Abstract Introduction The purpose of this study was to compare healing after root-end surgery by using grey mineral trioxide aggregate (MTA) and EndoSequence Root Repair Material (RRM) as root-end ...filling material in an animal model. Methods Apical periodontitis was induced in 55 mandibular premolars of 4 healthy beagle dogs. After 6 weeks, root-end surgeries were performed by using modern microsurgical techniques. Two different root-end filling materials were used, grey MTA and RRM. Six months after surgery, healing of the periapical area was assessed by periapical radiographs, cone-beam computed tomography (CBCT), micro computed tomography (CT), and histology. Results Minimal or no inflammatory response was observed in the majority of periapical areas regardless of the material. The degree of inflammatory infiltration and cortical plate healing were not significantly different between the 2 materials. However, a significantly greater root-end surface area was covered by cementum-like, periodontal ligament–like tissue, and bone in RRM group than in MTA group. When evaluating with periapical radiographs, complete healing rate in RRM and MTA groups was 92.6% and 75%, respectively, and the difference was not statistically significant ( P = .073). However, on CBCT and micro CT images, RRM group demonstrated significantly superior healing on the resected root-end surface and in the periapical area ( P = .000 to .027). Conclusions Like MTA, RRM is a biocompatible material with good sealing ability. However, in this animal model RRM achieved a better tissue healing response adjacent to the resected root-end surface histologically. The superior healing tendency associated with RRM could be detected by CBCT and micro CT but not periapical radiography.
Limb synovial joints are composed of distinct tissues, but it is unclear which progenitors produce those tissues and how articular cartilage acquires its functional postnatal organization ...characterized by chondrocyte columns, zone-specific cell volumes and anisotropic matrix. Using novel Gdf5CreERT2 (Gdf5-CE), Prg4-CE and Dkk3-CE mice mated to R26-Confetti or single-color reporters, we found that knee joint progenitors produced small non-migratory progenies and distinct local tissues over prenatal and postnatal time. Stereological imaging and quantification indicated that the columns present in juvenile-adult tibial articular cartilage consisted of non-daughter, partially overlapping lineage cells, likely reflecting cell rearrangement and stacking. Zone-specific increases in cell volume were major drivers of tissue thickening, while cell proliferation or death played minor roles. Second harmonic generation with 2-photon microscopy showed that the collagen matrix went from being isotropic and scattered at young stages to being anisotropic and aligned along the cell stacks in adults. Progenitor tracing at prenatal or juvenile stages showed that joint injury provoked a massive and rapid increase in synovial Prg4+ and CD44+/P75+ cells some of which filling the injury site, while neighboring chondrocytes appeared unresponsive. Our data indicate that local cell populations produce distinct joint tissues and that articular cartilage growth and zonal organization are mainly brought about by cell volume expansion and topographical cell rearrangement. Synovial Prg4+ lineage progenitors are exquisitely responsive to acute injury and may represent pioneers in joint tissue repair.
•Local progenitor cells emerging at distinct sites and times give rise to distinct joint tissues.•Articular cartilage zonal structure mainly reflects cell rearrangement into stacks.•Postnatal cartilage thickening is driven by zonal-specific increases in cell volume.•Synovial progenitors with a Prg4 lineage signature appear exquisitely responsive to tissue injury.
Heterotopic ossification (HO) is a common, potentially debilitating pathology that is instigated by inflammation caused by tissue damage or other insults, which is followed by chondrogenesis, ...osteogenesis, and extraskeletal bone accumulation. Current remedies are not very effective and have side effects, including the risk of triggering additional HO. The TGF-β family member activin A is produced by activated macrophages and other inflammatory cells and stimulates the intracellular effectors SMAD2 and SMAD3 (SMAD2/3). Because HO starts with inflammation and because SMAD2/3 activation is chondrogenic, we tested whether activin A stimulated HO development. Using mouse models of acquired intramuscular and subdermal HO, we found that blockage of endogenous activin A by a systemically administered neutralizing antibody reduced HO development and bone accumulation. Single-cell RNA-seq analysis and developmental trajectories showed that the antibody treatment reduced the recruitment of
skeletal progenitors, many of which also expressed the gene encoding activin A (
), to HO sites. Gain-of-function assays showed that activin A enhanced the chondrogenic differentiation of progenitor cells through SMAD2/3 signaling, and inclusion of activin A in HO-inducing implants enhanced HO development in vivo. Together, our data reveal that activin A is a critical upstream signaling stimulator of acquired HO in mice and could represent an effective therapeutic target against forms of this pathology in patients.
