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 tissue-engineered 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 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 2- to 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.
Discussion
This study clarified a gene expression profile of the cartilage-like tissue regenerated by using the PAMPS/PDMAAm DN gel in comparison with that of the normal mature cartilage. The present study showed that gene expression profiles of the tissues spontaneously regenerated at both 2 and 4 weeks by using the PAMPS/PDMAAm DN gel were similar to the normal cartilage. Type-2 collagen-related genes, MGP, SN6, and PH domain genes were highly expressed in both the tissues. Type-2 collagen-related genes are the most essential markers that upregulate upon hyaline cartilage differentiation, and MGP is a calcification inhibitor in the cartilage [
26]. SN6 and PH domain genes were highly expressed within all the tissues. Roles of these molecules in chondrogenesis have been unknown. However, we should note that mammalian constitutive photomorphogenesis 9 SN6 connects signaling with the ubiquitin-mediated proteasome degradation pathway and is implicated in cell cycle regulation and DNA damage response [
27], and that PH domains play a role in recruiting proteins to different membranes and enable proteins to interact with other components of the signal transduction pathways [
28]. Therefore, the present study suggested that the regenerated cells were genetically differentiated into the hyaline cartilage cells by 2 weeks. Histological and immunohistochemical examinations in the present study showed that type-2 collagen and proteoglycan molecules were highly expressed in the regenerated cells and matrix at 2 and 4 weeks, respectively. These results were supported by our previous study [
21]. Additionally, we took notice of the immune-related genes in Tables
1 and
3. The ratio of immune-related gene expression was identical among the normal cartilage and the regeneration tissues. These facts implied that the implantation of the PAMPS/PDMAAm DN gel plug did not induce any immune reactions in the rabbit, although it was a foreign body.
Secondly, the present study demonstrated that the cartilage marker gene profile of the tissues regenerated by the DN gel implantation was similar to that of the normal cartilage, concerning the types of highly expressed genes. Genetically, we can regard the regenerated tissue as the hyaline cartilage. However, the gene profile of the regenerated tissue has some differences concerning the quantity of each expression level. In the regenerated tissues, the expression degree of COL2A1, COL1A2, and COL10A1 genes in the genes encoding collagens, DCN and FMOD genes in the genes encoding proteoglycans, and SPARC, FLOD2, CHAD, CTGF, and COMPs genes in the genes encoding noncollagen/nonproteoglycan constituents of the extracellular matrix were obviously greater in comparison with the normal cartilage. COL10A1 is an important marker of prehypertrophic and hypertrophic chondrocytes. DCN is produced predominantly by mesenchymal stem cells, binds to type-2 collagen, and is involved in the control of fibrillogenesis [
29]. FMOD interacts with type-1 and type-2 collagen fibrils and inhibits fibrillogenesis
in vitro, and may participate in the assembly of the extracellular matrix
in vivo[
30]. SPARC modulates synthesis as well as turnover of the collagenous extracellular matrix. [
31]. PLOD2 is regulated with total collagen synthesis [
32]. CHAD binds to two sites on type-2 collagen. Both CHAD and collagen interact with chondrocytes, partly via the same receptor, but give rise to different cellular responses [
33]. CTGF is an important growth factor that coordinates chondrogenesis [
34]. COMPs are prominent in cartilage; however, it is also present in tendon and binds to type-1 and type-2 collagens with high affinity [
35,
36], although the functions is not sufficiently clarified. Therefore, the above-described results suggested that various genes concerning condrogenesis were strongly enhanced in the tissues spontaneously regenerated by implanting the DN gel. However, it is unclear whether such gene expression profile is specific characteristics of the cartilage tissue regenerated by the DN gel implantation or common characteristics of cartilage tissues regenerated with tissue-engineering techniques, because gene expression profiles of the tissue-engineered cartilage tissues have not been clarified as of yet.
