Maurizio Pacifici

Developmental regulation of Wnt/β-catenin signals is required for growth plate assembly, cartilage integrity, and endochondral ossification

Studies have suggested that continuous Wnt/β-catenin signaling in nascent cartilaginous skeletal elements blocks chondrocyte hypertrophy and endochondral ossification, whereas signaling starting at later stages stimulates hypertrophy and ossification, indicating that Wnt/β-catenin roles are developmentally regulated. To test this conclusion further, we created transgenic mice expressing a fusion mutant protein of β-catenin and LEF (CA-LEF) in nascent chondrocytes. Transgenic mice had severe skeletal defects, particularly in limbs. Growth plates were totally disorganized, lacked maturing chondrocytes expressing Indian hedgehog and collagen X, and failed to undergo endochondral ossification. Interestingly, the transgenic cartilaginous elements were ill defined, intermingled with surrounding connective and vascular tissues, and even displayed abnormal joints. However, when activated β-catenin mutant (Δ-β-catenin) was expressed in chondrocytes already engaged in maturation such as those present in chick limbs, chondrocyte maturation and bone formation were greatly enhanced. Differential responses to Wnt/β-catenin signaling were confirmed in cultured chondrocytes. Activation in immature cells blocked maturation and actually de-stabilized their phenotype, as revealed by reduced expression of chondrocyte markers, abnormal cytoarchitecture, and loss of proteoglycan matrix. Activation in mature cells instead stimulated hypertrophy, matrix mineralization, and expression of terminal markers such as metalloprotease (MMP)-13 and vascular endothelial growth factor. Because proteoglycans are crucial for cartilage function, we tested possible mechanisms for matrix loss. Δ-β-Catenin expression markedly increased expression of MMP-2, MMP-3, MMP-7, MMP-9, MT3-MMP, and ADAMTS5. In conclusion, Wnt/β-catenin signaling regulates chondrocyte phenotype, maturation, and function in a developmentally regulated manner, and regulated action by this pathway is critical for growth plate organization, cartilage boundary definition, and endochondral ossification.

A distinct cohort of progenitor cells participates in synovial joint and articular cartilage formation during mouse limb skeletogenesis

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.

Cellular and molecular mechanisms of synovial joint and articular cartilage formation.

Abstract:  Synovial joints and articular cartilage play crucial roles in the skeletal function, but relatively little is actually known about their embryonic development. Here we first focused on the interzone, a thin mesenchymal cell layer forming at future joint sites that is widely thought to be critical for joint and articular cartilage development. To determine interzone cell origin and fate, we microinjected the vital fluorescent dye DiI at several peri‐joint sites in chick limbs and monitored the behavior and fate of labeled cells over time. Peri‐joint mesenchymal cells located immediately adjacent to incipient joints migrated, became part of the interzone, and were eventually found in epiphyseal articular layer and joint capsule. Interzone cells isolated and reared in vitro expressed typical phenotypic markers, including GDF‐5, Wnt‐14, and CD‐44, and differentiated into chondrocytes over time. To determine the molecular mechanisms of articular chondrocyte formation, we carried out additional studies on the ets transcription factor family member ERG and its alternatively spliced variant C‐1‐1 that we previously found to be expressed in developing avian articular chondrocytes. We cloned the human counterpart of avian C‐1‐1 (ERGp55Δ81) and conditionally expressed it in transgenic mice under cartilage‐specific Col2 gene promotor‐enhancer control. The entire transgenic mouse limb chondrocyte population exhibited an immature articular‐like phenotype and a virtual lack of growth plate formation and chondrocyte maturation compared to wild‐type littermate. Together, our studies reveal that peri‐joint mesenchymal cells take part in interzone and articular layer formation, interzone cells can differentiate into chondrocytes, and acquisition of a permanent articular chondrocyte phenotype is aided and perhaps dictated by ets transcription factor ERG.

