Construction Of Triphasic Biomimetic Tissue Engineered Osteochondral Composite With Natural Calcified Zone Of Cartilage By A Novel Dual-chamber Bioreactor

BackgroundOsteochondral tissue is the mainly load-bearing system that provides excellent lubrication, stability and uniform distribution of loads. Osteochondral defects caused by traumatic injury or disease are problematic within the clinical setting and are leading causes of severe pain and functional impairment of joints, bringing heavy burden for patients and society. Curl et al. reported that cartilage defects were diagnosed in 63% of all knees subjected to knee arthroscopy in a retrospective study on 31516 patients. Due to the absence of vascularity and the interfacial nature of osteochondral tissue, cartilage is hard to repair once damaged. Current clinical interventions such as conservative treatment, joint debridement, microfrature, autogenous or allograft osteochondral transplant and joint replacement exit obvious deficiency, long term outcomes are less than satisfactory. Microfrature, aslo known as marrow stimulating technique, is the most frequently-used treatment in clinic. By drilling the bony base of chondral defect, the chondral defect will be filled by a blood clot, this clot includes multipotent stem cells from the underlying bone marrow. These cells are able to differentiate into fibrochondrocytes which stimulate fibrocartilage repair to fill the defect, relieve the symptoms and delay the happening of osteoarthritis. But this fibrocartilage repair does not restore the original cartilage structure and function, poor in long-term effect. Because of source limited and accompanied disadvantages including invasive surgery from healthy sites and donor sites morbidity, which limits the quantity of donor tissue, application of osteochondral transplant or autologous chondrocyte implantation is restricted too. As for joint replacement, operation trauma, postoperative complications and limited prosthetic long-term survivorship are challenges need to be overcome at this stage.Regenerative medicine, such as tissue engineered osteochondral technique, for restoring the original function and structure of osteochondral defects have been developed as a potential alternative to overcome the limitations of these current treatments. However, at this stage, tissue engineered osteochondral technique is less than satisfactory due to drawbacks including bad integration with host tissue, poor mechanics function and fibrocartilage-like degeneration. In our opinion, this is due to the disadvantages exist in structure and function of tissue engineered osteochondral graft, especially lack of the interface structure between hyaline cartilage and subchondral bone, the calcified zone of cartilage(CZC).The calcified zone of cartilage is defined as mineralized cartilage zone which lies between the hyaline cartilage and subchondral bone in mature synovial joints. It interlock s tightly with hyaline cartilage above in the manner of ―ravine-engomphosis‖ and interlocks tightly with underlying subchondral bone in the manner of ―comb-anchor‖. Its dense matrix consists of abundant collagenous fiber and mineral composition with mean thicknesses of 104.16 ± 20.87 μm. The CZC is important for force transmission between the compliant cartilage and the stiff subchondral bone, and may also act as a biochemical barrier between these two structures. During the pathological process of osteoarthritis, the thickness and permeability of CZC will chang; perforation and tidemarks duplication of CZC will happen, accompanied by the invasion of vessels and nerves. Hence, CZC plays an important role in the maintenance of normal physiological functions of osteochondral tissue. Except for mechanics function, its barrier effect can prevent the cells migration and provide an independent growing microenvironment for osteogenic and chondrogenesis differentiation. Therefore, maybe, it is of great significance to construct a biomimetic tissue engineered osteochondral composite with calcified zone of cartilage.Therefore, based on the deep insight of the structure and physiological function of CZC, we presented a novel concept for the construction of a triphasic biomimetic osteochondral scaffold by one-step method. This scaffold was constructed by decellularized extracellular matrix(ECM) and collegen type II gel through freezen-drying and genepin cross-linking. One-step means construct these three layers together by one-step. Then, in order to provide an independent growing microenvironment for cartilage layer and subchondral bone layer, we designed a noval dual-chamber bioreactor by computer aided design(CAD) and rapid prototyping 3D printing technology with CZC as the barrier of these two chambers, taking polylactic acid(PLA) as raw material. In order to test the feasibility of this dual-chamber bioreactor, amniotic mesenchymal stem cells were seeded in this scaffold, fluorescence labeling was used for cell tracking, and inducing medium was used to induce stem cells differentiation. Culture medium in these two chambers was circulated separately by peristaltic pumps. Cells distribution, adhesion and proliferation in the scaffold were tested by fluorescence cell tracking, pathological section examination and PCR 7 days later. Finally, these triphasic biomimetic osteochondral scaffolds were used for osteochondral defects repair in minipigs animal model after 14 days’ culture. Twelve and twenty-four weeks later, the repair effect was inspected.Section I: Fabrication and Test of Triphasic Biomimetic Osteochondral ScaffoldIn this section, cylindrical osteochondral grafts were harvested from adult pig knees, hyaline cartilage was cut away for COL II extraction, residual CZC and subchondral bone layer was decellularized. The type II collagen gel was poured onto the CZC layer, followed by freeze-drying and genipin cross-linking, then we acquired the triphasic biomimetic osteochondral scaffold. Related test of scafflod was applied.Materials and Methods1. Fabrication and test of decellularized bone graft(1) Cylindrical osteochondral grafts with 12 mm diameter were harvested from adult pig knees, then remove the hyaline cartilage layer.