Development Of Secondary Ossification Centers Research Articles

Skeletal and dental age estimation via postmortem computed tomography in Polish subadults group

This article is a retrospective analysis of postmortem computed tomography scans of ossification stages of the anterior and posterior intra-occipital sutures, the anterior arch of the atlas, and the neurocentral junction of the axis. We also analyzed the development of secondary ossification centers in the proximal humeral, femoral, and tibial epiphyses, and the distal femoral and tibial epiphyses. Additionally, the development of primary ossification centers in the wrist and metacarpals, and maxillary and mandibular deciduous tooth maturation. A total of 58 cadavers (35 males, 23 females), whose age ranged from 3rd month of pregnancy to 14 years, were analyzed. The results of this study show that analysis of synchondrosis closure, primary, and secondary ossification center development and deciduous tooth changes are a good tool for age estimation in subadults group (fetuses, newborns, infants, and children). The results of the study in a Polish population are consistent with those reported by other authors.

Ultrasonographic Assessment of the Proximal Femoral Ossification Center in Children Under 1 Year

Background. Developmental disorders of the secondary ossification center (SOC) of the proximal femoral epiphysis can be a marker of some childhood diseases that require timely diagnosis and treatment.
 The study aimed to assess the capabilities of ultrasonography in the diagnosis of ossification processes of the proximal femoral epiphysis.
 Material and Methods. The study is based on the results of a survey of 524 children aged 2 weeks to 1 year with normal hip joints, including 259 boys and 265 girls. All patients underwent ultrasound examination of the hip joints according to the method of R. Graf in a standard coronary section. In older children, to eliminate errors in measuring the size of the SOC, an additional cross-section was used.
 Results. The SOC was detected in isolated cases in boys aged up to 3 months and girls up to 2 months. At age 3 months, the SOC was visualized in 45% of girls and 5% of boys. By age 5 months, 81% of girls had a SOC compared with 46% of boys. By 7 months, SOC was determined in more than 90% of cases of both groups. Thus, girls showed an earlier formation of the SOC than boys. The SOC was located in the center of the femoral head in 82% of cases, lateral in 14%, and medial in 4%. In 95% of the examined children, SOC development occurred symmetrically in both joints. In addition, discrepancies were revealed with simultaneous ultrasonography and X-ray of the hip joints since the SOC becomes visible earlier during ultrasonography.
 Conclusion. Sonography is a highly informative method for determining the ossification processes of the proximal femur. Knowledge of the normal sonographic appearance of the femoral head SOC by age and sex will help clinicians diagnose and treat hip disorders.

Secondary ossification center induces and protects growth plate structure.

Growth plate and articular cartilage constitute a single anatomical entity early in development but later separate into two distinct structures by the secondary ossification center (SOC). The reason for such separation remains unknown. We found that evolutionarily SOC appears in animals conquering the land - amniotes. Analysis of the ossification pattern in mammals with specialized extremities (whales, bats, jerboa) revealed that SOC development correlates with the extent of mechanical loads. Mathematical modeling revealed that SOC reduces mechanical stress within the growth plate. Functional experiments revealed the high vulnerability of hypertrophic chondrocytes to mechanical stress and showed that SOC protects these cells from apoptosis caused by extensive loading. Atomic force microscopy showed that hypertrophic chondrocytes are the least mechanically stiff cells within the growth plate. Altogether, these findings suggest that SOC has evolved to protect the hypertrophic chondrocytes from the high mechanical stress encountered in the terrestrial environment.

Periarticular Mesenchymal Progenitors Initiate and Contribute to Secondary Ossification Center Formation During Mouse Long Bone Development.

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.

Normal Skeletal Maturation and Imaging Pitfalls in the Pediatric Shoulder.

A growing number of magnetic resonance (MR) imaging studies of the shoulder are being performed as a result of greater and earlier participation of children and adolescents in competitive sports such as softball and baseball. However, scant information is available regarding the MR imaging features of the normal sequential development of the shoulder. The authors discuss the radiographic and MR imaging appearances of the normal musculoskeletal maturation patterns of the shoulder, with emphasis on (a) development of secondary ossification centers of the glenoid (including the subcoracoid and peripheral glenoid ossification centers); (b) development of preossification and secondary ossification centers of the humeral head and the variable appearance and number of the secondary ossification centers of the distal acromion, with emphasis on the formation of the os acromiale; (c) development of the growth plates, glenoid bone plates, glenoid bare area, and proximal humeral metaphyseal stripe; and (d) marrow signal alterations in the distal humerus, acromion, and clavicle. In addition, the authors discuss various imaging interpretation pitfalls inherent to the normal skeletal maturation of the shoulder, examining clues that may help distinguish normal development from true disease (eg, osteochondral lesions, labral tears, abscesses, fractures, infection, tendon disease, acromioclavicular widening, and os acromiale). Familiarity with the timing, location, and appearance of maturation patterns in the pediatric shoulder is crucial for correct image interpretation.