Temporomandibular joint formation and condyle growth require Indian hedgehog signaling

The temporomandibular joint (TMJ) is essential for jaw function, but the mechanisms regulating its development remain poorly understood. Because Indian hedgehog (Ihh) regulates trunk and limb skeletogenesis, we studied its possible roles in TMJ development. In wild‐type mouse embryos, Ihh expression was already strong in condylar cartilage by embryonic day (E) 15.5, and expression of Ihh receptors and effector genes (Gli1, Gli2, Gli3, and PTHrP) indicated that Ihh range of action normally reached apical condylar tissue layers, including polymorphic chondroprogenitor layer and articular disc primordia. In Ihh−/− embryos, TMJ development was severely compromised. Condylar cartilage growth, polymorphic cell proliferation, and PTHrP expression were all inhibited, and growth plate organization and chondrocyte gene expression patterns were abnormal. These severe defects were partially corrected in double Ihh−/−/Gli3−/− mutants, signifying that Ihh action is normally modulated and delimited by Gli3 and Gli3R in particular. Both single and double mutants, however, failed to form an articular disc primordium, normally appreciable as an independent condensation between condylar apex and neighboring developing temporal bone in wild‐type. This failure persisted at later stages, leading to complete absence of a normal functional disc and lubricin‐expressing joint cavities. In summary, Ihh is very important for TMJ development, where it appears to regulate growth and elongation events, condylar cartilage phenotype, and chondroprogenitor cell function. Absence of articular disc and joint cavities in single and double mutants points to irreplaceable Ihh roles in formation of those critical TMJ components. Developmental Dynamics 236:426–434, 2007.

Concave pit-containing scaffold surfaces improve stem cell-derived osteoblast performance and lead to significant bone tissue formation.

Background Scaffold surface features are thought to be important regulators of stem cell performance and endurance in tissue engineering applications, but details about these fundamental aspects of stem cell biology remain largely unclear. Methodology and Findings In the present study, smooth clinical-grade lactide-coglyolic acid 85:15 (PLGA) scaffolds were carved as membranes and treated with NMP (N-metil-pyrrolidone) to create controlled subtractive pits or microcavities. Scanning electron and confocal microscopy revealed that the NMP-treated membranes contained: (i) large microcavities of 80–120 µm in diameter and 40–100 µm in depth, which we termed primary; and (ii) smaller microcavities of 10–20 µm in diameter and 3–10 µm in depth located within the primary cavities, which we termed secondary. We asked whether a microcavity-rich scaffold had distinct bone-forming capabilities compared to a smooth one. To do so, mesenchymal stem cells derived from human dental pulp were seeded onto the two types of scaffold and monitored over time for cytoarchitectural characteristics, differentiation status and production of important factors, including bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF). We found that the microcavity-rich scaffold enhanced cell adhesion: the cells created intimate contact with secondary microcavities and were polarized. These cytological responses were not seen with the smooth-surface scaffold. Moreover, cells on the microcavity-rich scaffold released larger amounts of BMP-2 and VEGF into the culture medium and expressed higher alkaline phosphatase activity. When this type of scaffold was transplanted into rats, superior bone formation was elicited compared to cells seeded on the smooth scaffold. Conclusion In conclusion, surface microcavities appear to support a more vigorous osteogenic response of stem cells and should be used in the design of therapeutic substrates to improve bone repair and bioengineering applications in the future.

Retinoic acid receptors are required for skeletal growth, matrix homeostasis and growth plate function in postnatal mouse

The retinoic acid receptors alpha, beta and gamma (RARalpha, RARbeta and RARgamma) 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 RARalpha and RARgamma (or RARbeta and RARgamma) 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 RARalpha and RARbeta, however, are virtually normal, suggesting that RARgamma is essential. In good correlation, we find that RARgamma 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 as indicated by previous RARE reporter mice and our direct biochemical measurements and thus, RARgamma is likely to exert ligand-less repressor function. Indeed, our data indicate that: aggrecan production is enhanced by RARgamma over-expression in chondrocytes under retinoid-free culture conditions; production is further boosted by co-repressor 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 RARgamma 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.

Transient Activation of Wnt/β-Catenin Signaling Induces Abnormal Growth Plate Closure and Articular Cartilage Thickening in Postnatal Mice

Wnt/beta-catenin signaling is required for skeletal development and organization and for function of the growth plate and articular cartilage. To further clarify these roles and their possible pathophysiological importance, we created a new transgenic mouse model in which Wnt/beta-catenin signaling can be activated in cartilage for specific periods of time. These transgenic mice expressed a constitutive active form of beta-catenin fused to a modified estrogen receptor ligand-binding domain under the control of cartilage-specific collagen 11alpha2 promoter/enhancer. Transient Wnt/beta-catenin signaling activation in young adult mice by tamoxifen injections induced growth retardation and severe deformities in knee joints. Tibial and femoral growth plates displayed an excessive number of apoptotic cells and eventually underwent abnormal regression. Articular cartilage exhibited an initial acute loss of proteoglycan matrix that was followed by increases in thickness, cell density, and cell proliferation. In reciprocal studies, we found that conditional ablation of beta-catenin in postnatal mice using a Col2-CreER strategy led to hypocellularity in articular cartilage, growth plate disorganization, and a severe reduction in bone volume. Together, these data provide evidence that Wnt/beta-catenin signaling has important and distinct roles in growth plate and articular cartilage and that postnatal dysregulation of this signaling pathway causes diverse structural and functional changes in the two cartilaginous structures.

