Aims

To explore the novel molecular mechanisms of histone deacetylase 4 (HDAC4) in chondrocytes via RNA sequencing (RNA-seq) analysis.

Aims

The study aimed to determine whether the microRNA miR21-5p (MiR21) mediates temporomandibular joint osteoarthritis (TMJ-OA) by targeting growth differentiation factor 5 (Gdf5).

Chondrocyte hypertrophy represents a crucial turning point during endochondral bone development. This process is tightly regulated by various factors, constituting a regulatory network that maintains normal bone development. Histone deacetylase 4 (HDAC4) is the most well-characterized member of the HDAC class IIa family and participates in different signalling networks during development in various tissues by promoting chromatin condensation and transcriptional repression. Studies have reported that HDAC4-null mice display premature ossification of developing bones due to ectopic and early-onset chondrocyte hypertrophy. Overexpression of HDAC4 in proliferating chondrocytes inhibits hypertrophy and ossification of developing bones, which suggests that HDAC4, as a negative regulator, is involved in the network regulating chondrocyte hypertrophy. Overall, HDAC4 plays a key role during bone development and disease. Thus, understanding the role of HDAC4 during chondrocyte hypertrophy and endochondral bone formation and its features regarding the structure, function, and regulation of this process will not only provide new insight into the mechanisms by which HDAC4 is involved in chondrocyte hypertrophy and endochondral bone development, but will also create a platform for developing a therapeutic strategy for related diseases.

Cite this article: Bone Joint Res. 2020;9(2):82–89.

Once damaged, articular cartilage has limited capacity for self-repair due to their avascular and acellular nature. Tissue engineering approaches using cultured chondrocytes and biomaterials as scaffoldings hold promises for repairing cartilage defects. However, the source of articular chondrocytes is limited and the chon-drocytes may de-differentiate when cultured for a prolonged period. Bone marrow derived mesenchymal stem cells (BMSCs) have multi-differentiation potentials and autologous BMSCs are easy to obtain and culture with no/little immunological reaction when re-implanted.

24 NZW rabbits were used. Rabbit autologous BMSCs were obtained through marrow aspirations and expanded in culture under the chondrogenic induction media (DMEM, 10% FCS, plus 10ng/ml TGF-β1) for 3 weeks. A full-thickness articular cartilage defect (3 mm in diameter and 3 mm in depth) was created on both medial condyles in the rabbit. For experimental group (16 joints), the defects were filled immediately with alginate capsules containing autologous chondrogenic cells (8.5 x 104); for the control groups, the defects were filled with either alginate capsules alone (16 joints) or left untreated (16 joints). All the animals were terminated at 6 and 12 weeks after surgery and the cartilage samples were harvested for histology, immunochemistry and in situ hybridization examinations.

For histology, in the experimental group the defects were filled with immature hyaline-like cartilaginous tissues at 6 weeks; by 12 weeks the newly formed cartilage showing signs of remodeling and integrating into the surrounding articular cartilage. The expression of type II collagen in the newly formed cartilaginous tissues was confirmed by immunohistochemistry and by in situ hybridization methods. In the control groups, the defects were mainly filled with fibrous tissues in all the animals at the two time points examined. We have used Wakitani cartilage grading system for semi-quantitative histological evaluation. Significant lower scores (with superior histology) were found in the experimental group comparing to the two control groups.

Our results confirmed that full-thickness articular cartilage defects can be repaired by chondrogenically differentiated autologous BMSCs seeded into alginate capsules. Further studies are ongoing to explore the long term outcome of this treatment approach as well as using new scaffolds for cartilage tissue engineering.

The repair of cartilage defects remains a significant clinical challenge. The use of mesenchymal stem cells for cell-based tissue-engineering strategies represents a promising alternative for the repair of the defects. In this study, we investigated the TGF-bate1 dose and cellular density-dependent effect on chondrogenic differentation of human bone marrow-derived mesenchymal stem cells (MSCs) cultured in alginate beads in vitro.