The joints are a diverse group of skeletal structures, and their genesis, morphogenesis, and acquisition of specialized tissues have intrigued biologists for decades. Here we review past and recent ...studies on important aspects of joint development, including the roles of the interzone and morphogenesis of articular cartilage. Studies have documented the requirement of interzone cells in limb joint initiation and formation of most, if not all, joint tissues. We highlight these studies and also report more detailed interzone dissection experiments in chick embryos. Articular cartilage has always received special attention owing to its complex architecture and phenotype and its importance in long-term joint function. We pay particular attention to mechanisms by which neonatal articular cartilage grows and thickens over time and eventually acquires its multi-zone structure and becomes mechanically fit in adults. These and other studies are placed in the context of evolutionary biology, specifically regarding the dramatic changes in limb joint organization during transition from aquatic to land life. We describe previous studies, and include new data, on the knee joints of aquatic axolotls that unlike those in higher vertebrates, are not cavitated, are filled with rigid fibrous tissues and resemble amphiarthroses. We show that when axolotls metamorph to life on land, their intra-knee fibrous tissue becomes sparse and seemingly more flexible and the articular cartilage becomes distinct and acquires a tidemark. In sum, there have been considerable advances toward a better understanding of limb joint development, biological responsiveness, and evolutionary influences, though much remains unclear. Future progress in these fields should also lead to creation of new developmental biology-based tools to repair and regenerate joint tissues in acute and chronic conditions.
Long bone development involves the embryonic formation of a primary ossification center (POC) in the incipient diaphysis followed by postnatal development of a secondary ossification center (SOC) at ...each epiphysis. Studies have elucidated major basic mechanisms of POC development, but relatively little is known about SOC development. To gain insights into SOC formation, we used Col2‐Cre Rosa‐tdTomato (Col2/Tomato) reporter mice and found that their periarticular region contained numerous Tomato‐positive lineage cells expressing much higher Tomato fluorescence (termed TomatoH) than underlying epiphyseal chondrocytes (termed TomatoL). With time, the TomatoH cells became evident at the SOC invagination site and cartilage canal, increased in number in the expanding SOC, and were present as mesenchymal lineage cells in the subchondral bone. These data were verified in two mouse lineage tracing models, Col2‐CreER Rosa‐tdTomato and Gli1‐CreER Rosa‐tdTomato. In vitro tests showed that the periarticular TomatoH cells from Col2/Tomato mice contained mesenchymal progenitors with multidifferentiation abilities. During canal initiation, the cells expressed vascular endothelial growth factor (VEGF) and migrated into epiphyseal cartilage ahead of individual or clusters of endothelial cells, suggesting a unique role in promoting vasculogenesis. Later during SOC expansion, chondrocytes in epiphyseal cartilage expressed VEGF, and angiogenic blood vessels preceded TomatoH cells. Gene expression analyses of microdissected samples revealed upregulation of MMPs in periarticular cells at the invagination site and suggested potential roles for novel kinase and growth factor signaling pathways in regulating SOC canal initiation. In summary, our data indicate that the periarticular region surrounding epiphyseal cartilage contains mesenchymal progenitors that initiate SOC development and form subchondral bone. Stem Cells 2019;37:677–689
Schematic of secondary ossification center canal formation. Secondary ossification center development is initiated by invasion of periarticular, multipotent mesenchymal progenitors from discrete sites along the epiphyseal surface into the underlying cartilage. (i) At the time of secondary ossification center initiation, these periarticular cells, but not surrounding chondrocytes, express vascular endothelial growth factor, important for recruiting individual endothelial cells (vasculogenesis) and existing vessels (angiogenesis) from the surrounding vasculature. (ii) Invasion of the secondary ossification center canal is associated with extensive proliferation within the canal, degradation of chondrocyte matrix and chondrocyte apoptosis. Transition from canal invasion to expansion is associated with a redistribution of vascular endothelial growth factor expression, from cells within the canal and along the leading edge, to surrounding hypertrophic chondrocytes. (iii) This transition is associated with a transformation from periarticular cell‐led invasion to blood vessel‐led secondary ossification center expansion. Secondary ossification center expansion continues, eventually giving rise to the epiphyseal marrow cavity. (iv) Mesenchymal lineage cells (i.e., osteoblasts, osteocytes, perivascular cells, adipocytes, etc.) within the epiphyseal region are derived from periarticular progenitor cells present within the invading secondary ossification center canal.
