Superior stemness of a rapidly growing subgroup of isolated human auricular chondrocytes and the potential for use in cartilage regenerative therapy.

INTRODUCTION: In cartilage regenerative medicine, transplanted chondrocytes contain a mixture of populations, that complicates the regeneration of uniform cartilage tissue. Our group previously reported that chondrocytes with higher chondrogenic ability could be enriched by selection of rapidly growing cells. In this study, the detailed properties of rapidly growing chondrocytes were examined and compared to slowly growing cells. METHODS: Human auricular chondrocytes were fluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE) and analyzed using flow cytometry, focusing on division rates as indicated by fluorescence intensity and cell morphology according to the forward scatter and side scatter. Rapid and slow growing cell groups were harvested on days 2 and 4 after CFSE labeling, and their ability to produce cartilage matrix in vitro was examined. To compare the chondrogenic ability in vivo, the cells were seeded on poly-l-lactic acid scaffolds and transplanted into nude mice. Gene expression differences between the rapid and slow cell groups were investigated by microarray analysis. RESULTS: On day 2 after CFSE labeling, the rapidly growing cell group showed the highest proliferation rate. The results of pellet culture showed that the rapid cell group produced more glycosaminoglycans per cell than the slow cell group. The amount of glycosaminoglycan production was highest in the rapid cell group on day 2 after CFSE labeling, indicating high chondrogenic ability. Furthermore, microarray, gene ontology, and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed upregulation of genes that promote cell division such as origin recognition complex subunit 1 and downregulation of genes that inhibit cell division such as cyclin dependent kinase inhibitor 1A. Besides cell cycle-related genes, chondrocyte-related genes such as serpin family B member 2, clusterin, bone morphogenetic protein 2, and matrix metalloproteinase 3 were downregulated, while fibroblast growth factor 5 which is involved in stem cell maintenance, and coiled-coil and C2 domain containing 2A, which is required for cilia formation, were upregulated. CONCLUSION: The results showed that the rapid cell group proliferated well and had more undifferentiated properties, suggesting a higher stemness. The present findings provide a basis for the use of the rapid cell group in cartilage regeneration.

Requirement of direct contact between chondrocytes and macrophages for the maturation of regenerative cartilage.

Regenerative cartilage prepared from cultured chondrocytes is generally immature in vitro and matures after transplantation. Although many factors, including host cells and humoral factors, have been shown to affect cartilage maturation in vivo, the requirement of direct cell-cell contact between host and donor cells remains to be verified. In this study, we examined the host cells that promote cartilage maturation via cell-cell contact. Based on analysis of the transplanted chondrocytes, we examined the contribution of endothelial cells and macrophages. Using a semiclosed device that is permeable to tissue fluids while blocking host cells, we selectively transplanted chondrocytes and HUVECs or untreated/M1-polarized/M2-polarized RAW264.7 cells. As a result, untreated RAW264.7 cells induced cartilage regeneration. Furthermore, an in vitro coculture assay indicated communication between chondrocytes and RAW264.7 cells mediated by RNA, suggesting the involvement of extracellular vesicles in this process. These findings provide insights for establishing a method of in vitro cartilage regeneration.

M1-like macrophage contributes to chondrogenesis in vitro.

Cartilage tissues have poor self-repairing abilities. Regenerative medicine can be applied to recover cartilage tissue damage in the oral and maxillofacial regions. However, hitherto it has not been possible to predict the maturity of the tissue construction after transplantation or to prepare mature cartilage tissues before transplantation that can meet clinical needs. Macrophages play an important role in cartilage tissue regeneration, although the exact mechanisms remain unknown. In this study, we established and verified an in vitro experimental system for the direct co-culture of cell pellets prepared from mouse auricular chondrocytes and macrophages polarized into four phenotypes (M1-like, M1, M2-like, and M2). We demonstrate that cartilage pellets co-cultured with M1-like promoted collagen type 2 and aggrecan production and induced the most significant increase in chondrogenesis. Furthermore, M1-like shifted to M2 on day 7 of co-culture, suggesting that the cartilage pellet supplied factors that changed the polarization of M1-like. Our findings suggest that cartilage regenerative medicine will be most effective if the maturation of cartilage tissues is induced in vitro by co-culture with M1-like before transplantation.

