A new protocol for validation of Chondro, Adipo and Osteo differentiation kit of Cultured Adipose-Derived Stem Cells (ADSC) by real-time rt-QPCR.

Mesenchymal stem cells (MSCs) are multipotent cells, originally derived from the embryonic mesenchyme, and able to differentiate into connective tissues such as bone, fat, cartilage, tendon, and muscle. Furthermore, MSCs derived from adipose tissue ADSC (Adipose derived Stem Cells) show a great potential for degenerative diseases treatment. In this study, we designed a series of experiments based on real-time rt-QPCR to validated a commercially available kit able to explore changes in gene expression under osteogenic, adipogenic and chondrogenic differentiation of human ADSC. Initially, we selected a better indicators of trilineage differentiation by using third passages of cultured ADSC from stromal vascular fraction (SVF) isolated from fresh adipose tissue by enzymatic digestion. On the basis of statistically significant results ACAN, FABP4A and Col11a1 were chosen as indicators of chondrogenic, adipogenic and osteogenic differentiation respectively. An in-vitro aging analysis was then performed to evaluate the ADSC passage with the highest differentiation potential. Total RNA extraction from induced differentiation and controls ADSC from passage 2-6 and relative quantifications of mRNA expression of selected genes were performed according to rt-PCR kits tested. The chondrogenic differentiation test showed equivalent ∆∆Ct values for ACAN detection for cell passages ranging from P3 to P6, proving that they can be considered as equivalent samples for differentiation assays evaluation. For what concerns adipogenic differentiation and FABP4 detection, similar results were observed in all the cell passages tested; on the contrary only passage P6 showed suitable ∆∆Ct values for Col11a1 detection for osteogenic differentiation evaluation. In conclusion, we have validated a suitable real-time rt-QPCR protocol for osteogenic, chondrogenic and adipogenic ADSC differentiation ability evaluation in-vitro.

Biopolymer Material from Human Spongiosa for Regenerative Medicine Application.

Natural biopolymers demonstrate significant bone and connective tissue-engineering application efficiency. However, the quality of the biopolymer directly depends on microstructure and biochemical properties. This study aims to investigate the biocompatibility and microstructural properties of demineralized human spongiosa Lyoplast® (Samara, Russian Federation). The graft's microstructural and biochemical properties were analyzed by scanning electron microscopy (SEM), micro-computed tomography, Raman spectroscopy, and proteomic analysis. Furthermore, the cell adhesion property of the graft was evaluated using cell cultures and fluorescence microscopy. Microstructural analysis revealed the hierarchical porous structure of the graft with complete removal of the cellular debris and bone marrow components. Moreover, the proteomic analysis confirmed the preservation of collagen and extracellular proteins, stimulating and inhibiting cell adhesion, proliferation, and differentiation. We revealed the adhesion of chondroblast cell cultures in vitro without any evidence of cytotoxicity. According to the study results, demineralized human spongiosa Lyoplast® can be effectively used as the bioactive scaffold for articular hyaline cartilage tissue engineering.

Bioprinting of Cartilage with Bioink Based on High-Concentration Collagen and Chondrocytes.

The study was aimed at the applicability of a bioink based on 4% collagen and chondrocytes for de novo cartilage formation. Extrusion-based bioprinting was used for the biofabrication. The printing parameters were tuned to obtain stable material flow. In vivo data proved the ability of the tested bioink to form a cartilage within five to six weeks after the subcutaneous scaffold implantation. Certain areas of cartilage formation were detected as early as in one week. The resulting cartilage tissue had a distinctive structure with groups of isogenic cells as well as a high content of glycosaminoglycans and type II collagen.

General Caspase Inhibition in Primary Chondrogenic Cultures Impacts Their Transcription Profile Including Osteoarthritis-Related Factors.