Infant cynomolgus monkeys exposed to denosumab in utero exhibit an osteoclast-poor osteopetrotic-like skeletal phenotype at birth and in the early postnatal period

RANKL is a key regulator of bone resorption and osteoclastogenesis. Denosumab is a fully human IgG2 monoclonal antibody that inhibits bone resorption by binding and inhibiting the activity of RANKL. To determine the effects of denosumab on pre- and postnatal skeletal growth and development, subcutaneous injections of 0 (control) or 50mg/kg/month denosumab were given to pregnant cynomolgus monkeys from approximately gestation day (GD) 20 until parturition (up to 6 doses). For up to 6months postpartum (birth day [BD] 180/181), evaluation of the infants included skeletal radiographs, bone biomarkers, and oral examinations for assessment of tooth eruption. Infant bones were collected at necropsy for densitometry, biomechanical testing, and histopathologic evaluation from control and denosumab-exposed infants on BD1 (or within 2weeks of birth) and BD181, and from infants that died or were euthanized moribund from BD5 to BD69.In all denosumab-exposed infants, biomarkers of bone resorption and formation were markedly decreased at BD1 and BD14 and slightly greater at BD91 vs control, then similar to control values by BD181. Spontaneous long bone fractures were detected clinically or radiographically in 4 denosumab-exposed infants at BD28 and BD60, with evidence of radiographic healing at ≥BD60. In BD1 infants exposed to denosumab in utero, radiographic evaluations of the skeleton revealed decreased long bone length; a generalized increased radio-opacity of the axial and appendicular skeleton and bones at the base of the skull with decreased or absent marrow cavities, widened growth plates, flared/club-shaped metaphysis, altered jaw/skull shape, and reduced jaw length; and delayed development of secondary ossification centers. Densitometric evaluations in these infants demonstrated a marked increase in bone mineral density at trabecular sites, but cortical bone mineral density was decreased. Histologically, long bone cortices were attenuated and there was an absence of osteoclasts. Bones with active endochondral ossification consisted largely of a dense network of retained primary spongiosa with reduced marrow space consistent with an osteopetrotic phenotype. A minimal increase in growth plate thickness largely due to the expansion of the hypertrophic zone was present. Retained woven bone was observed in bones formed by intramembranous ossification, consistent with absence of bone remodeling. These changes in bone tissue composition and geometry were reflected in reduced biomechanical strength and material properties of bones from denosumab-exposed infants. Material property changes were characterized by increased tissue brittleness reflected in reductions in calculated material toughness at the femur diaphysis and lack of correlation between energy and bone mass at the vertebra; these changes were likely the basis for the increased skeletal fragility (fractures).Although tooth eruption was not impaired in denosumab-exposed infants, the reduced growth and increased bone density of the mandible resulted in dental abnormalities consisting of tooth malalignment and dental dysplasia.Radiographic changes at BD1 persisted at BD28, with evidence of resumption of bone resorption and remodeling observed in most infants at BD60 and/or BD90. In 2 infants euthanized on BD60 and BD69, there was histologic and radiographic evidence of subphyseal/metaphyseal bone resorption accompanied by multiple foci of ossification in growth plates that were markedly increased in thickness. In infants necropsied at BD181, where systemic exposure to denosumab had been below limits of quantitation for approximately 3months, there was largely full recovery from all bone-related changes observed earlier postpartum, including tissue brittleness. Persistent changes included dental dysplasia, decreased bone length, reduced cortical thickness, and decreased peak load and ultimate strength at the femur diaphysis.In conclusion, the skeletal and secondary dental effects observed in infant monkeys exposed in utero to denosumab are consistent with the anticipated pharmacological activity of denosumab as a monoclonal antibody against RANKL and inhibitor of osteoclastogenesis. The resulting inhibition of resorption impaired both bone modeling and remodeling during skeletal development and growth. The skeletal phenotype of these infant monkeys resembles human infants with osteoclast-poor osteopetrosis due to inactivating mutations of RANK or RANKL.