Methods A volume of 6 ml bone marrow was collected from six volunteer donors respectively. MSCs were cultured in different cellular density (1×104, 1×105, 1×106 and 5×106/ml) and treated with different doses of TGF-beta1 (0, 1, 10, 50 and 100 ng/ml). Immunohistochem-istry and in situ hybridization were applied to detect the expression of collagen type II and assay proteoglycan in different time internal.

Results 95% cellss were alive after density gradient centrifugation. BMSCs had a similar spindle-like morphology. Type II collagen and proteoglycan were showed positive staining in the 10 ng/ml TGF-beta1 group, weakly positive in the 50 ng/ml and 100 ng/ml group, negative in the 0 ng/ml and 1 ng/ml group. With time, the proteoglycan quantity increased. All cell density groups except 1×104/ml showed positive expression of collagen type II and proteoglycan synthesis, and better staining with increase of cellular density. Proteoglycan synthesis did not increased until the fifth weeks.

Conclusions The chondrogenesis differentiation of human MSCs is dose-dependent. 10ng/ml TGF-beta1 is a suitable concentration for such inducing. The cellular density is also important for the differentiation of MSCs. Too small density is ineffective. The more cells, the better differentiation. And the time of in vitro culture should not be longer than 4 weeks

Studies have demonstrated that use of peptides including bone morphogenetic proteins, fibroblast growth factors, insulin-like growth factor (IGF), and transforming growth factor-beta (TGF-beta), may be pivotal in promoting chondrogenesis and matrix development. As a prelude to studies, it is necessary to determine which gene or combination of genes gives the best result to improve proliferation of chondrocytes and synthesis of extracellar matrix. We investigate the effect of transfec-tion of recombined rat TGF-beta1 and recombined rat IGF-1 on rabbit chondrocytes ex vivo.

Chondrocytes were isolated from articular cartilage of knee joint of mature New Zealand White rabbits. Cells were seeded at a density of 1×105 cells/ml into 6-well plates. Monolayer cultures were infected respectively with recombinant rat gene pcDNA3+TGF-beta 1, pAT153+IGF-1 and lac Z reporter gene by using lipo-fectamine, and were co-transfected by pcDNA3+TGF-beta 1, pAT153+IGF-1. The control group remained uninfected. To determine whether the genes transcript were translated and the gene products were released, the synthesis of TGF-beta 1, IGF-1,and type II collagen were measured by in situ hybridization, immunohisto-chemistry and immunofluoroscopy. The proliferation of chondrocytes was detected by flow cytometer and 3H-TdR radiolabeling.

The expression of TGF-beta1,IGF-1 and type II collagen in recombinant rat gene transfection groups was high beyond control levels and the lac Z gene levels (P< 0.05). The co-transfection elevated these factors synthesis beyond the levels of single gene transfection (P< 0.05). In pcDNA3 +TGF-beta1 transfection group, the level of TGF-beta1 and type II collagen were higher than the levels of pAT153+IGF-1 group (P< 0.05), while the content of IGF-1 has no significant difference with pAT153+IGF-1 group. By using flow cytometer, the chondrocytes ratio of S stage in pcDNA3+TGF-beta 1 group, pAT153+IGF-1 group and co-transfection group was 33.4%,28.7% and 40.1% respectively, which was higher than 5.6% and 4.8% of the control group and the lac Z gene group (P< 0.05). The 3H-TdR radiolabeling detection also indicated that the recombinant rat gene transfection groups improved the chondrocytes proliferation, and co-transfection group has the best effect.

The data presented support that transfection of genes of TGF-beta1 and IGF-1 into chondrocytes ex vivo can greatly increase cell proliferation and matrix synthesis, and the co-transfection can provoke more increase in the synthesis of TGF-beta1, IGF-1 and type II collagen, which encourages the further research of gene potential therapeutic use for osteoarthritis.