Table
3 and Table
5 show a category of the genes that were different in the expression level between the normal cartilage and the tissues regenerated by the DN gel implantation. Specifically, we paid attention to the fact that protein metabolism-related genes of more than 60 (26%) and cell growth-related genes of more than 40 (20%) were highly expressed 5 times or more than the normal articular cartilage in the regenerated cartilage tissue. We carefully checked these up-regulated genes, and did not find any genes related to tumor or abnormal cell growth, such as alpha-fetoprotein, beta-2-microglobulin, beta-HCG, Bladder tumor antigen, CA15-3, CA19-9, CA27, CA29, CA72-4, CA125, calcitonin, carcinoembryonic antigen, chromogranin A, epidermal growth factor receptor, hormone receptors, HER2, human chorionic gonadotropin, immunoglobulins, neuron-specific enolase, NMP22, prostate-specific antigen, prostatic acid phosphatase, prostate-specific membrane antigen, S-100, TA-90, thyroglobulin, and etc. These results indicated that the genes up-regulated in the present study were not related to differentiation to tumor cells, but to the chondrocyte itself. In addition, the gene profile shown in the present study suggested that the regeneration process continued over the 4 week period. In the histological findings, we observed a little unrepaired lesion in the middle of the regenerated cartilage. The 4 week period after implantation is considered to be insufficient to complete a comprehensive osteochondral regeneration in adult rabbit knees with a full-thickness defect. This information will contribute to an increase of basic database to create an effective protocol for regeneration of cartilage in the near future.
In the present study, we found that TFCP2, CITED, and elongation factor-1 (EF-1) alpha were highly expressed in the cartilage tissue regenerated by the DN gel implantation in comparison to the normal cartilage. It is known that TFCP2 regulates genes involved in development of mesenchymal stem cells [
37] and CITED controls the transcription activity of Cart1 involved in skeletal development [
38]. Therefore, we speculate that TFCP2 and CITED may play an important role in the cartilage regeneration induced by the DN gel implantation. In addition, it has been reported that EF-1 alpha is related to protect a cell from endoplasmic reticulum stress [
39], which reduces both chondrocyte growth and matrix expression and induces chondrocyte apoptosis [
40]. Therefore, we speculate that EF-1 alpha also may contribute to the DN gel-induced cartilage regeneration by suppressing endoplasmic reticulum stress.
In the list of the top 30 genes that were expressed in the regenerated tissues 5 times or more as compared with the normal cartilage, we have taken notice of fibronectin and vimentin. It has been known that fibronectin plays a critical role in prechondrogenic condensation in the early stage of chondrogenesis [
41] and that vimentin contributes to the maintenance of the chondrocyte phenotype [
42]. Therefore, the highy expressed fibronectin and vimentin may contribute to the spontaneous cartilage regeneration by stimulating the condensation process of mesenchymal stem cells and maintaining cartilage phenotype, respectively. In addition, we also found that GAPDH, which plays a significant role in glycolysis, was highly expressed in the regenerated cartilage in comparison with the normal cartilage. It is known that the energy is supplied by glycolysis in a hypoxia environment rather than by mitochondrial respiration [
43]. Escoubet et al [
44] reported that hypoxia increases GAPDH transcription in rat alveolar epithelial cells. Therefore, the high expression of GAPDH observed in the present study implies that glycolysis is enhanced in the cartilage regeneration induced by the DN gel implantation in order to respond to a high energy demand in a low-oxygen environment.
Finally, the present study has demonstrated that the tissue regenerated in vivo by using the PAMPS/PDMAAm DN gel can be genetically regarded as the hyaline cartilage, although it has some minor differences from the normal cartilage. Therefore, we can conclude that spontaneous articular cartilage regeneration can be induced in vivo by using the PAMPS/PDMAAm DN gel. However, biomechanical and biochemical studies are needed to develop a novel therapeutic method to induce the spontaneous articular cartilage regeneration. The present study includes a few limitations. First, we did not perform statistical analyses. Secondly, we did not evaluated functions of the highly expressed genes. Further studies are needed to clarify genetic mechanisms of the spontaneously regenerated cartilage. However, the genetic data shown in this study are considered to be useful for future studies to identify specific genes involved in spontaneous cartilage regeneration and to make their mechanisms clear.
Conclusions
The present study has demonstrated that the tissue spontaneously regenerated in vivo by using the PAMPS/PDMAAm DN gel can be genetically regarded as the hyaline cartilage, although it has some minor differences from the normal cartilage. 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, EF-1 alpha, TFCP2, and GAPDH genes. The genetic data shown in this study are useful for future studies to identify specific genes involved in spontaneous cartilage regeneration and to make their mechanisms clear.
Competing interests
We have no financial or non- financial competing interests. We do not hold or are not currently applying for any patents relating to the content of the manuscript.
Authors' contributions
RI performed the animal experiment and the microarray analysis. YO instructed the microarray analysis. HJK instructed isolation of total RNA and synthesis of the fluorescence-labelled cDNA. SO contributed to the data analysis. NK instructed the animal experiment and performed the histological examinations. TK and JPG created the DN-gel material. KY designed the study and drafted the manuscript. All authors read and approved the final manuscript.