OBJECTIVE: The knowledge about functions of caspases, usually associated with cell death and inflammation, keeps expanding also regarding cartilage. Active caspases are present in the growth plate, and caspase inhibition in limb-derived chondroblasts altered the expression of osteogenesis-related genes. Caspase inhibitors were reported to reduce the severity of cartilage lesions in osteoarthritis (OA), and caspase-3 might represent a promising biomarker for OA prognosis. The objective of this investigation was to decipher the transcriptomic regulation of caspase inhibition in chondrogenic cells. DESIGN: Limb-derived chondroblasts were cultured in the presence of 2 different inhibitors: Z-VAD-FMK (FMK) and Q-VD-OPH (OPH). A whole transcriptome RNA sequencing was performed as the key analysis. RESULTS: The analysis revealed a statistically significant increase in the expression of 252 genes in the FMK samples and 163 genes in the OPH samples compared with controls. Conversely, there was a significant decrease in the expression of 290 genes in the FMK group and 188 in the OPH group. Among the top up- and downregulated genes (more than 10 times changed), almost half of them were associated with OA. Both inhibitors displayed the highest upregulation of the inflammatory chemokine Ccl5, the most downregulated gene was the one for mannose receptors Mrc1. CONCLUSIONS: The obtained datasets pointed to a significant impact of caspase inhibition on the expression of several chondro-/osteogenesis-related markers in an in vitro model of endochondral ossification. Notably, the list of these genes included some encoding for factors associated with cartilage/bone pathologies such as OA.

Exercise as an Adjuvant to Cartilage Regeneration Therapy.

This article provides a brief review of the pathophysiology of osteoarthritis and the ontogeny of chondrocytes and details how physical exercise improves the health of osteoarthritic joints and enhances the potential of autologous chondrocyte implants, matrix-induced autologous chondrocyte implants, and mesenchymal stem cell implants for the successful treatment of damaged articular cartilage and subchondral bone. In response to exercise, articular chondrocytes increase their production of glycosaminoglycans, bone morphogenic proteins, and anti-inflammatory cytokines and decrease their production of proinflammatory cytokines and matrix-degrading metalloproteinases. These changes are associated with improvements in cartilage organization and reductions in cartilage degeneration. Studies in humans indicate that exercise enhances joint recruitment of bone marrow-derived mesenchymal stem cells and upregulates their expression of osteogenic and chondrogenic genes, osteogenic microRNAs, and osteogenic growth factors. Rodent experiments demonstrate that exercise enhances the osteogenic potential of bone marrow-derived mesenchymal stem cells while diminishing their adipogenic potential, and that exercise done after stem cell implantation may benefit stem cell transplant viability. Physical exercise also exerts a beneficial effect on the skeletal system by decreasing immune cell production of osteoclastogenic cytokines interleukin-1ß, tumor necrosis factor-α, and interferon-γ, while increasing their production of antiosteoclastogenic cytokines interleukin-10 and transforming growth factor-ß. In conclusion, physical exercise done both by bone marrow-derived mesenchymal stem cell donors and recipients and by autologous chondrocyte donor recipients may improve the outcome of osteochondral regeneration therapy and improve skeletal health by downregulating osteoclastogenic cytokine production and upregulating antiosteoclastogenic cytokine production by circulating immune cells.

Effect of activin A receptor type i on growth and development of mandibular condylar cartilage in mice and its mechanism / 吉林大学学报(医学版)

Objective: To observe the effect of type 1 bone morphogenetic protein (BMP) receptor activin A receptor type 1 ( ACVRl) on the morphology, proliferation and differentiation of the mandibular condylar cartilage (MCC) cells in the postnatal mice, and to provide the reference for the study on etiology and treatment of MCC-related disease. Methods: The C57BL/6J mouse model of conditional deletion of ACVRl gene was constructed by using the Cre-LoxP system. The female and male mice with Acvrl1" ; RS/RS and Acvrl ; Osterix (+)/( ) genotypes were paired off with each other; the offspring Osterix-Cre ( + ); Acvrl'∗ ; RS/+ genotype mice were selected as experiment group, and the Osterix-Cre ( + ); Acvrl1" ; RS/+ mice were selected as control group. The newborn (n-3). postnatal day 21 (n=4) and PN42 (n=5) male mice were selected. X-gal staining was used to detect the expressions of Osterix-Cre in MCC tissue of the mice in two groups. micro-CT was used to detect the condylar widths and condylar head lengths of mandible of the mice in two groups. HE and Toluidine blue staining were used to analyze the morphology of MCC cells and the thickness of caritilage in each layer of MCC tissue of the mice in two groups, immunohistochemical (1HC) staining was used to detect the number of proliferating cell nuclear antigen (PCNA)-positive cells and the level of type X collagen in MCC tissue of the mice in two groups. Results: The X-gal staining and 1HC results showed that the mouse model of ACVRl gene conditional deletion was successfully constructed. At PN21. compared with control group, the condylar width and the condylar head length of mandible of the mice in experiment group were significantly shortened ( P<0. 05); the morphology of the MCC cells of the mice in two groups had no significant difference. Compared with control group, the number of PCNA-positive cells in the MCC cells of hypertrophic chondrocyte zone (Hy) and chondroblastic zone (Ch) and single Hy of the mice in experiment group were significantly increased ( P<0. 05 or P-<0. 01). At PN42. compared with control group, the shape of parts of the mandibular condylar cartilage cells of the mice in experiment group was abnormal, and the arrangement of some condylar chondrocytes was disordered, the cell thickness of the Ar. Pr and Ch in intermediate part and Hy in anterior part of the condylar cartilage of the mice in experiment group were significantly increased ( P<0. 05 or P<.0. 01); compared with control group, the number of PCNA-positive cells in each zone and the level of type X collagen in Ch of MCC tissue of the mice in experiment group were incresed. Conclusion: ACVRl affects the morphology of MCC cells and structure of MCC tissue by inhibiting the proliferation of MCC cells and the differentiation of chondroblasts into hypertrophic chondrocytes.

