In situ hybridisation study of type I, II, X collagens and aggrecan mRNAs in the developing condylar cartilage of fetal mouse mandible

The aim of this study was to investigate the developmental characteristics of the mandibular condyle in sequential phases at the gene level using in situ hybridisation. At d 14.5 of gestation, although no expression of type II collagen mRNA was observed, aggrecan mRNA was detected with type I collagen mRNA in the posterior region of the mesenchymal cell aggregation continuous with the ossifying mandibular bone anlage prior to chondrogenesis. At d 15.0 of gestation, the first cartilaginous tissue appeared at the posterior edge of the ossifying mandibular bone anlage. The primarily formed chondrocytes in the cartilage matrix had already shown the appearance of hypertrophy and expressed types I, II and X collagens and aggrecan mRNAs simultaneously. At d 16.0 of gestation, the condylar cartilage increased in size due to accumulation of hypertrophic chondrocytes characterised by the expression of type X collagen mRNA, whereas the expression of type I collagen mRNA had been reduced in the hypertrophic chondrocytes and was confined to the periosteal osteogenic cells surrounding the cartilaginous tissue. At d 18.0 of gestation before birth, cartilage-characteristic gene expression had been reduced in the chondrocytes of the lower half of the hypertrophic cell layer. The present findings demonstrate that the initial chondrogenesis for the mandibular condyle starts continuous with the posterior edge of the mandibular periosteum and that chondroprogenitor cells for the condylar cartilage rapidly differentiate into hypertrophic chondrocytes. Further, it is indicated that sequential rapid changes and reductions of each mRNA might be closely related to the construction of the temporal mandibular ramus in the fetal stage.     

Osteopontin distribution in the canine skeleton during growth and structural maturation

The distribution of osteopontin (OPN) was studied immunohistochemically in cells and extracellular matrix in the humerus, scapula, and lumbar vertebrae of growing (age: 6 weeks, 12 weeks, 4.5 months) and adult dogs. OPN was expressed in hypertrophic chondrocytes of epiphyseal cartilage and in chondrocytes of the deep zone of mature articular cartilage, where extracellular matrix was also stained. OPN expression was strongest in 4.5-month-old puppies in cells of the osteoblastic lineage. It also varied with microlocation and was pronounced in areas prone to resorption due to modelling and remodelling activities. Osteoclasts were always strongly labelled with OPN. OPN deposition in extracellular bone matrix was detected particularly as a delineation of cartilage cores within secondary trabeculae and as a lining of the trabecular surfaces in resorption microlocations. The OPN distribution pattern is discussed here for each cell population with regard to its functional implications.     

Human chondrocytes differentially express matrix modulators during in vitro expansion for tissue engineering

Cartilage tissue engineering plays an important role in the generation of grafts for reconstructive surgery. In cultured chondrocytes, the dedifferentiation of cells seems unavoidable for multiplication. Dedifferentiated cells produce matrix of less quality, and the molecular basis is still not well understood. Therefore, the aim of our study was to investigate the expression of matrix modulators in human chondrocytes during expansion. Human chondrocytes were isolated from septal cartilage (n=32) and held in primary cell culture. Cells were harvested after 1, 6 and 21 days. The differentiation of cells using light microscopy, the expression patterns of various proteins (MMPs, BMPs, and TIMPs) using immunohistochemistry, and the expression of distinct genes using microarray technique, were investigated. The chondrocytes showed strong in vitro proliferation. After 6 and 21 days, BMP-5 and -8 were up-regulated, BMP-2 was down-regulated and BMP-6 was inactivated. Other BMPs were not expressed. The expression of MMP-2, -3 and -13 was up-regulated from day 1 to 21, and MMP-12 and -20 were down-regulated. Other MMPs were not expressed. TIMP-1 was up-regulated and TIMP-3 was down-regulated during expansion. Differential expression of matrix modulators might influence the matrix composition of engineered cartilage. Improving the basic knowledge in this area may ultimately help clinicians to identify and proactively intervene in an attempt to prevent bioartificial cartilage from losing stability.     