To explore the relationship of hyaluronan level in synovial fluid of the knee with the degree of synovitis and cartilage injury.

A total of 104 knees in 102 patients with knee osteoarthritis or other knee diseases was studied. The hyaluronan level in the synovial fluid of the knees was measured with enzyme linked immunoassay. The pathology of the synovium and articular cartilage was evaluated with Ayral’s score system and Outerbridge’s score system under arthroscopy. The data were analyzed by t’-test or nonparametric test, ANOVA, Pearson or Spearman correlation and multiple liner regression.

The results showed that the hyaluronan level in the synovial fluid of the knees was correlated positively with Ayral’s score (beta’A=0.497, P< 0.001) and negatively with accumulative Outerbridge’s score (beta’O=-0.364, P< 0.001), especially Ayral’s synovitis score in 104 cases. The hyaluronan level in the synovial fluid of the knees was higher in those with Ayral’s score > and = 60 than in those with the score< 60 (P< 0.001). The hyaluronan level in the synovial fluid of the knees was lower in those with accumulative Outerbridge’s score > and = 10 than in those with the score < 10 (P< 0.05). The level of hyaluro-nan in the synovial fluid in the knees with Ayral’s score > and = 60 was correlated negatively with accumulative Outerbridge’s score (beta’O=-0.437, P< 0.001) and positively with Ayral’s score (beta’A=0.339, P< 0.01), especially accumulative Outerbridge’s score.

Compared with other knee diseases, the hyaluronan level of OA knees was lower (P< 0.05). However, Ayral’s score and accumulative Outerbridge’s score were higher in OA knees (P< 0.001).

The hyaluronan level in the synovial fluid of the knee can reflect the degree of synovitis and accumulative cartilage injury, especially synovitis. It reflects the degree of accumulative cartilage injury mainly when synovitis is more severe. The decrease of the hyaluronan level in the synovial fluid of OA knee is results of integrating effect of the synovitis and cartilage injury.

Objective To decide whether recombined rat transforming growth factor beta-1 gene and insulin-like growth factor-1 gene have positive influences on ACLT-induced osteoarthritis-like changes in NZW rabbit articular cartilage.

Methods Twenty-four NZW rabbits, with osteoarthritis caused by anterior cruciate ligament transection£^..ACLT£©, were distributed to 4 groups randomly and another six rabbits were taken as normal control group (group 1). Chondrocytes which had been transfected with TGF-¦Â1 gene, IGF-1 gene (group 3–5) were injected into the knee of these NZW rabbits. Experimental control group (group 2) was only suffered ACLT but nothing injected. After 4, 8 weeks, rabbits were sacrificed and evaluated by morphological grades, histological examination, examination of in situ hybridization, immunohistochemistry, and transmission electron microscopy (TEM).

Results The data of morphological grades showed that the normal control showed a significant difference compared with experimental control group (P< 0.01). The groups with injected chondrocytes carring TGF-¦Â1 gene and double genes (group 3,5) had a significant difference compared with experimental control group (P< 0.05). The in situ hybridization and immunohis-tochemistry examination showed the same results as above, and the group carring double genes (group 5) had a significant difference with that single gene (group 3,4) (P< 0.05). After 8 weeks, the examination data showed that all groups lower than the data of 4 weeks except the normal control group and experimental control group (P< 0.05). Ultrastructural examination indicated that the ultrastructure of experimental control group was more turbulent than that of normal control group. The ultra-structure of the gene therapy groups was more normal than that of experimental control group after gene therapy, but it turned to be turbulent again after 8 weeks.

Conclusion It is effectual on osteoarthritis to inject chondrocytes carring recombined TGF-¦Â1,IGF-1 genes into NZW rabbits knee joints. It was obvious that the therapy effect of double genes was better than single gene. The fact that gene expression was decreased gradually after 4 weeks makes out that gene therapy is limited by time. These results suggest that therapeutic TGF-¦Â1 and IGF-1 gene transfer may be applicable for the treatment of OA.