Automated digital image quantification of histological staining for the analysis of the trilineage differentiation potential of mesenchymal stem cells.

BACKGROUND: Multipotent mesenchymal stem cells (MSCs) have the potential to repair and regenerate damaged tissues and are considered as attractive candidates for the development of cell-based regenerative therapies. Currently, there are more than 200 clinical trials involving the use of MSCs for a wide variety of indications. However, variations in their isolation, expansion, and particularly characterization have made the interpretation of study outcomes or the rigorous assessment of therapeutic efficacy difficult. An unbiased characterization of MSCs is of major importance and essential to guaranty that only the most suitable cells will be used. The development of standardized and reproducible assays to predict MSC potency is therefore mandatory. The currently used quantification methodologies for the determination of the trilineage potential of MSCs are usually based on absorbance measurements which are imprecise and prone to errors. We therefore aimed at developing a methodology first offering a standardized way to objectively quantify the trilineage potential of MSC preparations and second allowing to discriminate functional differences between clonally expanded cell populations. METHOD: MSCs originating from several patients were differentiated into osteoblasts, adipocytes, and chondroblasts for 14, 17, and 21 days. Differentiated cells were then stained with the classical dyes: Alizarin Red S for osteoblasts, Oil Red O for adipocytes, and Alcian Blue 8GX for chondroblasts. Quantification of differentiation was then performed with our newly developed digital image analysis (DIA) tool followed by the classical absorbance measurement. The results from the two techniques were then compared. RESULT: Quantification based on DIA allowed highly standardized and objective dye quantification with superior sensitivity compared to absorbance measurements. Furthermore, small differences between MSC lines in the differentiation potential were highlighted using DIA whereas no difference was detected using absorbance quantification. CONCLUSION: Our approach represents a novel method that simplifies the laboratory procedures not only for the quantification of histological dyes and the degree of differentiation of MSCs, but also due to its color independence, it can be easily adapted for the quantification of a wide range of staining procedures in histology. The method is easily applicable since it is based on open source software and standard light microscopy.

The osteoarthritis-associated gene PAPSS2 promotes differentiation and matrix formation in ATDC5 chondrogenic cells.

3'-Phosphoadenosine 5'-phosphosulfate synthetase 2 (PAPSS2) has been shown to be important in the development of normal skeletal structure. The aim of the present study was to evaluate the role of PAPSS2 in the differentiation of chondrocytes as well as their mechanisms. Using RNA interference-mediated via a lentivirus and a retrovirus, PAPSS2 gene silence and overexpression in ATDC5 chondrogenic cells were performed. Chondrocyte differentiation and chondrogenic-related gene markers associated with extracellular matrix formation were noted. The mRNA and protein expression for Wnt4, ß-catenin and SOX9 genes were observed. The PAPSS2 transcript expression levels progressively decline in ATDC5-induced chondrocyte-like cells during differentiation. Silencing of PAPSS2 expression had a significantly attenuating effect on cell differentiation and decreased expression of collagen II and X. In contrast, over-expression of PAPSS2 promoted the differentiation of ATDC5 chondrogenic cells. The mRNA expression levels of Wnt4 and SOX9 decreased significantly in PAPSS2 knock down cells vs. control cells. However, this expression was increased in the cells over-expressing PAPSS2. These data indicate that PAPSS2 regulates aggrecan activity as well as cell differentiation. The findings favor a mechanism by which PAPSS2 induces differentiation in ATDC5 cells via direct regulation of early signaling events that promote formation of collagenous matrix components. This control is probably mediated via extracellular matrix formation Wnt/ß-catenin signaling pathways.