Cartilage-specific matrix protein,chondromodulin-I (ChM-I), is a strong angio-inhibitor in endochondral ossification of human neonatal vertebral tissues in vivo: relationship with angiogenic factors in the cartilage

Although cartilage contains many angiogenic factors during endochondral ossification, it is an avascular tissue. The cartilage-specific non-collagenous matrix protein chondromodulin-I (ChM-I) has been shown to be a strong angio-inhibitor. To elucidate whether ChM-I plays an essential role in angio-inhibition during endochondral ossification in man, we investigated the expression and localization of ChM-I in comparison with those of angiogenic factors and the endothelial cell marker CD34 in human neonatal vertebral tissues. Although invasion of CD34-positive endothelial cells was observed in primary subchondral spongiosa, expression of the marker of endothelial cells, CD34, was not found in neonatal vertebral cartilage matrix. Type II collagen was deposited in all matrices during endochondral ossification, whereas aggrecan was deposited in the matrix of hypertrophic cartilage, especially around lacunae. Vascular endothelial growth factor (VEGF), which is known to be a strong angiogenic factor, was localized in chondrocytes in mature to hypertrophic cartilage and also in bone marrow. Fibroblast growth factor-2 (FGF-2; basic fibroblast growth factor), which is also known to be a strong angiogenic factor, was localized in the cytoplasm of chondrocytes of mature cartilage in human vertebral cartilage tissues. Transforming growth factor (TGF)-beta has been reported to have many functions including angiogenesis, and TGF-beta1 was also localized in mature chondrocytes in endochondral tissues undergoing ossification. On the other hand, the novel cartilage-specific matrix protein ChM-I was localized in interterritorial regions of the matrix in mature to hypertrophic cartilage, especially around lacunae. In conclusion, these observations indicate that ChM-I may serve as a barrier against the angiogenic properties of VEGF, FGF-2 and TGF-beta1 during endochondral ossification, and this matrix molecule may play an essential role in determining the avascular nature of cartilage in vivo.     

In situ hybridization and immunohistochemistry of bone sialoprotein and secreted phosphoprotein 1 (osteopontin) in the developing mouse mandibular condylar cartilage compared with limb bud cartilage

Mandibular condylar cartilage is often classified as a secondary cartilage, differing from the primary cartilaginous skeleton in its rapid progress from progenitor cells to hypertrophic chondrocytes. In this study we used in situ hybridization and immunohistochemistry to investigate whether the formation of primary (tibial) and secondary (condylar) cartilage also differs with respect to the expression of two major non‐collagenous glycoproteins of bone matrix, bone sialoprotein (BSP) and secreted phosphoprotein 1 (Spp1, osteopontin). The mRNAs for both molecules were never expressed until hypertrophic chondrocytes appeared. In the tibial cartilage, hypertrophic chondrocytes first appeared at E14 and the expression of BSP and Spp1 mRNAs was detected in the lower hypertrophic cell zone, but the expression of BSP mRNA was very weak. In the condylar cartilage, hypertrophic chondrocytes appeared at E15 as soon as cartilage tissue appeared. The mRNAs for both molecules were expressed in the newly formed condylar cartilage, although the proteins were not detected by immunostaining; BSP mRNA in the condylar cartilage was more extensively expressed than that in the tibial cartilage at the corresponding stage (first appearance of hypertrophic cell zone). Endochondral bone formation started at E15 in the tibial cartilage and at E16 in the condylar cartilage. At this stage (first appearance of endochondral bone formation), BSP mRNA was also more extensively expressed in the condylar cartilage than in the tibial cartilage. The hypertrophic cell zone in the condylar cartilage rapidly extended during E15–16. These results indicate that the formation process of the mandibular condylar cartilage differs from that of limb bud cartilage with respect to the extensive expression of BSP mRNA and the rapid extension of the hypertrophic cell zone at early stages of cartilage formation. Furthermore, these results support the hypothesis that, in vivo, BSP promotes the initiation of mineralization.     