Indian hedgehog and syndecans-3 coregulate chondrocyte proliferation and function during chick limb skeletogenesis

Hedgehog proteins exert critical roles in embryogenesis and require heparan sulfate proteoglycans (HS‐PGs) for action. Indian hedgehog (Ihh) is produced by prehypertrophic chondrocytes in developing long bones and regulates chondrocyte proliferation and other events, but it is not known whether it requires HS‐PGs for function. Because the HS‐PG syndecan‐3 is preferentially expressed by proliferating chondrocytes, we tested whether it mediates Ihh action. Primary chick chondrocyte cultures were treated with recombinant Ihh (rIhh‐N) in absence or presence of heparinase I or syndecan‐3 neutralizing antibodies. While rIhh‐N stimulated proliferation in control cultures, it failed to do so in heparinase‐ or antibody‐treated cultures. In reciprocal gain‐of‐function studies, chondrocytes were made to overexpress syndecan‐3 by an RCAS viral vector. Cells became more responsive to rIhh‐N, but even this response was counteracted by heparinase or antibody treatment. To complement the in vitro data, RCAS viral particles were microinjected in day 4–5 chick wing buds and effects of syndecan‐3 misexpression were monitored over time. Syndecan‐3 misexpression led to widespread chondrocyte proliferation and, interestingly, broader expression and distribution of Ihh. In addition, the syndecan‐3 misexpressing skeletal elements were short, remained cartilaginous, lacked osteogenesis, and exhibited a markedly reduced expression of collagen X and osteopontin, products characteristic of hypertrophic chondrocytes and bone cells. The data are the first to indicate that Ihh action in chondrocyte proliferation involves syndecan‐3 and to identify a specific member of the syndecan family as mediator of hedgehog function. Developmental Dynamics 229:607–617, 2004.

Indian Hedgehog Roles in Post-natal TMJ Development and Organization:

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.

Synovial Joint Formation during Mouse Limb Skeletogenesis: Roles of Indian Hedgehog Signaling

Abstract:  Indian hedgehog (Ihh) has been previously found to regulate synovial joint formation. To analyze mechanisms, we carried out morphological, molecular, and cell fate map analyses of interzone and joint development in wild‐type and Ihh−/− mouse embryo long bones. We found that Ihh−/− cartilaginous digit anlagen remained fused and lacked interzones or mature joints, whereas wrist skeletal elements were not fused but their joints were morphologically abnormal. E14.5 and E17.5 wild‐type digit and ankle prospective joints expressed hedgehog target genes including Gli1 and Gli2 and interzone‐associated genes including Gdf5, Erg, and tenascin‐C, but expression of all these genes was barely detectable in mutant joints. For cell fate map analysis of joint progenitor cells, we mated Gdf5‐Cre+/−/Rosa R26R+/− double transgenic mice with heterozygous Ihh+/− mice and monitored reporter β‐galactosidase activity and gene expression in triple‐transgenic progeny. In control Gdf5‐Cre+/−/R26R+/−/Ihh+/− limbs, reporter‐positive cells were present in developing interzones, articulating layers, and synovial lining tissue and absent from underlying growth plates. In mutant Gdf5‐Cre+/−/R26R+/−/Ihh−/− specimens, reporter‐positive cells were present also. However, the cells were mostly located around the prospective and uninterrupted digit joint sites and, interestingly, still expressed Erg, tenascin‐C, and Gdf5. Topographical analysis revealed that interzone and associated cells were not uniformly distributed, but were much more numerous ventrally. A similar topographical bias was seen for cavitation process and capsule primordia formation. In sum, Ihh is a critical and possibly direct regulator of joint development. In its absence, distribution and function of Gdf5‐expressing interzone‐associated cells are abnormal, but their patterning at prospective joint sites still occurs. The joint‐forming functions of the cells appear to normally involve a previously unsuspected asymmetric distribution along the ventral‐to‐dorsal plane of the developing joint.