Influence of Damage-Associated Molecular Patterns from Chondrocytes in Tissue-Engineered Cartilage.

To obtain stable outcomes in regenerative medicine, the quality of cells for transplantation is of great importance. Cellular stress potentially results in the release of damage-associated molecular patterns (DAMPs) and activates immunological responses, affecting the outcome of transplanted tissue. In this study, we intentionally prepared necrotic chondrocytes that would gradually die and release DAMPs and investigated how the maturation of tissue-engineered cartilage was affected. Necrotic chondrocytes were prepared by a conventional heat-treatment method, by which their viability started to decrease after 24 h. When tissue-engineered cartilage containing necrotic chondrocytes was subcutaneously transplanted into C57BL/6J mice, accumulation of cartilage matrix was decreased compared to the control. Meanwhile, immunohistochemical staining demonstrated that localization of macrophages and neutrophils was more apparent in the constructs of necrotic chondrocytes, suggesting that DAMPs from necrotic chondrocytes could prompt migration of more immune cells. Two-dimensional electrophoresis and mass spectrometry identified prelamin as a significant biomolecule released from necrotic chondrocytes. Also, when prelamin was added to a culture of RAW264, Inos and Il1b were increased in accordance with the content of added prelamin. It was suggested that DAMPs from dying chondrocytes could induce inflammatory properties in surrounding macrophages, impairing the maturation of tissue-engineered cartilage. In conclusion, maturation of tissue-engineered cartilage was hampered when less viable chondrocytes releasing DAMPs were included. Impact statement In regenerative medicine, the quality of cells is of great importance to secure clinical safety. During culture, damage of cells could occur, if not critical enough to cause immediate cell death, but still inducing a less viable status. Damage-associated molecular patterns (DAMPs) are released from necrotic cells, but their influence in regenerative medicine has yet to be clarified. In this study, we elucidated how DAMPs from chondrocytes could affect the maturation of tissue-engineered cartilage. Also, possible DAMPs from necrotic chondrocytes were comprehensively analyzed, and prelamin was identified as a significant molecule, which may serve for detecting the existence of necrotic chondrocytes.

Establishment of a new technique for the fabrication of regenerative cartilage with a microslicer device to prepare three dimensional diced cartilage.

Chondrocytes are utilized to cartilage regeneration by being harvested through enzymatic digestion and expanded by monolayer culture. However, these procedures will cause deterioration and dedifferentiation of the chondrocytes. In addition, scaffolds are often needed to provide the cartilage with mechanical strength and three-dimensional structures. We tried to use diced cartilage prepared using a micro-slicer without digestion, monolayer culture or scaffolds. In this study, an appropriate culture condition to induce the fusion of diced cartilage in vitro and cartilage regeneration in vitro and in vivo was determined to realize a scaffold-free cartilage regeneration. As a result, diced cartilages aggregated when they were cultured more than 5 weeks in the media containing 10% fetal bovine serum (FBS). Diced cartilage cultured for 7 weeks with the media containing 10%, followed by the culture with the media containing insulin-like growth factor-1 for 5 weeks in the ultralow attachment plate showed most prominent cartilage formation both in vitro and in vivo. The volume of regenerated cartilage was 2.14 times larger than that of the original cartilage. These results indicated that large regenerative cartilage from a small amount of cartilage was achieved without deterioration or dedifferentiation.

Glial Fibrillary Acidic Protein as Biomarker Indicates Purity and Property of Auricular Chondrocytes.