Deboning broiler chicken legs and wings by dislocation of articular cartilage followed by stripping periosteum

The yield of deboned meat is an important economic factor affecting the profit of the meat industry. This study was undertaken to determine whether the yield of boneless meat from broiler chicken leg (thigh and drumstick) and wing (drumette and winglet) is improved by introducing a new deboning method consisting of articular cartilage dislocation followed by stripping periosteum. A total of 44 broiler chicken carcasses were used in the deboning experiment. Right and left legs or wings from the first 22 carcasses were assigned to the new and ordinary hand deboning methods, respectively. For the remaining 22 carcasses, right and left legs or wings were assigned to the ordinary and new methods, respectively. The weight of residue, composed of bone and small amounts of cartilage and noncartilaginous tissues obtained after deboning, was then compared between the right and left legs or wings to see the difference between the 2 methods. The removal of tibia, fibula, humerus, radius, or ulna resulted in formation of a hollow in boneless meat obtained. There was no difference (P > 0.05) between the right and left legs or wings in the weight of residue obtained after deboning as expected. The weight of residue was less (P < 0.05) with the new method compared with the ordinary method in all chicken parts examined. The difference of residue weight between the 2 methods accounted for 10, 12, 14, and 21% of the weight of residue obtained by the ordinary method in thigh, drumstick, drumette, and winglet, respectively. The new method may be useful to deboners at home kitchens as well as the poultry meat industry. The present study also showed the development of a secondary ossification center at the proximal end of the carpometacarpus of chickens. This is, to our knowledge, the first report of development of secondary ossification center in chicken wings.

Cartilage-specific β-catenin signaling regulates chondrocyte maturation, generation of ossification centers, and perichondrial bone formation during skeletal development

The WNT/β-catenin signaling pathway is a critical regulator of chondrocyte and osteoblast differentiation during multiple phases of cartilage and bone development. Although the importance of β-catenin signaling during the process of endochondral bone development has been previously appreciated using a variety of genetic models that manipulate β-catenin in skeletal progenitors and osteoblasts, genetic evidence demonstrating a specific role for β-catenin in committed growth-plate chondrocytes has been less robust. To identify the specific role of cartilage-derived β-catenin in regulating cartilage and bone development, we studied chondrocyte-specific gain- and loss-of-function genetic mouse models using the tamoxifen-inducible Col2Cre(ERT2) transgene in combination with β-catenin(fx(exon3)/wt) or β-catenin(fx/fx) floxed alleles, respectively. From these genetic models and biochemical data, three significant and novel findings were uncovered. First, cartilage-specific β-catenin signaling promotes chondrocyte maturation, possibly involving a bone morphogenic protein 2 (BMP2)-mediated mechanism. Second, cartilage-specific β-catenin facilitates primary and secondary ossification center formation via the induction of chondrocyte hypertrophy, possibly through enhanced matrix metalloproteinase (MMP) expression at sites of cartilage degradation, and potentially by enhancing Indian hedgehog (IHH) signaling activity to recruit vascular tissues. Finally, cartilage-specific β-catenin signaling promotes perichondrial bone formation possibly via a mechanism in which BMP2 and IHH paracrine signals synergize to accelerate perichondrial osteoblastic differentiation. The work presented here supports the concept that the cartilage-derived β-catenin signal is a central mediator for major events during endochondral bone formation, including chondrocyte maturation, primary and secondary ossification center development, vascularization, and perichondrial bone formation.

The role of Akt1 in terminal stages of endochondral bone formation: Angiogenesis and ossification

Longitudinal bone growth is the result of endochondral bone formation which takes place in the growth plate. The rate of chondrocyte proliferation and hypertrophy, vascular invasion with the formation of primary ossification centers and cartilage replacement by bone tissue are all important processes required for normal growth. We have shown a role for the PI3K signaling pathway in chondrocyte hypertrophy and bone growth in tibia explant cultures. In this current study, we aimed to investigate the role of Akt1, an important target of PI3K, in endochondral ossification. Akt1 KO mice showed reduced size compared to their littermates throughout life, but the largest difference in body size was observed around 1 week of age. Focusing on this specific developmental stage, we discovered delayed secondary ossification in the long bones of Akt1 KO mice. A delay in formation of a structure resembling a secondary ossification center was also seen in tibia organ cultures treated with the PI3K inhibitor LY294002. The expression of matrix metalloproteinase-14 (MMP-14), the main protease responsible for development of secondary ossification centers, was decreased in the epiphysis of Akt1 KO mice, possibly explaining the delay in secondary ossification centers seen in the Akt1 KO mice. Bone mineral density (BMD) and bone mineral content (BMC) measured in the proximal tibia of 1-year-old mice were decreased in Akt1 KO mice, suggesting that the original delay in ossification might affect bone quality in older animals.

