartilage and bone component analysis(1) To analyse collagen protein of osteochondral composite by immunohistochemistry and amino acid detection technology qualitatively and quantitatively.(2) To analyse hydroxyapatite of osteochondral composite by X-ray diffraction technique qualitatively and quantitatively.Results1. Adult articular cartilage composite can be divided into three-layer structure: hyaline cartilage, calcified cartilage and subchondral bone zone. Hyaline cartilage is about 3 mm in thickness; alcified cartilage zone is only a few microns to 300 microns in thickness and its up interface showed a wave-like structure, the bottom interface of the calcified cartilage zone showed different sizes of comb teeth to anchor with subchondral bone.2. The main component of hyaline cartilage is collagen typeⅡ, which account 61.39%±0.38% of the dry weight, The rest component is proteoglycan; calcified cartilage zone mainly composited by typeⅡcollagen protein and hydroxyapatite, they account 20.16±0.96% and 65.09%±2.31% of the dry weight respectively; the main component of subchondral bone is typeⅠcollagen protein and hydroxyapatite, they account 13.69%±0.45% and 85.78%±3.42 % of the dry weight respectively.ConclusionAdult articular cartilage composite can be divided into three layer structure: hyaline cartilage, calcified cartilage and subchondral bone zone. the main component of hyaline cartilage is collagen type , calcified cartilage zone mainly composited by typeⅡcollagen protein and hydroxyapatiteⅡand the main component of subchondral bone is typeⅠcollagen protein and hydroxyapatite and the bottom interface of the calcified cartilage zone showed different sizes of comb teeth to anchor with subchondral bone. SECOND PART: Production of bionic materials of osteochondral matrixThis part the study was based on the results of the first part study, the first the composition and ratio of bionic materials of osteochondral matrix composite were identified, then modified the matrix composite by the way of grafting technique, collagen mineralization technology, as well as spray-drying technology, the last detected the physical and chemical properties, compatibility of cells and organizations and biological security of the matrix composite.Methods1. Production of matrix materials of tissue engineered bionic osteochondral composite(1) Cartilage matrix material was produced by collagen and chitosan graft technology; calcified layer and subchondral bone material was produced by collagen protein and mineralized hydroxyapatite, then micron powder was prepared by spray-dried technique.(2) Test the matrix material by Fourier Transform Infrared Spectroscopy.2. Biocompatibility of bionic materials of osteochondral matrix(1) Co-cultured matrix material with rabbit bone marrow-derived mesenchymal stem cells.(2) Implant matrix materials subcutaneously to the rabbit back to test the tissue compatibility and bio-safetyResults1. Successfully production three osteochondral matrix material: cartilage matrix materials -Ⅱcollagen protein / chitosan polymer powder; matrix material of the calcified zone -Ⅱcollagen protein / hydroxyapatite polymer powder; Matrix materials of subchondral bone.Ⅰcollagen protein / hydroxyapatite polymer powder. Fourier transforms infrared spectroscopy shows that the composition of the polymer material link in the molecular bonding.2. Cytocompatibility experiment showed that cocultured cells grow well, illustrate normal morphology and adhere on materials. Tissue compatibility experiment showed that implant material was wrapped by fibrous tissue, and did not happen inflammatory response, the organ pathological biopsy showed normal structure; which showed good cell, tissue compatibility and good bio-security. ConclusionCollagen grafting techniques, mineralization and spray drying technology can successfully produce the three osteochondral matrix material, physical and chemical properties of the structure showed that the biological material have good cells and tissues compatibility and biological security.THIRD PART: Design and Preparation of Tissue Engineered Bionic Osteochondral compositeThe design of tissue engineered bionic osteochondral composite is based on the first part of the research. Then use the second part of the development of the biological material to construct tissue engineered bionic osteochondral composite in vivo. The preliminary preparation of the tissue engineered bionic osteochondral composite by the method of rapid prototyping 3D printing technology.Methods1. Preparation of Engineered osteochondral composite tissue in vivo(1) Establish osteochondral defect model on the rabbit knee femoral trochlea(2) Prepare composite of materials and MSCs, implant the composite in defect model layerly then observe it.2. Preparation of bionic integrated engineering osteochondral scaffolds(1) Establish CAD model of bionic integrated engineering osteochondral scaffolds(2) Produce bionic integrated engineering osteochondral scaffolds by 3D printing technologyResults1. Control group :defect had not been repaired; scaffold group : a small portion of the edge repaired; material group without calcified zone:defect is shallow, area is narrow, but the surface is cracking; material group without calcified zone : repair tissue was white and elastic, defect is shallow, area is narrow, interface integration are continuous, but there is still a central defect.2. Bone cartilage bionic scaffold model produced by rapid prototyping 3D printing technology, the parameters of the model conform to that of the computer model. At present rapid prototyping 3D printing technology mainly used in industrial model and can not be used for the biomedical field now. Binder should improve to meet the requirements of 3D printing technology and have good biocompatibility.ConclusionUsing osteochondral composite to repair bone cartilage defect in vivo, we observed repair tissue was white and elastic, defect is shallow, defect is narrow, interface integration are continuous, but there is still a central defect.A small number of new cartilage were observed in tissue section, but most of that exfoliated to joint cavity for unfast fixation, therefore this technology require an advance study. Bone cartilage bionic scaffold model was produced by rapid prototyping 3D printing technology, the parameters of the model conformed to that of the computer model. At present rapid prototyping 3D printing technology mainly used in industrial model and cannot be used for the biomedical field now. Binder should improve to meet the requirements of 3D printing technology and have good biocompatibility.