Instructions for use Title Gene expression profile of the cartilage tissue spontaneously regenerated in vivo by using a noveldouble-network gel : Comparisons with the normal articular cartilage

Background: We have recently found a phenomenon that spontaneous regeneration of a hyaline cartilage-like tissue can be induced in a large osteochondral defect by implanting a double-network (DN) hydrogel plug, which was composed of poly-(2-Acrylamido-2-methylpropanesulfonic acid) and poly-(N, N’-Dimetyl acrylamide), at the bottom of the defect. The purpose of this study was to clarify gene expression profile of the regenerated tissue in comparison with that of the normal articular cartilage. Methods: We created a cylindrical osteochondral defect in the rabbit femoral grooves. Then, we implanted the DN gel plug at the bottom of the defect. At 2 and 4 weeks after surgery, the regenerated tissue was analyzed using DNA microarray and immunohistochemical examinations. Results: The gene expression profiles of the regenerated tissues were macroscopically similar to the normal cartilage, but showed some minor differences. The expression degree of COL2A1, COL1A2, COL10A1, DCN, FMOD, SPARC, FLOD2, CHAD, CTGF, and COMP genes was greater in the regenerated tissue than in the normal cartilage. The top 30 genes that expressed 5 times or more in the regenerated tissue as compared with the normal cartilage included type-2 collagen, type-10 collagen, FN, vimentin, COMP, EF1alpha, TFCP2, and GAPDH genes. Conclusions: The tissue regenerated by using the DN gel was genetically similar but not completely identical to articular cartilage. The genetic data shown in this study are useful for future studies to identify specific genes involved in spontaneous cartilage regeneration. Background Articular (hyaline) cartilage is a highly organized soft tissue [1]. Articular cartilage is frequently damaged due to trauma, and treatment of damaged cartilage is a significant health care concern. It has been a common belief up to now that hyaline cartilage tissue cannot spontaneously regenerate in vivo [2,3]. Therefore, the most prevalent strategy to repair the articular cartilage defect is to fill an osteochondral defect with a tissueengineered cartilage-like tissue or a cell-seeded scaffold material [2,4-6]. However, recent studies have pointed out various practical problems in this strategy, including zoonosis transmission, the need for two-staged surgery, a long period until weight bearing after implantation, an enormous amount of money to establish a therapeutic system [7-11]. Thus, functional repair of articular cartilage defects remains a major challenge in the field of joint surgery and tissue regeneration medicine. We have paid special attention to the clinical fact that the fibrocartilage tissue can be regenerated in an osteochondral defect by creating many small holes penetrating into the subchondral bone at the bottom of the defect space in order to enhance bleeding from the bone marrow [12]. Namely, the clot formed from bone * Correspondence: yasukaz@med.hokudai. ac.jp † Contributed equally Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan Full list of author information is available at the end of the article Imabuchi et al. BMC Musculoskeletal Disorders 2011, 12:213 http://www.biomedcentral. com/1471-2474/12/213 © 2011 Imabuchi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. marrow blood contains mesenchymal stem cells, which can differentiate into cartilage tissues. In addition, recent studies have shown that, in autologous chondrocyte transplantation, quality of the tissue located just beneath the transplanted cells significantly affects quality of the regenerated cartilage [13-15]. In an ex vivo study, Engler et al [16] reported that elasticity of the microenvironment in a culture system directs stem-cell differentiation. Therefore, we have considered that, if we implant any bioactive elastic hydrogel at the bottom of an osteochondral defect under conditions similar to in the above-described multiple-penetration surgery, we may be able to induce hyaline cartilage regeneration in vivo in the defect space. We have focused on an originally developed PAMPS/PDMAAm double-network (DN) hydrogel [17,18], which was composed of poly-(2-Acrylamido-2-methylpropanesulfonic acid) (PAMPS) and poly-(N, N’-Dimetyl acrylamide) (PDMAAm). In DN gel, the two independently cross-linked polymer networks are physically entangled with each other. The PAMPS network in this DN gel is negatively charged and has a sulphonic acid base, being similar to proteoglycans in normal cartilage. This bioactive DN gel has the elastic modulus of 0.20 MPa [19,20]. In addition, the PAMPS/PDMAAm DN gel surface can enhance differentiation of chondrogenic ATDC5 cells into chondrocytes in the in vitro condition [21,22]. Thus, we have recently found a noteworthy phenomenon that, when we implant the PAMPS/PDMAAm DN hydrogel plug at the bottom of an osteochondral defect in the rabbit so that a 2to 3-mm deep vacant space is intentionally left in the defect, a hyaline cartilage-like tissue rich in type-2 collagen and proteoglycan is spontaneously regenerated in vivo in the defect within 4 weeks [21]. Because this phenomenon has a potential that may lead to development of a novel therapeutic method to spontaneously regenerate a hyaline cartilage-like tissue, we should perform multidisciplinary evaluations of the quantity and quality of the regenerated tissue to increase a scientific database of this phenomenon. We have performed histological and immunohistological evaluations [23,24]. However, no biomechanical, biochemical, and genetic studies to evaluate the regenerated tissue have not been reported as of yet. Thus, the present study using DNA microarray has been conducted to genetically clarify what gene expression is induced in the regenerated tissue by the DN gel implantation, and to ask whether the regenerated tissue is genetically identical to the normal articular cartilage. The purpose of the present in vivo study using the microarray analysis is to clarify a gene expression profile of the cartilage-like tissue spontaneously regenerated by using the PAMPS/PDMAAm DN gel in comparison with the normal articular cartilage. Methods 1) Hydrogel preparation The PAMPS/PDMAAm DN gel was synthesized using the previously reported two-step sequential polymerization method [17]. After polymerization, the PAMPS-PDMAAm DN gel was immersed in pure water for 1 week and the water was changed 2 times every day to remove any unreacted materials. From the PAMPS/PDMAAm DN gel, we created cylindrical plugs having a 4.5-mm diameter and an 8-mm length. 2) Study design A total of 17 mature female Japanese white rabbits weighing 3.6 ± 0.5 kg were used in this study. Animal experiments were carried out in the Institute of Animal Experimentation, Hokkaido University School of Medicine under the Rules and Regulation of the Animal Care and Use Committee, Hokkaido University School of Medicine. Fourteen of the 17 rabbits underwent the previously reported operation [21] in the bilateral knees under intravenous anesthesia (pentobarbital, 25 mg/kg) and sterile conditions. Briefly, we created a cylindrical osteochondral defect having a diameter of 4.3-mm and a depth of 10.0 mm in the femoral groove of the patellofemoral joint, using a drill. Then, we implanted the DN gel plug into the defect so that a defect having approximately 2-mm depth from the articular surface remained in place after surgery (Figure 1). The depth of 2 mm was chosen because this depth was the most effective to induce the spontaneous cartilage regeneration in our previous preliminary study [21]. The incised joint capsule and the skin wound were closed in layers with 3-0 nylon sutures, and an antiseptic spray dressing was applied. Postoperatively, each animal was allowed unrestricted activity in a cage (310 × 550 × 320 mm) without any joint immobilization. Seven animals were sacrificed at 2 and 4 weeks after surgery, respectively. Figure 1 How to induce cartilage regeneration. A cylindrical osteochondral defect having a diameter of 4.3-mm was created in the femoral groove. Then, a DN gel plug was implanted into the defect so that a defect having approximately 2-mm depth from the articular surface remained in place after surgery. Imabuchi et al. BMC Musculoskeletal Disorders 2011, 12:213 http://www.biomedcentral. com/1471-2474/12/213 Page 2 of 11