(2) The CZC and subchondral bone composite was decellularized in 1% Triton X-100.(3) HE and DAPI staining were carried out to test if the cells were totally decellularized.2. Extraction of COL II gel(1) Obtain cartilage powder.(2) Cartilage powder digestion, salting out and dialysis.3. Fabrication and test of scaffold(1) Construction of mould for scaffold fabrication by 3D printing.(2) Type II collagen gel was poured onto the CZC layer, and then apply freeze-dry and genipin cross-link.(3) Measure the pore diameter, porosity, cytotoxicity and architectural of the scaffold.ResultsThe scaffold was dark blue and the subchondral bone was decellularized completely. The mean diameter of random and interconnected pores of collagen layer was 102.3 ± 35.27 μm, the mean diameter of pores of subchondral bone was 470.2 ± 158.8 μm. The mean porosity of the collagen layer was(96.1 ± 3.8) %, subchondral layer was(73.5 ± 2.6) %. Scaffold leaching liquor didn’t affect the growth of cells.Section II: Fabrication of the Double-Chamber BioreactorIn this section, in order to construct an independent culture microenvironment for chondral layer and subchondral layer, we constructed a novel dual-chamber bioreactor take advantage of the barrier effect of CZC by computer-aided design and 3D printing technology. Chondrogenic and osteogenic medium was applied in cartilage chamber and subchondral bone chamber respectively to induce the differentiation of mesenchymal stem cell. This dual-chamber bioreactor was more in line with the physiological feature of osteochondral tissue; hence, it’s likely to construct a more biomimetic repair material for osteochondral defects.Materials and Methods1. Fabrication of the double-chamber bioreactor(1) Design the double-chamber bioreactor model by computer aided design(CAD).(2) Fabricate the double-chamber parts by 3D printing.(3) Assemble the double-chamber, apply the impermeability test, sterilize the scaffold and the double-chamber bioreactor by irradiation(Co60).2. Culture of human amniotic mesenchymal(HAM) cellsHuman amniotic mesenchymal cells were harvested by collagenase type I, cells purification was applied by differential digestion method.3. Cells cultured with scaffold(1) Cells labeled by CFDA SE and Di I.(2) Cells suspension drip infusion and type Ⅱcollagen gel inoculation were applied for cells seeding.(3) Pathological section examination, PCR and total DNA content analysis were applied to test the cells adhension, proliferation, distribution and differentiation.ResultsAfter 7 days culture, the seeded cells distributed in the scaffold regularly, cell adhesion and proliferation was not be affected. CFDA SE labeled cells distributed in collagen scaffold, and Di I labeled cells distributed in subchondral bone layer respectiv ely. No cells migrated across the CZC. Chondrogenic and osteogenic medium could induce stem cells in cartilage chamber and subchondral bone chamber to differentiate into chondrocytes and osteocytes, respectively. Total DNA content analysis showed that cells in scaffold increased in a time-dependentmanner.Section III: Animal Experiment for Osteochondral ScaffoldIn this section, in order to test the repair effect of the scaffold for osteochondral defect, we applied the animal experiment. Osteochondral defects were created at the weight-bearing regions in femur condyle of Guizhou mini-pigs. Biomimetic osteochondral scaffolds with natural CZC were used to repair the osteochondral defects; repair effect was compared with control groups.Materials and Methods1. Cells cultured with biomimetic osteochondral scaffolds with natural CZC in double-chamber bioreactor(1) BMSCs acquired from Guizhou mini-pigs by Percoll density gradient centrifugation.(2) BMSCs cultured with scaffolds.2. Create and repair osteochondral defects(1) 30 Guizhou mini-pigs were divided into 3 groups(Group A, Group B and Group C), 20 knees for each group(60 knees in total). Osteochondral defects were created at the weight-bearing regions in femur condyle of Guizhou mini-pigs.(2) Group A was blank control group(no scaffold repair), osteochondral defects from Group B were treated with osteochondral scaffolds which didn’t have CZC, osteochondral defects from Group C were treated with biomimetic osteochondral scaffolds with natural CZC.3. Test of osteochondral defects repair effectStereoscopic microscope, MRI, HE staining, Fast Green-Safranin O staining, Sirius-Red staining and O’Driscoll histological evaluation were used to evaluate the repair effect.ResultsRepair effect of group C was better than that of group A and B, the structure and composition of repair tissue was familiar with host tissue, and intact CZC was found in group C. However, partial cartilage defects were not repaired completely in group C.ConclusionTriphasic biomimetic tissue engineered osteochondral scaffold could be fabricated by collegen type II gel and decellularized extracellular matrix(ECM) of bone with natural CZC through freezen-drying and genepin cross-linking. This scaffold had intact CZC structure, well pore structure, without obvious cytotoxicity. CZC had the function of physiological barrier; it was feasible to construct an independent culture microenvironment for chondral layer and subchondral layer with dual-chamber bioreactor by the barrier effect of CZC. Chondrogenic and osteogenic medium could induce stem cells in cartilage chamber and subchondral bone chamber to differentiate into chondrocytes and osteocytes, respectively. Total DNA content analysis showed that cells in scaffold increased in a time-dependentmanner. In the experiment of osteochondral defect repair, biomimetic osteochondral scaffolds with natural CZC had better repair effect for osteochondral defects when compared to blank control and osteochondral scaffolds without CZC. The structure and component of repair tissue was similar to native osteochondral tissue.