Gene Expression and Distribution of Connective Tissue Growth Factor (CCN2/CTGF) During Secondary Ossification Center Formation

CCN2/connective tissue growth factor (CCN2/CTGF) is a critical signaling modulator of mesenchymal tissue development. This study investigated the localization and expression of CCN2/CTGF as a factor supporting angiogenesis and chondrogenesis during development of secondary ossification centers in the mouse tibial epiphysis. Formation of the secondary ossification center was initiated by cartilage canal formation and blood vessel invasion at 7 days of age, and onset of ossification was observed at 14 days. In situ hybridization showed that CCN2/CTGF mRNA was distinctively expressed in the region of the cartilage canal and capsule-attached marginal tissues at 7 days of age, and distinct expression was also observed in proliferating chondrocytes around the marrow space at 14 days of age. Immunostaining showed that CCN2/CTGF was distributed broadly around the expressed cells located in the central region of the epiphysis, where the chondrocytes become hypertrophic and the cartilage canal enters into the hypertrophic mass. Furthermore, an overlapping distribution of metalloproteinase (MMP)9 and CCN2/CTGF was found in the secondary ossification center. These findings suggest that the CCN2/CTGF is involved in establishing epiphyseal vascularization and remodeling, which eventually determines the secondary ossification center in the developing epiphysial cartilage.

A RADIOGRAPHIC STUDY ON THE DEVELOPMENT OF THE ANTEBRACHIUM IN GREAT DANE PUPS ON DIFFERENT CALCIUM INTAKES

Radiographic examination of the radius and ulna was performed in three groups of Great Danes fed diets containing high, normal, and low calcium contents from the ages of 7 to 21 weeks. Development and maturation of secondary ossification centers, architecture of the distal ulnar metaphysis, and growth in length of the radius were noted. In the dogs fed a high‐calcium diet, there was retardation in the development of the ulnar styloid process, the humeral medial epicondyle, the olecranal apophysis, and the anconeal process. In addition, these dogs developed severe changes over the entire width of the distal ulnar metaphysis, whereas the other groups developed only focal changes. The dogs fed a low‐calcium diet developed severe signs of nutritional hyperparathyroidism. It is concluded that the development of the secondary ossification centers and the growth in length of the radius are inversely related to calcium intake.

Bone growth aud development of secondary ossification centers of extremities in the cynomolgus monkey (Macaca fascicularis).

The development of so-called long bones in the extremity has been studied roentgenographically in forty-seven males and fifty-one females cynomolgus monkeys bred and reared at the National Institute of Health. The age of the females ranged from five months to eight years and nine months, and that of the males was from four months to seven years. In addition, the fetuses of six to twenty weeks of gestation age were examined for the time of appearance of ossification centers. As the biological parameters concerning body growth, the body weight and the bone length were measured and the secondary ossification centers were scrutinized and assessed the maturity process on the basis of the criteria that divided the state into eleven stages. Also the allometric analyses of body weight against bone length was conducted. Most of the secondary ossification centers except the proximal fibulal epiphysis appeared during the period from the prenatal stage (15-20 weeks of gestationage) to the postnatal one (several months of age). From four to five months of age, many ossification centers had developed to some extent. But, the appearance of proximal fibulal epiphysis was delayed and often lacking until 10 months of age in female and one year and three months of age in male. The earliest epiphyseal fusion was observed at the distal humeral epiphysis in both sexes. The latest epiphyseal fusion was observed at the distal ulnal epiphysis in both sexes and at the distal ulnal and radial epiphyses in female. From this study, the time of fusion was at five and three guarters years of age in females and at six and a half years of age in males. As a result, it is suggested that the estimation of animal's age might be put to practical use by introducing the assessing method that the score was given from the observation of the secondary ossification center.