Human chondrogenic paraxial mesoderm, directed specification and prospective isolation from pluripotent stem cells

Directed specification and prospective isolation of chondrogenic paraxial mesoderm progeny from human pluripotent stem (PS) cells have not yet been achieved. Here we report the successful generation of KDR−PDGFRα+ progeny expressing paraxial mesoderm genes and the mesendoderm reporter MIXL1-GFP in a chemically defined medium containing the canonical WNT signaling activator, BMP-inhibitor, and the Nodal/Activin/TGFβ signaling controller. Isolated (GFP+)KDR−PDGFRα+ mesoderm cells were sensitive to sequential addition of the three chondrogenic factors PDGF, TGFβ and BMP. Under these conditions, the cells showed robust chondrogenic activity in micromass culture, and generated a hyaline-like translucent cartilage particle in serum-free medium. In contrast, both STRO1+ mesenchymal stem/stromal cells from adult human marrow and mesenchymal cells spontaneously arising from hPS cells showed a relatively weaker chondrogenic response in vitro, and formed more of the fibrotic cartilage particles. Thus, hPS cell-derived KDR−PDGFRα+ paraxial mesoderm-like cells have potential in engineered cartilage formation and cartilage repair

In-vivo generation of bone via endochondral ossification by in-vitro chondrogenic priming of adult human and rat mesenchymal stem cells

Background: Bone grafts are required to repair large bone defects after tumour resection or large tr

Association between expression of the Bone morphogenetic proteins 2 and 7 in the repair of circumscribed cartilage lesions with clinical outcome

<p>Abstract</p> <p>Background</p> <p>Although there is much known about the role of BMPs in cartilage metabolism reliable data about the <it>in vivo </it>regulation in natural and surgically induced cartilage repair are still missing.</p> <p>Methods</p> <p>Lavage fluids of knee joints of 47 patients were collected during surgical therapy. 5 patients had no cartilage lesion and served as a control group, the other 42 patients with circumscribed cartilage defects were treated by microfracturing (19) or by an Autologous Chondrocyte Implantation (23). The concentrations of BMP-2 and BMP-7 were determined by ELISA. The clinical status was evaluated using the IKDC Score prior to and 1 year following the operation.</p> <p>Results</p> <p>High level expression in the control group was found for BMP-2, concentrations of BMP-7 remained below detection levels. No statistical differences could be detected in concentrations of BMP-2 or BMP-7 in the lavage fluids of knees with cartilage lesions compared to the control group. Levels of BMP-7 did not change after surgical cartilage repair, whereas concentrations of BMP-2 statistically significant increased after the intervention (p < 0.001). The clinical outcome following cartilage regenerating surgery increased after 1 year by 29% (p < 0.001). The difference of the IKDC score after 1 year and prior to the operation was used to quantify the degree of improvement following surgery. This difference statistically significant correlated with initial BMP-2 (R = 0.554, p < 0.001) but not BMP-7 (R = 0.031, n.s.) levels in the knee joints.</p> <p>Conclusions</p> <p>BMP-2 seems to play an important role in surgically induced cartilage repair; synovial expression correlates with the clinical outcome.</p

Cryopreservation Effect on Proliferative and Chondrogenic Potential of Human Chondrocytes Isolated from Superficial and Deep Cartilage

[Abstract] Objectives: To compare the proliferative and chondrogenic potential of fresh and frozen chondrocytes isolated from superficial and deep articular cartilage biopsies. Materials and Methodology: The study included 12 samples of fresh and frozen healthy human knee articular cartilage. Cell proliferation was tested at 3, 6 and 9 days. Studies of mRNA quantification, protein expression and immunofluorescence for proliferation and chondrogenic markers were performed. Results: Stimulation of fresh and frozen chondrocytes from both superficial and deep cartilage with fetal bovine serum produced an increase in the proliferative capacity compared to the non-stimulated control group. In the stimulated fresh cells group, the proliferative capacity of cells from the deep biopsy was greater than that from cells from the superficial biopsy (0.046 vs 0.028, respectively, p<0.05). There was also a significant difference between the proliferative capacity of superficial zone fresh (0.028) and frozen (0.051) chondrocytes (p<0.05). CCND1 mRNA and protein expression levels, and immunopositivity for Ki67 revealed a higher proliferative capacity for fresh articular chondrocytes from deep cartilage. Regarding the chondrogenic potential, stimulated fresh cells showed higher SOX9 and Col II expression in chondrocytes from deep than from superficial zone (p<0.05, T student test). Conclusions: The highest rate of cell proliferation and chondrogenic potential of fresh chondrocytes was found in cells obtained from deep cartilage biopsies, whereas there were no statistically significant differences in proliferative and chondrogenic capacity between biopsy origins with frozen chondrocytes. These results indicate that both origin and cryopreservation affect the proliferative and chondrogenic potential of chondrocytes.Servizo Galego de Saúde; PS07/84Instituto de Salud Carlos III; CIBER BBN CB06-01-0040Ministerio Ciencia e Innovacion; PLE2009-0144Ministerio Ciencia e Innovación; PI 08/202