Chondromodulin-I expression in rat articular cartilage

The localization and expression of chondromodulin-I (ChM-I), an angiogenesis inhibitor, in the rat articular cartilage during maturation from 2 to 10 weeks of age were examined by immunohistochemistry, Western blot analysis and ribonuclease protection assay, and the results were compared with those in the epiphyseal cartilage. ChM-I was found to be diffusely immunostained in the inter-territorial space of the cartilage matrix from the intermediate to the deep layers at the immature stage. As the articular cartilage matured, the immunoreactivity was localized around the hypertrophic chondrocytes in the deep layer and the immunoreactivity became weak after maturation. In contrast, the ChM-I immunoreactivity was intense in the epiphyseal cartilage at all ages examined. ChM-I was detected by Western blotting as a broad band or occasionally as a cluster of multiple bands (approximately 25 kDa) in both the articular and the epiphyseal cartilage. The intensity of the bands decreased gradually with age in the articular cartilage, but was unchanged in the epiphyseal cartilage at all ages. Ribonuclease protection assay revealed that ChM-I mRNA also decreased gradually with age in the articular cartilage in parallel with the maturation of the articular cartilage, while no decrease in ChM-I mRNA was found in the epiphyseal cartilage. The expression of ChM-I mRNA in the articular cartilage was less than that in the epiphyseal cartilage at all ages. The decrease in amount of ChM-I in the mature articular cartilage suggests that ChM-I plays a more important role in the maintenance of avascularity in the immature articular cartilage than in the mature one. The avascular condition may be preserved by angiogenic inhibitors or mechanisms other than ChM-I in the mature articular cartilage.     

无纺网与多孔海绵状材料作为软骨组织工程支架的适用性及体内降解性

背景：聚羟基乙酸无纺网与聚羟基丁酸酯-聚羟基己酸酯共聚物多孔海绵具有良好的塑形适应性、生物降解性与生物相容性。 目的：观察聚羟基乙酸无纺网与聚羟基丁酸酯-聚羟基己酸酯共聚物多孔海绵作为软骨组织工程支架的适用性及体内降解性。 方法：分别制备乳兔软骨细胞-聚羟基乙酸无纺网复合物、乳兔软骨细胞-聚羟基丁酸酯-聚羟基己酸酯共聚物多孔海绵复合物。在实验组成年兔两侧背部皮下分别植入制备的两种复合物,在对照组成年兔两侧背部皮下分别植入聚羟基乙酸无纺网与聚羟基丁酸酯-聚羟基己酸酯共聚物。 结果与结论：组织学观察显示,以聚羟基乙酸无纺网获取的组织工程软骨,植入4 周时软骨细胞较小,软骨内有较多聚羟基乙酸纤维残留,8周时软骨细胞较成熟,包埋在陷窝内,聚羟基乙酸纤维消失,12周时软骨细胞成熟,基质分泌丰富,无聚羟基乙酸存留;以聚羟基丁酸酯-聚羟基己酸酯共聚物多孔海绵获取的组织工程软骨,植入4周时软骨细胞不成熟,软骨基质内似“杂质”样材料残留物较多,8周时软骨细胞较成熟,软骨基质内仍可见材料残留,12周时软骨基质材料残留基本消失。两组组织工程软骨特殊染色与免疫组织化学检测均显示再生软骨胶原与基质黏多糖生成良好,软骨中均检测出Ⅱ型胶原。表明两种材料作为软骨组织工程支架具有良好的适用性,其降解时间均达到组织工程软骨构建的要求。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Cartilage engineering using chondrocyte cell sheets and its application in reconstruction of microtia

The imperfections of scaffold materials have hindered the clinical application of cartilage tissue engineering. The recently developed cell-sheet technique is adopted to engineer tissues without scaffold materials, thus is considered being potentially able to overcome the problems concerning the scaffold imperfections. This study constructed monolayer and bilayer chondrocyte cell sheets and harvested the sheets with cell scraper instead of temperature-responsive culture dishes. The properties of the cultured chondrocyte cell sheets and the feasibility of cartilage engineering using the chondrocyte cell sheets was further investigated via in vitro and in vivo study. Primary extracellular matrix (ECM) formation and type II collagen expression was detected in the cell sheets during in vitro culture. After implanted into nude mice for 8 weeks, mature cartilage discs were harvested. The morphology of newly formed cartilage was similar in the constructs originated from monolayer and bilayer chondrocyte cell sheet. The chondrocytes were located within evenly distributed ovoid lacunae. Robust ECM formation and intense expression of type II collagen was observed surrounding the evenly distributed chondrocytes in the neocartilages. Biochemical analysis showed that the DNA contents of the neocartilages were higher than native human costal cartilage; while the contents of the main component of ECM, glycosaminoglycan and hydroxyproline, were similar to native human costal cartilage. In conclusion, the chondrocyte cell sheet constructed using the simple and low-cost technique is basically the same with the cell sheet cultured and harvested in temperature-responsive culture dishes, and can be used for cartilage tissue engineering.     

