The origin, roles and fate of progenitor cells forming synovial joints during limb skeletogenesis remain largely unclear. Here we produced prenatal and postnatal genetic cell fate-maps by mating ROSA-LacZ-reporter mice with mice expressing Cre-recombinase at prospective joint sites under the control of Gdf5 regulatory sequences (Gdf5-Cre). Reporter-expressing cells initially constituted the interzone, a compact mesenchymal structure representing the first overt sign of joint formation, and displayed a gradient-like distribution along the ventral-to-dorsal axis. The cells expressed genes such as Wnt9a, Erg and collagen IIA, remained predominant in the joint-forming sites over time, gave rise to articular cartilage, synovial lining and other joint tissues, but contributed little if any to underlying growth plate cartilage and shaft. To study their developmental properties more directly, we isolated the joint-forming cells from prospective autopod joint sites using a novel microsurgical procedure and tested them in vitro. The cells displayed a propensity to undergo chondrogenesis that was enhanced by treatment with exogenous rGdf5 but blocked by Wnt9a over-expression. To test roles for such Wnt-mediated anti-chondrogenic capacity in vivo, we created conditional mutants deficient in Wnt/beta-catenin signaling using Col2-Cre or Gdf5-Cre. Synovial joints did form in both mutants; however, the joints displayed a defective flat cell layer normally abutting the synovial cavity and expressed markedly reduced levels of lubricin. In sum, our data indicate that cells present at prospective joint sites and expressing Gdf5 constitute a distinct cohort of progenitor cells responsible for limb joint formation. The cells appear to be patterned along specific limb symmetry axes and rely on local signaling tools to make distinct contributions to joint formation.

To investigate the effectiveness of autologous culture-expanded bone marrow mesenchymal cell transplantation for repairing articular cartilage defects, we transplanted autologous culture-expanded bone marrow mesenchymal cells into nine full-thickness articular cartilage defects of the patello-femoral joints (including two kissing lesions) in the knees of three patients, a 31 year-old female, a 44 year-old male and a 45 year-old male. Three weeks before transplantation, bone marrow blood was aspirated from the iliac crest. Adherent cells were cultured with media containing autologous serum. Single-passaged cells were collected, embedded in a collagen solution (5 x 10(6) cells/ml), placed on a collagen sheet, gelated, transplanted into the defect and covered with autologous periosteum or synovium. Six months after transplantation, the patients' clinical symptoms had improved and the improvements have been maintained over the follow-up periods (17-27 months). Histology of the first patient 12 months after the transplantation revealed that the defect had been repaired with the fibrocartilaginous tissue. Magnetic resonance imaging of the second patient 1 year after transplantation revealed complete coverage of the defect, but we were unable to determine whether or not the material that covered the defects was hyaline cartilage. Autologous bone marrow mesenchymal cells transplantation may be an effective approach to promote the repair of articular cartilage defects.

Among autologous somatic stem cells, bone marrow-derived mesenchymal stem cells (BMSCs) are the most widely used worldwide to repair not only mesenchymal tissues (bone, cartilage) but also many other kinds of tissues, including heart, skin, and liver. Autologous BMSCs are thought to be safe because of the absence of immunological reaction and disease transmission. However, it is possible that they will form tumours during long-term follow-up. In 1988, we transplanted autologous BMSCs to repair articular cartilage, which was the first such trial ever reported. Subsequently we performed this procedure in about 40 patients. Demonstration that neither partial infections nor tumours appeared in these patients provided strong evidence for the safety of autologous BMSC transplantation. Thus, in this study we checked these patients for tumour development and infections. Between January 1998 and November 2008, 41 patients received 45 transplantations. We checked their records until their last visit. We telephoned or mailed the patients who had not visited the clinics recently to establish whether there were any abnormalities in the operated joints. Neither tumours nor infections were observed between 5 and 137 (mean 75) months of follow-up. Autologous BMSC transplantation is a safe procedure and will be widely used around the world.