The temporomandibular joint (TMJ) is an intricate structure composed of the mandibular condyle, articular disc, and glenoid fossa in the temporal bone. Apical condylar cartilage is classified as a ...secondary cartilage, is fibrocartilaginous in nature, and is structurally distinct from growth plate and articular cartilage in long bones. Condylar cartilage is organized in distinct cellular layers that include a superficial layer that produces lubricants, a polymorphic/progenitor layer that contains stem/progenitor cells, and underlying layers of flattened and hypertrophic chondrocytes. Uniquely, progenitor cells reside near the articular surface, proliferate, undergo chondrogenesis, and mature into hypertrophic chondrocytes. During the past decades, there has been a growing interest in the molecular mechanisms by which the TMJ develops and acquires its unique structural and functional features. Indian hedgehog (Ihh), which regulates skeletal development including synovial joint formation, also plays pivotal roles in TMJ development and postnatal maintenance. This review provides a description of the many important recent advances in Hedgehog (Hh) signaling in TMJ biology. These include studies that used conventional approaches and those that analyzed the phenotype of tissue-specific mouse mutants lacking Ihh or associated molecules. The recent advances in understanding the molecular mechanism regulating TMJ development are impressive and these findings will have major implications for future translational medicine tools to repair and regenerate TMJ congenital anomalies and acquired diseases, such as degenerative damage in TMJ osteoarthritic conditions.
Meniscal tears are associated with a high risk of osteoarthritis but currently have no disease-modifying therapies. Using a Gli1 reporter line, we found that Gli1
cells contribute to the development ...of meniscus horns from 2 weeks of age. In adult mice, Gli1
cells resided at the superficial layer of meniscus and expressed known mesenchymal progenitor markers. In culture, meniscal Gli1
cells possessed high progenitor activities under the control of Hh signal. Meniscus injury at the anterior horn induced a quick expansion of
cells. Normally, meniscal tissue healed slowly, leading to cartilage degeneration. Ablation of Gli1
cells further hindered this repair process. Strikingly, intra-articular injection of Gli1
meniscal cells or an Hh agonist right after injury accelerated the bridging of the interrupted ends and attenuated signs of osteoarthritis. Taken together, our work identified a novel progenitor population in meniscus and proposes a new treatment for repairing injured meniscus and preventing osteoarthritis.
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/β-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.