Haem oxygenase-1 regulates catabolic and anabolic processes in osteoarthritic chondrocytes

Pro-inflammatory cytokines, matrix metalloproteinases (MMPs) and other catabolic factors participate in the pathogenesis of cartilage damage in osteoarthritis (OA). Pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) mediate cartilage degradation and might be involved in the progression of OA. Previously, we found that haem oxygenase-1 (HO-1) is down-regulated by pro-inflammatory cytokines and up-regulated by IL-10 in OA chondrocytes. The aim of this study was to determine whether HO-1 can modify the catabolic effects of IL-1beta in OA cartilage and chondrocytes. Up-regulation of HO-1 by cobalt protoporphyrin IX significantly reduced glycosaminoglycan degradation elicited by IL-1beta in OA cartilage explants but increased glycosaminoglycan synthesis and the expression of collagen II in OA chondrocytes in primary culture, as determined by radiometric procedures, immunoblotting and immunocytochemistry. HO-1 decreased the activation of extracellular signal-regulated kinase 1/2. This was accompanied by a significant inhibition in MMP activity and expression of collagenases MMP-1 and MMP-13 at the protein and mRNA levels. In addition, HO-1 induction caused a significant increase in the production of insulin-like growth factor-1 and a reduction in the levels of insulin-like growth factor binding protein-3. We have shown in primary culture of chondrocytes and articular explants from OA patients that HO-1 counteracts the catabolic and anti-anabolic effects of IL-1beta. Our data thus suggest that HO-1 may be a factor regulating the degradation and synthesis of extracellular matrix components in OA.     

Novel type II collagen reporter mice: New tool for assessing collagen 2α1 expression in vivo and in vitro

We report the generation of a new mouse strain harboring a Col2‐pd2EGFP reporter transgene; pd2EGFP has a much shorter half‐life than EGFP, making it a near real‐time reporter for Col2α1 expression in vivo and in vitro. In the post‐natal growth plate, pd2EGFP fluorescence was expressed in almost all proliferative chondrocytes and in some hypertrophic chondrocytes based on localization with type X collagen. In articular cartilage, pd2EGFP fluorescence diminished over time, nicely illustrating the decrease of type II collagen synthesis in articular chondrocytes during growth. Monolayers of FACS‐sorted chondrocytes from P1‐2 mice showed faster loss of pd2EGFP compared to EGFP, reflecting rapid chondrocyte de‐differentiation. High‐density culture of FACS‐pd2EGFP‐ growth plate chondrocytes revealed the typical temporal expression pattern in which type II collagen preceded type X collagen matrix deposition. The Col2‐pd2EGFP reporter mouse will be a valuable tool for studies of growth plate chondrocyte biology. Developmental Dynamics 240:663–673, 2011. © 2011 Wiley‐Liss, Inc.     

Rabbit articular chondrocytes seeded on collagen-chitosan-GAG scaffold for cartilage tissue engineering in vivo

In this study, we prepared a tri-copolymer porous matrices by natural polymer, collagen (Col), Chitosan (Chi) and Chondroitin (CS). Rabbit articular chondrocytes were isolated from the shoulder articular joints of a rabbit, seeded in Col-Chi-CS scaffold, and implanted subcutaneously in the dorsum of athymic nude mice to tissue engineer articular cartilage in vivo. In vitro studies show that Chondrocytes adhered to the scaffold, where they proliferated and secreted extracellular matrices with time, filling the space within the scaffold. The results of hematoxylin and eosin staining scanning electron microscopy revealed that most of the chondrocytes maintained their typically rounded morphology. After 28 days of culture within Col-Chi-CS scaffold in vitro, the results of histological staining showed forming of cartilage-specific morphological appearance and structural characteristics such as lacunae. Subcutaneous implantation studies in nude mice demonstrated that a homogeneous cartilaginous tissue, which was similar to those of natural cartilage, formed when chondrocytes were seeded in Col-Chi-CS matrix after implant 12 weeks. The tri-copolymer matrix could therefore have potential applications as a three-dimensional scaffold for cartilage tissue engineering.     