Instead of the silicone implants previously used for repair and reconstruction of the auricle and nose lost due to accidents and disease, a new treatment method using tissue-engineered cartilage has been attracting attention. The quality of cultured cells is important in this method because it affects treatment outcomes. However, a marker of chondrocytes, particularly auricular chondrocytes, has not yet been established. The objective of this study was to establish an optimal marker to evaluate the quality of cultured auricular chondrocytes as a cell source of regenerative cartilage tissue. Gene expression levels were comprehensively compared using the microarray method between human undifferentiated and dedifferentiated auricular chondrocytes to investigate a candidate quality control index with an expression level that is high in differentiated cells, but markedly decreases in dedifferentiated cells. We identified glial fibrillary acidic protein (GFAP) as a marker that decreased with serial passages in auricular chondrocytes. GFAP was not detected in articular chondrocytes, costal chondrocytes, or fibroblasts, which need to be distinguished from auricular chondrocytes in cell cultures. GFAP mRNA expression was observed in cultured auricular chondrocytes, and GFAP protein levels were also measured in the cell lysates and culture supernatants of these cells. However, GFAP levels detected from mRNA and protein in cell lysates were significantly decreased by increases in the incubation period. In contrast, the amount of protein in the cell supernatant was not affected by the incubation period. Furthermore, the protein level of GFAP in the supernatants of cultured cells correlated with the in vitro and in vivo production of the cartilage matrix by these cells. The productivity of the cartilage matrix in cultured auricular chondrocytes may be predicted by measuring GFAP protein levels in the culture supernatants of these cells. Thus, GFAP is regarded as a marker of the purity and properties of cultured auricular chondrocytes.

The usefulness of the decellularized matrix from three-dimensional regenerative cartilage as a scaffold material.

In cartilage tissue engineering, research on materials for three-dimensional (3D) scaffold has attracted attention. Decellularized matrix can be one of the candidates for the scaffold material. In this study, decellularization of regenerated cartilage was carried out and its effectiveness as a scaffold material was examined. Three-dimensionally-cultured cartilage constructs in the differentiation medium containing IGF-1 produced more cartilage matrix than those in the proliferation medium. Detergent-enzymatic method (DEM) could decellularize 3D-cultured cartilage constructs only by 1 cycle without breaking down the structure of the constructs. In vitro, newly-seeded chondrocytes were infiltrated and engrafted into decellularized constructs in the proliferation medium, and newly formed fibers were observed around the surface where newly-seeded cells were attached. Recellularized constructs could mature similarly as those without decellularization in vivo. The decellularized 3D-cultured matrix from regenerative cartilage is expected to be used as a scaffold material in the future.

Proliferation medium in three-dimensional culture of auricular chondrocytes promotes effective cartilage regeneration in vivo.

INTRODUCTION: Cartilage regeneration have been attracted attentions because of the poor ability of cartilage tissues to regenerate. Three-dimensional (3D) culture of chondrocytes is considered to be advantageous for cartilage regeneration. Although it is plausible that maturation of the constructs before transplantation positively affects the chondrogenesis, matured constructs after cultures for longer periods do not necessarily result in effective cartilage regeneration. In this study, we compared different types of culture media including growth factors which are clinically available. We prepared differentiation medium containing insulin-like growth factor-1 (IGF-1), proliferation medium containing fibroblast growth factor-2 (FGF-2) and insulin, and combination of them, and compared their efficacies on chondrogenesis when used in 3D culture of engineered cartilage constructs. METHODS: Cartilage constructs were fabricated by auricular chondrocytes and atelocollagen, and they were 3D-cultured with four types of media: control medium, differentiation medium, proliferation medium, and combination medium. After 3 weeks of culture, the constructs were analyzed for cell number, gene and protein expressions and mechanical properties. The constructs were also transplanted into nude mice. After 8 weeks, the degree of cartilage regeneration was evaluated. Constructs manufactured with canine auricular chondrocytes were subjected to autologous transplantation into beagles and examined for cartilage regeneration. RESULTS: During 3D culture, remarkably high gene expression of type II collagen was detected in the construct cultured with the differentiation medium whereas cell apoptosis were suppressed in the proliferation medium. When transplanted into nude mice, the constructs 3D-cultured in the proliferation medium produced abundant cartilage matrices. In autologous implantation model, the construct cultured in the proliferation medium again showed better chondrogenesis than those in other media. CONCLUSIONS: The present study indicates that 3D culture with the proliferation medium maintains the cell viability to potentiate the subsequent cartilage regeneration. Here, we propose that not only differentiation but also high cell viability accompanied by proliferation factors should be taken into account to improve cartilage regeneration.

Biological aspects of tissue-engineered cartilage.

