Cdc42 in osterix-expressing cells alters osteoblast behavior and myeloid lineage commitment.

Osteoblasts are not only responsible for bone formation. They also support hematopoiesis. This requires responding to cues originating from several signaling pathways, a task performed by Rho GTPases. We therefore examined several transgenic mouse models and used inhibitors of Cdc42 in vitro. Deletion of Cdc42 in vivo using the Osterix promoter suppressed osteoblast function, while its deletion in differentiating osteoblasts using the Collagen-α1(I) promoter decreased osteoblast numbers. In both cases, bone mineral density diminished confirming the importance of Cdc42. Evaluation of hematopoiesis revealed that deletion of Cdc42 using the Osterix, but not the Collagen-α1(I) promoter increased the common myeloid progenitors (CMPs) in the bone marrow as well as the erythrocytes and the thrombocytes/platelets in peripheral blood. Causality between Cdc42 loss in early osteoblasts and increased myelopoiesis was confirmed in vitro. Work in vitro supported the conclusion that interleukin-4 mediated the increase in myelopoiesis. Thus, Cdc42 is required for healthy bone through regulation of bone formation in Osterix-expressing osteoblasts and the number of osteoblasts in differentiating osteoblasts. In addition, its expression in early osteoblasts/stromal cells modulates myelopoiesis. This highlights the importance of osteoblasts in regulating hematopoiesis.

The effect of medical ozone treatment on cartilage chondrocyte autophagy in a rat model of osteoarthritis.

Many studies have shown that ozone (O3) can inhibit inflammation in osteoarthritis (OA) and regulate the metabolic balance of articular cartilage, but the mechanisms of this process are not well understood. Our study investigated the therapeutic mechanism of O3 in OA. OA models were established, and the MWT and PWL were measured. HE staining and safranin O-fast green staining were used to observe cartilage degeneration. The levels of MMP-13, collagen-2, LC3II and P62 were measured by immunohistochemistry, and the levels of TNF-α and IL-6 were measured by ELISA. The results showed that intra-articular injection of O3 can effectively alleviate pain and inhibit cartilage degeneration in OA rats. O3 can also reduce the concentrations of TNF-α and IL-6, inhibit the expression of MMP-13 and the degradation of collagen-2, upregulate the autophagy-related protein LC3II and inhibit P62. This effect is associated with the upregulation of chondrocyte autophagy in OA.

Activation of Hes1 and Msx1 in Transgenic Mouse Embryonic Stem Cells Increases Differentiation into Neural Crest Derivatives.

The neural crest (NC) comprises a multipotent cell population that produces peripheral neurons, cartilage, and smooth muscle cells, among other phenotypes. The participation of Hes1 and Msx1 when expressed in mouse embryonic stem cells (mESCs) undergoing NC differentiation is unexplored. In this work, we generated stable mESCs transfected with constructs encoding chimeric proteins in which the ligand binding domain of glucocorticoid receptor (GR), which is translocated to the nucleus by dexamethasone addition, is fused to either Hes1 (HGR) or Msx1 (MGR), as well as double-transgenic cells (HGR+MGR). These lines continued to express pluripotency markers. Upon NC differentiation, all lines exhibited significantly decreased Sox2 expression and upregulated Sox9, Snai1, and Msx1 expression, indicating NC commitment. Dexamethasone was added to induce nuclear translocation of the chimeric proteins. We found that Collagen IIa transcripts were increased in MGR cells, whereas coactivation of HGR+MGR caused a significant increase in Smooth muscle actin (α-Sma) transcripts. Immunostaining showed that activation in HGR+MGR cells induced higher proportions of ß-TUBULIN III⁺, α-SMA⁺ and COL2A1⁺ cells. These findings indicate that nuclear translocation of MSX-1, alone or in combination with HES-1, produce chondrocyte-like cells, and simultaneous activation of HES-1 and MSX-1 increases the generation of smooth muscle and neuronal cells.

Isolation and characterization of human articular chondrocytes from surgical waste after total knee arthroplasty (TKA).

BACKGROUND: Cartilage tissue engineering is a fast-evolving field of biomedical engineering, in which the chondrocytes represent the most commonly used cell type. Since research in tissue engineering always consumes a lot of cells, simple and cheap isolation methods could form a powerful basis to boost such studies and enable their faster progress to the clinics. Isolated chondrocytes can be used for autologous chondrocyte implantation in cartilage repair, and are the base for valuable models to investigate cartilage phenotype preservation, as well as enable studies of molecular features, nature and scales of cellular responses to alterations in the cartilage tissue. METHODS: Isolation and consequent cultivation of primary human adult articular chondrocytes from the surgical waste obtained during total knee arthroplasty (TKA) was performed. To evaluate the chondrogenic potential of the isolated cells, gene expression of collagen type 2 (COL2), collagen 1 (COL1) and aggrecan (ACAN) was evaluated. Immunocytochemical staining of all mentioned proteins was performed to evaluate chondrocyte specific production. RESULTS: Cartilage specific gene expression of COL2 and ACAN has been shown that the proposed protocol leads to isolation of cells with a high chondrogenic potential, possibly even specific phenotype preservation up to the second passage. COL1 expression has confirmed the tendency of the isolated cells dedifferentiation into a fibroblast-like phenotype already in the second passage, which confirms previous findings that higher passages should be used with care in cartilage tissue engineering. To evaluate the effectiveness of our approach, immunocytochemical staining of the evaluated chondrocyte specific products was performed as well. DISCUSSION: In this study, we developed a protocol for isolation and consequent cultivation of primary human adult articular chondrocytes with the desired phenotype from the surgical waste obtained during TKA. TKA is a common and very frequently performed orthopaedic surgery during which both femoral condyles are removed. The latter present the ideal source for a simple and relatively cheap isolation of chondrocytes as was confirmed in our study.