During limb skeletogenesis the cartilaginous long bone anlagen and their growth plates become delimited by perichondrium with which they interact functionally. Yet, little is known about how, despite ...being so intimately associated with cartilage, perichondrium acquires and maintains its distinct phenotype and exerts its border function. Because perichondrium becomes deranged and interrupted by cartilaginous outgrowths in Hereditary Multiple Exostoses (HME), a pediatric disorder caused by EXT mutations and consequent heparan sulfate (HS) deficiency, we asked whether EXT genes and HS normally have roles in establishing its phenotype and function. Indeed, conditional Ext1 ablation in perichondrium and lateral chondrocytes flanking the epiphyseal region of mouse embryo long bone anlagen – a region encompassing the groove of Ranvier – caused ectopic cartilage formation. A similar response was observed when HS function was disrupted in long bone anlagen explants by genetic, pharmacological or enzymatic means, a response preceded by ectopic BMP signaling within perichondrium. These treatments also triggered excess chondrogenesis and cartilage nodule formation and overexpression of chondrogenic and matrix genes in limb bud mesenchymal cells in micromass culture. Interestingly, the treatments disrupted the peripheral definition and border of the cartilage nodules in such a way that many nodules overgrew and fused with each other into large amorphous cartilaginous masses. Interference with HS function reduced the physical association and interactions of BMP2 with HS and increased the cell responsiveness to endogenous and exogenous BMP proteins. In sum, Ext genes and HS are needed to establish and maintain perichondrium's phenotype and border function, restrain pro-chondrogenic signaling proteins including BMPs, and restrict chondrogenesis. Alterations in these mechanisms may contribute to exostosis formation in HME, particularly at the expense of regions rich in progenitor cells including the groove of Ranvier.
► Conditional deletion of Ext1 in perichondrium causes ectopic cartilage formation. ► Interfering with heparan sulfate function increases chondrogenic potential in vitro. ► Responsiveness to signaling factors such as BMP2 can increase after HS interference. ► Ext genes and heparan sulfate are needed to maintain the perichondrium phenotype.
Condylar articular cartilage in mouse temporomandibular joint develops from progenitor cells near the articulating surface that proliferate, undergo chondrogenesis and mature into hypertrophic ...chondrocytes. However, it remains unclear how these processes are regulated, particularly postnatally. Here we focused on the apical polymorphic layer rich in progenitors and asked whether the phenotype and fate of the cells require signaling by Indian hedgehog (Ihh) previously studied in developing long bones. In condyles in newborn mice, the apical polymorphic/progenitor cell layer was ~10 cell layer-thick and expressed the articular matrix marker Tenascin-C (Tn-C), and the underlying thick cell layer expressed Tn-C as well as the chondrogenic master regulator Sox9. By 1 month, condylar cartilage had gained its full width, but became thinner along its main longitudinal axis and displayed hypertrophic chondrocytes. By 3 months, articular cartilage consisted of a 2–3 cell layer-thick zone of superficial cells and chondroprogenitors expressing both Tn-C and Sox9 and a bottom zone of chondrocytes displaying vertical matrix septa. EdU cell tracing in juvenile mice revealed that conversion of chondroprogenitors into chondrocytes and hypertrophic chondrocytes required about 48 and 72 h, respectively. Notably, EdU injection in 3 month-old mice labeled both progenitors and maturing chondrocytes by 96 h. Conditional ablation of Ihh in juvenile/early adult mice compromised chondroprogenitor organization and function and led to reduced chondroprogenitor and chondrocyte proliferation. The phenotype of mutant condyles worsened over time as indicated by apoptotic chondrocyte incidence, ectopic chondrocyte hypertrophy, chondrocyte column derangement and subchondral bone deterioration. In micromass cultures of condylar apical cells, hedgehog (Hh) treatment stimulated chondrogenesis and alkaline phosphatase (APase) activity, while treatment with HhAntag inhibited both. Our findings indicate that the chondroprogenitor layer is continuously engaged in condylar growth postnatally and its organization and functioning depend on hedgehog signaling.
•Development of progentor layer and structure of condylar articular cartilage are clarified during postnatal TMJ development.•Chondroprogenitors and their fate are clarified in postnatal condylar articular cartilage.•We evaluate gene recombination in postnatal TMJs using Aggrecan (Agc)-CreER;R26-tdTomato reporter mice.•The role of Ihh signaling is clarified in postnatal TMJs using compound Agc-CreER;Ihh f/f;Gli1-nLacZ mice.•Ablation of Ihh signaling in postnatal Agc-CreER;Ihh f/f mice disrupts chondroprogenitor function.•Hh signaling promotes chondrogenesis and chondrocyte maturation in condylar apical cell culture.
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