Clinically Translatable Cell Tracking and Quantification by MRI in Cartilage Repair Using Superparamagnetic Iron Oxides

Background: Articular cartilage has very limited intrinsic regenerative capacity, making cell-based therapy a tempting approach for cartilage repair. Cell tracking can be a major step towards unraveling and improving the repair process of these therapies. We studied superparamagnetic iron oxides (SPIO) for labeling human bone marrow-derived mesenchymal stem cells (hBMSCs) regarding effectivity, cell viability, long term metabolic cell activity, chondrogenic differentiation and hBMSC secretion profile. We additionally examined the capacity of synovial cells to endocytose SPIO from dead, labeled cells, together with the use of magnetic resonance imaging (MRI) for intra-articular visualization and quantification of SPIO labeled cells. Methodology/Prinicipal Findings: Efficacy and various safety aspects of SPIO cell labeling were determined using appropriate assays. Synovial SPIO re-uptake was investigated in vitro by co-labeling cells with SPIO and green fluorescent protein (GFP). MRI experiments were performed on a clinical 3.0T MRI scanner. Two cell-based cartilage repair techniques were mimicked for evaluating MRI traceability of labeled cells: intra-articular cell injection and cell implantation in cartilage defects. Cells were applied ex vivo or in vitro in an intra-articular environment and immediately scanned. SPIO labeling was effective and did not impair any of the studied safety aspects, including hBMSC secretion profile. SPIO from dead, labeled cells could be taken up by synovial cells. Both injected and implanted SPIO-labeled cells could accurately be visualized by MRI in a clinically relevant sized joint model using clinically applied cell doses. Finally, we quantified the amount of labeled cells seeded in cartilage defects using MR-based relaxometry. Conclusions: SPIO labeling appears to be safe without influencing cell behavior. SPIO labeled cells can be visualized in an intra-articular environment and quantified when seeded in cartilage defects.Biomechanical EngineeringMechanical, Maritime and Materials Engineerin

The Inhibitory Effect of Salmon Calcitonin on Tri-Iodothyronine Induction of Early Hypertrophy in Articular Cartilage

Salmon calcitonin has chondroprotective effect both in vitro and in vivo, and is therefore being tested as a candidate drug for cartilage degenerative diseases. Recent studies have indicated that different chondrocyte phenotypes may express the calcitonin receptor (CTR) differentially. We tested for the presence of the CTR in chondrocytes from tri-iodothyronin (T3)-induced bovine articular cartilage explants. Moreover, investigated the effects of human and salmon calcitonin on the explants.Early chondrocyte hypertrophy was induced in bovine articular cartilage explants by stimulation over four days with 20 ng/mL T3. The degree of hypertrophy was investigated by molecular markers of hypertrophy (ALP, IHH, COLX and MMP13), by biochemical markers of cartilage turnover (C2M, P2NP and AGNxII) and histology. The expression of the CTR was detected by qPCR and immunohistochemistry. T3-induced explants were treated with salmon or human calcitonin. Calcitonin down-stream signaling was measured by levels of cAMP, and by the molecular markers.Compared with untreated control explants, T3 induction increased expression of the hypertrophic markers (p<0.05), of cartilage turnover (p<0.05), and of CTR (p<0.01). Salmon, but not human, calcitonin induced cAMP release (p<0.001). Salmon calcitonin also inhibited expression of markers of hypertrophy and cartilage turnover (p<0.05).T3 induced early hypertrophy of chondrocytes, which showed an elevated expression of the CTR and was thus a target for salmon calcitonin. Molecular marker levels indicated salmon, but not human, calcitonin protected the cartilage from hypertrophy. These results confirm that salmon calcitonin is able to modulate the CTR and thus have chondroprotective effects