Hydroxylation of methylated DNA by TET1 in chondrocyte differentiation of C3H10T1/2 cells.

DNA methylation is closely involved in the regulation of cellular differentiation, including chondrogenic differentiation of mesenchymal stem cells. Recent studies showed that Ten-eleven translocation (TET) family proteins converted 5-methylcytosine (5mC) to 5-hydroxymethylcytosine, 5-formylcytosine and 5carboxylcytosine by oxidation. These reactions constitute potential mechanisms for active demethylation of methylated DNA. However, the relationship between the DNA methylation patterns and the effects of TET family proteins in chondrocyte differentiation is still unclear. In this study, we showed that DNA hydroxylation of 5mC was increased during chondrocytic differentiation of C3H10T1/2 cells and that the expression of Tet1 was particularly enhanced. Moreover, knockdown experiments revealed that the downregulation of Tet1 expression caused decreases in chondrogenesis markers such as type 2 and type 10 collagens. Furthermore, we found that TET proteins had a site preference for hydroxylation of 5mC on the Insulin-like growth factor 1 (Igf1) promoter in chondrocytes. Taken together, we showed that the expression of Tet1 was specifically facilitated in chondrocyte differentiation and Tet1 can regulate chondrocyte marker gene expression presumably through its hydroxylation activity for DNA.

Low Molecular Weight Fraction of Commercial Human Serum Albumin Induces Morphologic and Transcriptional Changes of Bone Marrow-Derived Mesenchymal Stem Cells.

Osteoarthritis (OA) is the most common chronic disease of the joint; however, the therapeutic options for severe OA are limited. The low molecular weight fraction of commercial 5% human serum albumin (LMWF5A) has been shown to have anti-inflammatory properties that are mediated, in part, by a diketopiperazine that is present in the albumin preparation and that was demonstrated to be safe and effective in reducing pain and improving function when administered intra-articularly in a phase III clinical trial. In the present study, bone marrow-derived mesenchymal stem cells (BMMSCs) exposed to LMWF5A exhibited an elongated phenotype with diffuse intracellular F-actin, pronounced migratory leading edges, and filopodia-like projections. In addition, LMWF5A promoted chondrogenic condensation in "micromass" culture, concurrent with the upregulation of collagen 2α1 mRNA. Furthermore, the transcription of the CXCR4-CXCL12 axis was significantly regulated in a manner conducive to migration and homing. Several transcription factors involved in stem cell differentiation were also found to bind oligonucleotide response element probes following exposure to LMWF5A. Finally, a rapid increase in PRAS40 phosphorylation was observed following treatment, potentially resulting in the activation mTORC1. Proteomic analysis of synovial fluid taken from a preliminary set of patients indicated that at 12 weeks following administration of LMWF5A, a microenvironment exists in the knee conducive to stem cell infiltration, self-renewal, and differentiation, in addition to indications of remodeling with a reduction in inflammation. Taken together, these findings imply that LMWF5A treatment may prime stem cells for both mobilization and chondrogenic differentiation, potentially explaining some of the beneficial effects achieved in clinical trials.

Chondrocyte source for cartilage regeneration in an immature animal: Is iliac apophysis a good alternative?

BACKGROUND: Autologous articular cartilage at present forms the main source of chondrocytes for cartilage tissue engineering. In children, iliac apophysis is a rich and readily accessible source of chondrocytes. This study compares the growth characteristics and phenotype maintenance of goat iliac apophysis growth plate chondrocytes with those sourced from goat articular cartilage, and thereby assesses their suitability for autologous chondrocyte transplantation in immature animals for growth plate and articular cartilage regeneration. MATERIALS AND METHODS: Four sets of experiments were carried out. Cartilage samples were harvested under aseptic conditions from goat iliac apophysis and knee articular cartilage. The chondrocytes were isolated in each set and viable cells were counted and subsequently cultured as a monolayer in tissue culture flasks containing chondrogenic media at 2.5 × 10(3)cells/cm(2). The growth was periodically assessed with phase contrast microcopy and the cells were harvested on 8(th) and 15(th) days for morphology, cell yield, and phenotype assessment. Student's t-test was used for comparison of the means. RESULTS: Confluence was reached in the iliac apophysis growth plate chondrocytes flasks on the 10(th) day and the articular cartilage chondrocytes flasks on the 14(th) day. Mean cell count of growth plate chondrocytes on the 8(th) day was 3.64 × 10(5) (SD = 0.601) and that of articular cartilage chondrocytes was 1.40 × 10(5) (SD = 0.758) per flask. The difference in the means was statistically significant (P = 0.003). On the 15(th) day, the mean cell number had increased to 1.35 × 10(6)(SD = 0.20) and 1.19 × 10(6) (SD = 0.064) per flask, respectively. This difference was not statistically significant (P = 0.26). The population doubling time on the 8(th) day of cell culture was 3.18 and 6.24 days respectively, for iliac apophyseal and articular cartilage chondrocytes, which was altered to 3.59 and 3.1 days, respectively, on the 15(th) day. The immunocytochemistry showed 100% retention of collagen 2 positive and collagen 1 negative cells in both sets of cultures in all samples. CONCLUSION: Iliac apophysis is a rich source of chondrocytes with a high growth rate and ability to retain phenotype when compared to articular cartilage derived chondrocytes. Further in vivo studies may determine the efficacy of physeal and articular repair in children with apophyseal chondrocytes.