Cartilage regenerative medicine has been progressed well, and it reaches the stage of clinical application. Among various techniques, tissue engineering, which incorporates elements of materials science, is investigated earnestly, driven by high clinical needs. The cartilage tissue engineering using a poly lactide scaffold has been exploratorily used in the treatment of cleft lip-nose patients, disclosing good clinical results during 3-year observation. However, to increase the reliability of this treatment, not only accumulation of clinical evidence on safety and usefulness of the tissue-engineered products, but also establishment of scientific background on biological mechanisms, are regarded essential. In this paper, we reviewed recent trends of cartilage tissue engineering in clinical practice, summarized experimental findings on cellular and matrix changes during the cartilage regeneration, and discussed the importance of further studies on biological aspects of tissue-engineered cartilage, especially by the histological and the morphological methods.

Optimal conditions of collagenase treatment for isolation of articular chondrocytes from aged human tissues.

INTRODUCTION: There are various types of cartilage, including the auricular and articular cartilages. These cartilages have different functions, and their matrix volume and density of chondrocytes may differ. Thus, different protocols may be required to digest different types of cartilage. METHODS: In this study, we examined protocols for the digestion of articular and auricular cartilages and determined the optimal conditions for articular cartilage digestion. RESULTS: Our histological findings showed that the articular cartilage has a larger matrix area and fewer cells than the auricular cartilage. In 1-mm2 areas of articular and auricular cartilages, the average numbers of cells were 44 and 380, respectively, and the average matrix areas were 0.94 and 0.77 mm2, respectively. The maximum numbers of viable cells (approximately 1 × 105 cells/0.1 g of tissue) were obtained after digestion in 0.15, 0.3, or 0.6% collagenase for 24 h, in 1.2% collagenase for 6 h, or in 2.4% collagenase for 4 h. In tissues incubated in 0.15 or 0.3% collagenase, the cell numbers were lower than 1 × 105, even at 24 h, possibly reflecting incomplete digestion of cartilage. No significant differences were observed in the results of apoptosis assays for all collagenase exposure times and concentrations. However, cell damage appeared to be greater when collagenase concentrations were high. When cells obtained after digestion with different concentrations of collagenase were seeded at a density of 3000 cells/cm2, they yielded the maximum cell numbers after 1 week. CONCLUSIONS: We recommend a 24-h incubation in 0.6% collagenase as the optimal condition for chondrocyte isolation from articular cartilage. Moreover, we found that the optimum cell-seeding density is approximately 3000 cells/cm2. Conditions determined in this study would maximize the yield of isolated articular chondrocytes and enable the generation of a large quantity of cultured cells.

Three-dimensional changes of noses after transplantation of implant-type tissue-engineered cartilage for secondary correction of cleft lip-nose patients.

INTRODUCTION: We have developed an implant-type tissue-engineered cartilage using a poly-l-lactide scaffold. In a clinical study, it was inserted into subcutaneous areas of nasal dorsum in three patients, to correct cleft lip-nose deformity. The aim of this study was to helping evaluation on the efficacy of the regenerative cartilage. METHODS: 3D data of nasal shapes were compared between before and after surgery in computed tomography (CT) images. Morphological and qualitative changes of transplants in the body were also evaluated on MRI, for one year. RESULTS: The 3D data from CT images showed effective augmentation (>2 mm) of nasal dorsum in almost whole length, observed on the medial line of faces. It was maintained by 1 year post-surgery in all patients, while affected curves of nasal dorsum was not detected throughout the observation period. In magnetic resonance imaging (MRI), the images of transplanted cartilage had been observed until 1 year post-surgery. Those images were seemingly not straight when viewed from the longitudinal plain, and may have shown gentle adaptation to the surrounding nasal bones and alar cartilage tissues. CONCLUSION: Those findings suggested the potential efficacy of this cartilage on improvement of cleft lip-nose deformity. A clinical trial is now being performed for industrialization.

Tissue responses against tissue-engineered cartilage consisting of chondrocytes encapsulated within non-absorbable hydrogel.