Recently, novel chiral interactions on 3(10)-helical peptides, of which the helicity is controlled by external chiral stimulus operating on the N-terminus, were proposed as a "noncovalent chiral domino effect (NCDE)" (Inai, Y.; et al. J. Am. Chem. Soc. 2000, 122, 11731. Inai, Y.; et al. J. Am. Chem. Soc. 2002, 124, 2466). The present study clarifies the mechanism for generating the NCDE. For this purpose, achiral nonapeptide (1), H-beta-Ala-(Delta(Z)Phe-Aib)(4)-OMe [Delta(Z)Phe = (Z)-didehydrophenylalanine, Aib = alpha-aminoisobutyric acid], was synthesized. Peptide 1 alone adopts a 3(10)-helical conformation in chloroform. On the basis of the induced CD signals of peptide 1 with chiral additives, chiral acid enabling the predominant formation of a one-handed helix was shown to need at least both carboxyl and urethane groups; that is, Boc-l-amino acid (Boc = tert-butoxycarbonyl) strongly induces a right-handed helix. NMR studies (NH resonance variations, low-temperature measurement, and NOESY) were performed for a CDCl(3) solution of peptide 1 and chiral additive, supporting the view that the N-terminal H-beta-Ala-Delta(Z)Phe-Aib, including the two free amide NH's, captures effectively a Boc-amino acid molecule through three-point interactions. The H-beta-Ala's amino group binds to the carboxyl group to form a salt bridge, while the Aib(3) NH is hydrogen-bonded to either oxygen of the carboxylate group. Subsequently, the free Delta(Z)Phe(2) NH forms a hydrogen bond to the urethane carbonyl oxygen. A semiempirical molecular orbital computation explicitly demonstrated that the dynamic looping complexation is energetically permitted and that the N-terminal segment of a right-handed 3(10)-helix binds more favorably to a Boc-l-amino acid than to the corresponding d-species. In conclusion, the N-terminal segment of a 3(10)-helix, ubiquitous in natural proteins and peptides, possesses the potency of chiral recognition in the backbone itself, furthermore enabling the conversion of the terminally acquired chiral sign and power into a dynamic control of the original helicity and helical stability.

The retinoic acid receptors α, β and γ (RARα, RARβ and RARγ) are nuclear hormone receptors that regulate fundamental processes during embryogenesis, but their roles in skeletal development and growth remain unclear. To study skeletal-specific RAR function, we created conditional mouse mutants deficient in RAR expression in cartilage. We find that mice deficient in RARα and RARγ (or RARβ and RARγ) exhibit severe growth retardation obvious by about 3 weeks postnatally. Their growth plates are defective and, importantly, display a major drop in aggrecan expression and content. Mice deficient in RARα and RARβ, however, are virtually normal, suggesting that RARγ is essential. In good correlation, we find that RARγ is the most strongly expressed RAR in mouse growth plate and its expression characterizes the proliferative and pre-hypertrophic zones where aggrecan is strongly expressed also. By being avascular, those zones lack endogenous retinoids and thus RARγ is likely to exert ligand-less repressor function. Indeed, our data indicate that: aggrecan production is enhanced by RARγ over-expression in chondrocytes under retinoid-free culture conditions; production is further boosted by corepressor Zac1 or pharmacologic agents that enhance RAR repressor function; and RAR/Zac1 function on aggrecan expression may involve Sox proteins. In sum, our data reveal that RARs, and RARγ in particular, exert previously unappreciated roles in growth plate function and skeletal growth and regulate aggrecan expression and content. Since aggrecan is critical for growth plate function, its deficiency in RAR-mutant mice is likely to have contributed directly to their growth retardation.

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Indian hedgehog (Ihh) is essential for embryonic mandibular condylar growth and disc primordium formation. To determine whether it regulates those processes during post-natal life, we ablated Ihh in cartilage of neonatal mice and assessed the consequences on temporomandibular joint (TMJ) growth and organization over age. Ihh deficiency caused condylar disorganization and growth retardation and reduced polymorphic cell layer proliferation. Expression of Sox9, Runx2, and Osterix was low, as was that of collagen II, collagen I, and aggrecan, thus altering the fibrocartilaginous nature of the condyle. Though a disc formed, it exhibited morphological defects, partial fusion with the glenoid bone surface, reduced synovial cavity space, and, unexpectedly, higher lubricin expression. Analysis of the data shows, for the first time, that continuous Ihh action is required for completion of post-natal TMJ growth and organization. Lubricin overexpression in mutants may represent a compensatory response to sustain TMJ movement and function.