Moderate Joint Loading Reduces Degenerative Actions of Matrix Metalloproteinases in the Articular Cartilage of Mouse Ulnae

Joint loading is a recently developed loading modality, which can enhance bone formation and accelerate healing of bone fracture. Since mechanical stimulation alters expression of matrix metalloproteinases (MMPs) in chondrocytes, a question addressed herein was, does joint loading alter actions of MMPs in the articular cartilage? We hypothesized that expression and activity of MMPs are regulated in a load–intensity-dependent manner and that moderate load scan downregulates MMPs. To test this hypothesis, a mouse elbow-loading model was employed. In the articular cartilage of an ulna, the mRNA levels of a group of MMPs as well as their degenerative activities were determined. The result revealed that elbow loading altered the expression and activities of MMPs depending on its loading intensity. Collectively, the data in this study indicate that 0.2 and 0.5 N joint loading significantly reduced the expression of multiple MMPs, that is, MMP-1, MMP-3, MMP-8, and MMP-13, and overall activities of collagenases or gelatinases in articular cartilage, while higher loads increased the expression and activity of MMP-1 and MMP-13. Furthermore, moderate loads at 1 N elevated the mRNA level of CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), but higher loads at 4 N did not induce a detectable amount of CITED2 mRNA. Since CITED2 is known to mediate the downregulation of MMP-1 and MMP-13, the result indicates that joint loading at moderate intensity reduces MMP activities through potential induction of CITED2. MMPs such as MMP-1 and MMP-13 are predominant collagenases in the pathology of osteoarthritis. Therefore, joint loading could offer an interventional regimen for maintenance of joint tissues.     

Remobilization causes site‐specific cyst formation in immobilization‐induced knee cartilage degeneration in an immobilized rat model

An understanding of the articular cartilage degenerative process is necessary for the prevention and treatment of joint disease. The present study aimed to examine how long‐term immobilization‐induced cartilage degeneration is aggravated by remobilization. Sixty 8‐week‐old male Wistar rats were used in this study. The unilateral knee joint was immobilized using an external fixator for 8 weeks. The rats were killed at 0 and 3 days, and at 1, 2, 4 and 8 weeks after removing the fixator. After the rats were killed, the maximum knee extension angles were measured. Histological sections at the medial mid‐condylar region (non‐contact, transitional and contact regions of the femur and tibia) were prepared and scored. The cartilage thickness and number of chondrocytes were measured, and CD44 and Col2‐3/4c expression levels were assessed immunohistochemically. The histological assessment revealed progressive aggravation of cartilage degeneration in the transitional region, with a decreased number of chondrocytes and CD44‐positive chondrocytes as well as poor scoring over time, particularly in the tibia. Cyst formation was confirmed in the transitional region of the tibia at 8 weeks post‐remobilization. The cartilage thickness in the transitional region was thicker than that in the contact region, particularly in the tibia. Col2‐3/4c expression was observed in the non‐contact and transitional regions, and the knee extension angle was recovered. In conclusion, immobilization‐induced cartilage degeneration was aggravated by remobilization over time in the transitional region, followed by observations of a decreased number of chondrocytes and morphological disparity between different cartilage regions.     