To disclose the influence of foreign body responses raised against a non-absorbable hydrogel consisting of tissue-engineered cartilage, we embedded human/canine chondrocytes within agarose and transplanted them into subcutaneous pockets in nude mice and donor beagles. One month after transplantation, cartilage formation was observed in the experiments using human chondrocytes in nude mice. No significant invasion of blood cells was noted in the areas where the cartilage was newly formed. Around the tissue-engineered cartilage, agarose fragments, a dense fibrous connective tissue and many macrophages were observed. On the other hand, no cartilage tissue was detected in the autologous transplantation of canine chondrocytes. Few surviving chondrocytes were observed in the agarose and no accumulation of blood cells was observed in the inner parts of the transplants. Localizations of IgG and complements were noted in areas of agarose, and also in the devitalized cells embedded within the agarose. Even if we had inhibited the proximity of the blood cells to the transplanted cells, the survival of the cells could not be secured. We suggest that these cytotoxic mechanisms seem to be associated not only with macrophages but also with soluble factors, including antibodies and complements.

Bone and cartilage repair by transplantation of induced pluripotent stem cells in murine joint defect model.

The establishment of cartilage regenerative medicine has been an important issue in the clinical field, because cartilage has the poor ability of self-repair. Currently, tissue engineering using autologous chondrocytes has risen, but we should investigate more appropriate cell sources that can be obtained without any quantitative limitation. In this study, we focused on induced pluripotent stem (iPS) cells, in which the ethical hurdle does not seem higher than that of embryonic stem cells. Mouse iPS cells were transplanted into the mouse joint defect model of the knee. Strains of the transplants and hosts were arranged to be either closest (homology 75% in genetic background) or identical (100%). For transplantation, we embedded the iPS cells within the collagen hydrogel in order to obtain the effective administration of the cells into defects, which induced the differentiation of the iPS cells. At 8 weeks of transplantation, although the iPS cells with a 75% homology to the host in the genetic background tended to form teratoma, those of 100% showed a joint regeneration. GFP immunohistochemistry proved that the transplanted iPS cells were responsible for the bone and cartilage repair. Taking these results together, the iPS cells are regarded as a promising cell source for the cartilage tissue engineering.

Application of floating cells for improved harvest in human chondrocyte culture.

Cell culture medium, which must be discarded during medium change, may contain many cells that do not attach to culture plates. In the present study, we focused on these floating cells and attempted to determine their usefulness for cartilage regeneration. We counted the number of floating cells discarded during medium change and compared the proliferation and differentiation between floating cells and their adherent counterparts. Chondrocyte monolayer culture at a density of 5 × 103 cells/cm(2) produced viable floating cells at a rate of 2.7-3.2 × 10(3) cells/cm(2) per primary culture. When only the floating cells from one dish were harvested and replated in another dish, the number of cells was 2.8 × 10(4) cells/cm(2) (approximately half confluency) on culture day 7. The number of cells was half of that obtained by culturing only adherent cells (5 × 10(4) cells/cm(2)). The floating and adherent cells showed similar proliferation and differentiation properties. The recovery of floating cells from the culture medium could provide an approximately 1.5-fold increase in cell number over conventional monolayer culture. Thus, the collection of floating cells may be regarded as a simple, easy, and reliable method to increase the cell harvest for chondrocytes.

Early stage foreign body reaction against biodegradable polymer scaffolds affects tissue regeneration during the autologous transplantation of tissue-engineered cartilage in the canine model.

To overcome the weak points of the present cartilage regenerative medicine, we applied a porous scaffold for the production of tissue-engineered cartilage with a greater firmness and a 3D structure. We combined the porous scaffolds with atelocollagen to retain the cells within the porous body. We conducted canine autologous chondrocyte transplants using biodegradable poly-L-lactic acid (PLLA) or poly-DL-lactic-co-glycolic acid (PLGA) polymer scaffolds, and morphologically and biochemically evaluated the time course changes of the transplants. The histological findings showed that the tissue-engineered constructs using PLLA contained abundant cartilage 1, 2, and 6 months after transplantation. However, the PLGA constructs did not possess cartilage and could not maintain their shapes. Biochemical measurement of the proteoglycan and type II collagen also supported the superiority of PLLA. The biodegradation of PLGA progressed much faster than that of PLLA, and the PLGA had almost disappeared by 2 months. The degraded products of PLGA may evoke a more severe tissue reaction at this early stage of transplantation than PLLA. The PLLA scaffolds were suitable for cartilage tissue engineering under immunocompetent conditions, because of the retarded degradation properties and the decrease in the severe tissue reactions during the early stage of transplantation.