[New experimental strategies in cartilage surgery]. / Neue experimentelle Ansätze in der Knorpelchirurgie.

BACKGROUND: Cell and growth factor based strategies bear great potential to support the healing processes in cartilage repair and the therapy of osteoarthritic joints. OBJECTIVES: The following review provides an overview of novel experimental strategies for the therapy of focal cartilage defects and osteoarthritis, with emphasis on cell and growth factor based approaches. MATERIALS AND METHODS: The authors summarize their own data regarding the intraarticular injection of stem cells to treat osteoarthritis of the knee and provide a synopsis of the available literature discussing the most significant publications. RESULTS: The development of novel strategies for the treatment of focal and arthrotic cartilage lesions focuses on the application of growth factors, platelet rich plasma (PRP), bone marrow (BMSAC) or adipose derived (stromal vascular fraction - SVF) cell concentrates, and ex vivo expanded mesenchymal stem cells (MSC). First clinical data on the use of expanded MSCs show the potential of this innovative therapeutic strategy. These approaches, however, are governed by EU law and often require approval by regulatory bodies. CONCLUSION: Currently, only a limited number of published, randomized, controlled trials available. Therefore, it is not possible to finally assess the efficacy of these strategies at this point in time.

[Cartilage regeneration surgery on the hip : What is feasible?] / Knorpelregenerative Eingriffe am Hüftgelenk : Was ist machbar?

Localized cartilage defects at the hip are mainly caused by pre-arthritic deformities, particularly by cam-type femoroacetabular impingement (FAI). Timely elimination of symptomatic deformities can prevent further progression such as cartilage defects. As the defects mostly occur in the anterolateral part of the acetabulum, they can be easily treated either by open surgery or by arthroscopy. To date the most effective methods of treatment are bone marrow stimulation, with or without a covering of biomaterials, and autologous chondrocyte transplantation. In selected cases, readaptation of the damaged cartilage can be attempted by biological procedures. In the present article, the findings reported in current studies on these procedures are summarized and discussed in detail. An outlook is given regarding possible future treatment concepts.

[Current overview of cartilage regeneration procedures]. / Aktuelle Übersicht knorpelregenerativer Verfahren.

BACKGROUND: Cartilage is an avascular, alymphatic and non-innervated tissue with limited intrinsic repair potential. The high prevalence of cartilage defects and their tremendous clinical importance are a challenge for all treating physicians. AIM: This article provides the reader with an overview about current cartilage treatment options and their clinical outcome. METHODS: Microfracture is still considered the gold standard in the treatment of small cartilage lesions. Small osteochondral defects can be effectively treated with the autologous osteochondral transplantation system. Larger cartilage defects are successfully treated by autologous membrane-induced chondrogenesis (AMIC) or by membrane-assisted autologous chondrocyte implantation (MACI). CONCLUSION: Despite limitations of current cartilage repair strategies, such procedures can result in short- and mid-term clinical improvement of the patients. Further developments and clinical studies are necessary to improve the long-term outcome following cartilage repair.

MicroRNAs and post-transcriptional regulation of skeletal development.

MicroRNAs (miRNAs) have become integral nodes of post-transcriptional control of genes that confer cellular identity and regulate differentiation. Cell-specific signaling and transcriptional regulation in skeletal biology are extremely dynamic processes that are highly reliant on dose-dependent responses. As such, skeletal cell-determining genes are ideal targets for quantitative regulation by miRNAs. So far, large amounts of evidence have revealed a characteristic temporal miRNA signature in skeletal cell differentiation and confirmed the essential roles that numerous miRNAs play in bone development and homeostasis. In addition, microarray expression data have provided evidence for their role in several skeletal pathologies. Mouse models in which their expression is altered have provided evidence of causal links between miRNAs and bone abnormalities. Thus, a detailed understanding of the function of miRNAs and their tight relationship with bone diseases would constitute a powerful tool for early diagnosis and future therapeutic approaches.