Integrative repair of cartilage with articular and nonarticular chondrocytes

Articular chondrocytes can synthesize new cartilaginous matrix in vivo that forms functional bonds with native cartilage. Other sources of chondrocytes may have a similar ability to form new cartilage with healing capacity. This study evaluates the ability of various chondrocyte sources to produce new cartilaginous matrix in vivo and to form functional bonds with native cartilage. Disks of articular cartilage and articular, auricular, and costal chondrocytes were harvested from swine. Articular, auricular, or costal chondrocytes suspended in fibrin glue (experimental), or fibrin glue alone (control), were placed between disks of articular cartilage, forming trilayer constructs, and implanted subcutaneously into nude mice for 6 and 12 weeks. Specimens were evaluated for neocartilage production and integration into native cartilage with histological and biomechanical analysis. New matrix was formed in all experimental samples, consisting mostly of neocartilage integrating with the cartilage disks. Control samples developed fibrous tissue without evidence of neocartilage. Ultimate tensile strength values for experimental samples were significantly increased (p < 0.05) from 6 to 12 weeks, and at 12 weeks they were significantly greater (p < 0.05) than those of controls. We conclude that articular, auricular, and costal chondrocytes have a similar ability to produce new cartilaginous matrix in vivo that forms mechanically functional bonds with native cartilage.     

Moderate joint loading reduces degenerative actions of matrix metalloproteinases in the articular cartilage of mouse ulnae

Joint loading is a recently developed loading modality, which can enhance bone formation and accelerate healing of bone fracture. Since mechanical stimulation alters expression of matrix metalloproteinases (MMPs) in chondrocytes, a question addressed herein was, does joint loading alter actions of MMPs in the articular cartilage? We hypothesized that expression and activity of MMPs are regulated in a load-intensity-dependent manner and that moderate load scan downregulates MMPs. To test this hypothesis, a mouse elbow-loading model was employed. In the articular cartilage of an ulna, the mRNA levels of a group of MMPs as well as their degenerative activities were determined. The result revealed that elbow loading altered the expression and activities of MMPs depending on its loading intensity. Collectively, the data in this study indicate that 0.2 and 0.5?N joint loading significantly reduced the expression of multiple MMPs, that is, MMP-1, MMP-3, MMP-8, and MMP-13, and overall activities of collagenases or gelatinases in articular cartilage, while higher loads increased the expression and activity of MMP-1 and MMP-13. Furthermore, moderate loads at 1 N elevated the mRNA level of CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), but higher loads at 4 N did not induce a detectable amount of CITED2 mRNA. Since CITED2 is known to mediate the downregulation of MMP-1 and MMP-13, the result indicates that joint loading at moderate intensity reduces MMP activities through potential induction of CITED2. MMPs such as MMP-1 and MMP-13 are predominant collagenases in the pathology of osteoarthritis. Therefore, joint loading could offer an interventional regimen for maintenance of joint tissues.     

Single vs. repeated matrix metalloproteinase-13 knockdown with intra-articular short interfering RNA administration in a murine osteoarthritis model

Purpose: Our aims were 1) to estimate the duration of short interfering RNA (siRNA) effect on matrix metalloproteinase-13 (Mmp-13) levels by a single intra-articular injection using a mouse knee osteoarthritis (OA) model and 2) to test whether repeated injections results in any additional suppressive effect on cartilage degradation compared to a single injection. Materials and Methods: OA was induced in 9 weeks old male C57BL/6 mice by destabilization of medial meniscus (DMM). Chemically modified siRNA targeted for Mmp-13 was injected into the knee joint at 1 week post-DMM surgery. Control group of knees received that for non-targeted genes. Synovial tissue was collected to measure Mmp-13 expression levels by quantitative polymerase chain reaction (qPCR) at 2, 3, and 6 weeks after surgery in each group. To test the effect of multiple injections, we created four experiment groups according to the number of injections. Histological assessment of articular cartilage was performed at 8 weeks post-DMM surgery. Results: In the Mmp-13 siRNA-treated group, expression levels of Mmp-13 mRNA were decreased by 40% compared to the control group at 2 weeks after surgery (p = 0.04), before returning to baseline at 3 weeks after surgery. A significant improvement in the histological score was observed in all Mmp-13 siRNA-treated groups compared to the control group (p < 0.05). However, no significant differences were seen between the single and multiple injection group. Conclusions: Our results suggested that the duration of siRNA effect in the knee joint lasts for at least 1 week, and that no further benefit is achieved by multiple injections.