Matrix remodeling and cytological changes during spontaneous cartilage repair.

During the repair of articular cartilage, type I collagen (COL1)-based fibrous tissues change into a mixture of COL1 and type II collagen (COL2) and finally form hyaline cartilaginous tissues consisting of COL2. In order to elucidate the changes that occur in the matrix during cartilage repair and the roles of fibroblasts and chondrocytes in this process, we generated a minimal cartilage defect model that could be spontaneously repaired. Defects of 0.3 mm were created on the patellofemoral articular cartilage of rats using an Er:YAG laser and were observed histologically, ultrastructurally and histochemically. At week 2 after this operation, fibroblastic cells were found to be surrounded by COL1 throughout the area of the defect. These cells became acid phosphatase positive by week 4, both taking in and degrading collagen fibrils. Thereafter, the cells became rounded, with both COL1 and 2 evident in the matrix, and showed immunolocalized matrix metalloproteinase-1 or -9. In the region of the bone marrow, the cells became hypertrophic and were surrounded mainly by COL2 and proteoglycans. By the eighth week, the cartilaginous matrix was found to contain abundant COL2, in which collagen fibrils of various diameters were arranged irregularly. These morphological changes suggested that the fibroblastic cells both produce and resolve the matrix and undertake remodeling to become chondrocytes by converting from a COL1- into a COL2-dominant matrix. This process eventually forms new articular cartilage, but this is not completely identical to normal articular cartilage at the ultrastructural level.

The development of a serum-free medium utilizing the interaction between growth factors and biomaterials.

To promote clinical application of cartilage tissue engineering, we should establish a serum-free chondrocyte growth medium. The serum-free medium would increase the cell numbers by more than 20-fold within one week, which proliferation ability almost matches that of serum-based one. For that, we examined the combinations of growth factors and the methods to enhance their effects by making use of the interaction with biomaterials. From various growth factors that are contained within the serum, we made the cocktail of FGF-2 (100 ng/mL), insulin (5 µg/mL), EGF (10 pg/mL), PDGF (625 pg/mL) and TGF-ß (5 pg/mL), which increased the chondrocyte numbers by approximately 3-fold for 7 days. Moreover, we used the biomaterials including albumin and hyaluronan as the carrier of those factors. By direct mixing of those factors with biomaterials before the administration to the medium, the medium containing those mixture showed the chondrocyte growth of approximately a 25-fold increase by day 10. In this medium, the FGF-2 or insulin concentration hardly decreased, and rather enhanced the activation of ERK. Due to the optimal usage of biomaterials, this serum-free medium will realize a constant harvest of chondrocytes and could contribute to the safety and quality in regenerative medicine.

Evaluation of the implant type tissue-engineered cartilage by scanning acoustic microscopy.

The tissue-engineered cartilages after implantation were nonuniform tissues which were mingling with biodegradable polymers, regeneration cartilage and others. It is a hard task to evaluate the biodegradation of polymers or the maturation of regenerated tissues in the transplants by the conventional examination. Otherwise, scanning acoustic microscopy (SAM) system specially developed to measure the tissue acoustic properties at a microscopic level. In this study, we examined acoustic properties of the tissue-engineered cartilage using SAM, and discuss the usefulness of this devise in the field of tissue engineering. We administered chondrocytes/atelocollagen mixture into the scaffolds of various polymers, and transplanted the constructs in the subcutaneous areas of nude mice for 2 months. We harvested them and examined the sound speed and the attenuation in the section of each construct by the SAM. As the results, images mapping the sound speed exhibited homogenous patterns mainly colored in blue, in all the tissue-engineered cartilage constructs. Contrarily, the images of the attenuation by SAM showed the variation of color ranged between blue and red. The low attenuation area colored in red, which meant hard materials, were corresponding to the polymer remnant in the toluidine blue images. The localizations of blue were almost similar with the metachromatic areas in the histology. In conclusion, the SAM is regarded as a useful tool to provide the information on acoustic properties and their localizations in the transplants that consist of heterogeneous tissues with various components.