Title: Self-assembled artificial cartilage-hydroxyapatite conjugate for combined articular cartilage and subchondral bone repair
Abstract: Purpose: Recently, subchondral bone deterioration has been widely recognized as a hallmark of OA. OA subchondral bone is known to be hypomineralized and to show abnormal bone metabolism, resulting in histopathological degeneration of subchondral bone. A functional joint unit comprising articular cartilage and subchondral bone may regulate the homeostasis and maintenance potential of articular cartilage against the progression of OA. Thus, histopathological changes of subchondral bone may be involved in the pathogenesis and pathophysiology of cartilage degeneration. Consequently, for cartilage repair in OA, regeneration of subchondral bone as well as articular cartilage is necessary. We have recently created three types of cartilage-like biomaterial combined with HAP, for use in cartilage tissue engineering, which were developed through self-assembled cartilage component molecules and HAP at the nanometer scale by manipulating intermolecular relations. In the current study, we demonstrate that it is possible to create a novel cartilage-like biomaterial combined with a HAP layer for cartilage repair, with high performance as well as microstructures comparable to hyaline articular cartilage and subchondral bone in vitro and in an in vivo rat model. Methods: In the present study, we prepared three types of self-assembled artificial cartilage: [[Unsupported ANSI Character - ]] self-organized cartilage-like material, ‘ self-organized cartilage-like tissue combined with a HAP bone block, ƒ self-organized cartilage-like tissue coated with HAP powder. Self-organized cartilage-like material formed from HA, proteoglycan, and type II collagen. Solutions generated by mixing aggrecan (AG) and hyaluronic acid (HA) were prepared with distilled-deionized water (DDW) at room temperature. Type II collagen was dissolved in DDW at room temperature. The pH of the AG + HA solutions and the collagen solutions were adjusted to values from 8.0 in increments of 0.5. Then, equal volumes of the AG + HA and the collagen solutions were mixed at pH 7.5, and were incubated at 37°C in a humidified 95% air and 5% CO2 atmosphere for 2 hours. After an incubation periods followed by 30 min centrifugation at 15,000 rpm. Formation of the self-organized cartilage-like material combined with a HAP bone block. We have newly created the self-organized cartilage-like material combined with a HAP bone block. The self-organized AG/HA/collagen complex was formed by the method described above, and then the complex was placed onto a HAP block and centrifuged at 50,000 × g for 30 minutes. Specimens of the self-organized AG/HA/collagen complex combined with the HAP block were then prepared for each experiment. Formation of the self-organized cartilage-like material coated with HAP powder. HAP powders (particle size: 40 nm) were suspended in DDW (1.0 mg/mL). The self-organized AG/HA/collagen complex was formed by the method described above, then carefully put into a 1.0 mg/mL suspension of one of the HAP powders and incubated at 4°C overnight. Only the bottom surface of the complex was coated with the HAP solution. In vitro studies: The viscoelasticity of the self-assembled artificial cartilage described above was measured using an elasticity measuring device. SEM analysis was also performed to verify the surface structure of biomaterials. In vivo study: An articular cartilage biomaterial was implanted into the cartilage defect of knee joint surface (femoral chondyle) in male rats, and the tissues of the implanted site were examined 4 and 8 weeks after implantation. The tissues were examined macroscopically. The histological quality of the repair tissue was assessed and scored using the International Cartilage Repair Society (ICRS) II visual analogue scale. Results: The viscoelasticity of the HAP-coated biomaterials tended to increase in comparison with the original (HAP-uncoated) biomaterials. This suggests that conjugation of the HAP with the self-organized cartilage-like material may induce improvements of its viscoelasticity. SEM analysis reveals that both HAP powders with 40 nm and HAP block were regularly bound to the self-organized AG/HA/collagen complex in the form of laminae. Histologic analysis showed that the biomaterial alone, without conjugated HAP, was not enough to regenerate damaged subchondral bone tissue, which is functionally connected with articular cartilage. Although the self-organized cartilage-like AG/HA/collagen complex alone (without HAP) showed a tendency to induce articular cartilage regeneration in vivo, even at 8 weeks after implantation, no significant difference in the ICRS II score for cartilage repair was observed between the group implanted with the self-organized AG/HA/collagen complex and its control. In contrast, the self-organized AG/HA/collagen complexes with a HAP layer (40 nm powder size HAP od HAP block) were superior to the self-organized AG/HA/collagen complex alone without a HAP layer in terms of articular cartilage regeneration as well as subchondral bone regeneration. Conclusions: Numerous reports and reviews have already demonstrated that biomaterials for cartilage repair are very usable for the intervention against cartilage defects. However, there are few reports concerning whether or not biomaterials can induce regeneration following subchondral bone degeneration. Our in vitro and in vivo data reveal that self-organized cartilage-like biomaterials conjugated to HAP are superior to the same biomaterial with no HAP in both cartilage regeneration and subchondral bone regeneration. Our present study indicates that the self-organized cartilage-like biomaterial, which is conjugated with a HAP layer, may have the potential to repair not only articular cartilage defects but also the degenerated subchondral bone in OA.