Femoral morphology and epiphyseal growth plate changes of the hip during maturation: MR assessments in a 1-year follow-up on a cross-sectional asymptomatic cohort in the age range of 9-17 years

The goal of this prospective study was to characterize the morphology and physeal changes of the femoral head during maturation using MRI in a population-based group of asymptomatic volunteers.

Loss of alpha10beta1 integrin expression leads to moderate dysfunction of growth plate chondrocytes

Integrin alpha10beta1 is a collagen-binding integrin expressed on chondrocytes. In order to unravel the role of the alpha10 integrin during development, we generated mice carrying a constitutive deletion of the alpha10 integrin gene. The mutant mice had a normal lifespan and were fertile but developed a growth retardation of the long bones. Analysis of the skeleton revealed defects in the growth plate after birth characterized by a disturbed columnar arrangement of chondrocytes, abnormal chondrocyte shape and reduced chondrocyte proliferation. Electron microscopy of growth plates from newborn mice revealed an increased number of apoptotic chondrocytes and reduced density of the collagen fibrillar network compared to these structures in control mice. These results demonstrate that integrin alpha10beta1 plays a specific role in growth plate morphogenesis and function.

CCN2/CTGF is required for matrix organization and to protect growth plate chondrocytes from cellular stress

CCN2 (connective tissue growth factor (CTGF/CCN2)) is a matricellular protein that utilizes integrins to regulate cell proliferation, migration and survival. The loss of CCN2 leads to perinatal lethality resulting from a severe chondrodysplasia. Upon closer inspection of Ccn2 mutant mice, we observed defects in extracellular matrix (ECM) organization and hypothesized that the severe chondrodysplasia caused by loss of CCN2 might be associated with defective chondrocyte survival. Ccn2 mutant growth plate chondrocytes exhibited enlarged endoplasmic reticula (ER), suggesting cellular stress. Immunofluorescence analysis confirmed elevated stress in Ccn2 mutants, with reduced stress observed in Ccn2 overexpressing transgenic mice. In vitro studies revealed that Ccn2 is a stress responsive gene in chondrocytes. The elevated stress observed in Ccn2-/- chondrocytes is direct and mediated in part through integrin α5. The expression of the survival marker NFκB and components of the autophagy pathway were decreased in Ccn2 mutant growth plates, suggesting that CCN2 may be involved in mediating chondrocyte survival. These data demonstrate that absence of a matricellular protein can result in increased cellular stress and highlight a novel protective role for CCN2 in chondrocyte survival. The severe chondrodysplasia caused by the loss of CCN2 may be due to increased chondrocyte stress and defective activation of autophagy pathways, leading to decreased cellular survival. These effects may be mediated through nuclear factor κB (NFκB) as part of a CCN2/integrin/NFκB signaling cascade.

Growth plate alteration precedes cam-type deformity in elite basketball players

BACKGROUND Vigorous sporting activity during the growth years is associated with an increased risk of having a cam-type deformity develop. The underlying cause of this osseous deformity is unclear. One may speculate whether this is caused by reactive bone apposition in the region of the anterosuperior head-neck junction or whether sports activity alters the shape of and growth in the growth plate. If the latter is true, then one would expect athletes to show an abnormal shape of the capital growth plate (specifically, the epiphyseal extension) before and/or after physeal closure. QUESTIONS/PURPOSES We therefore raised three questions: (1) Do adolescent basketball players show abnormal epiphyseal extension? (2) Does the epiphyseal extension differ before and after physeal closure? (3) Is abnormal epiphyseal extension associated with high alpha angles? METHODS We performed a case-control comparative analysis of young (age range, 9-22 years) male elite basketball athletes with age-matched nonathletes, substratified by whether they had open or closed physes. We measured epiphyseal extension on radial-sequence MRI cuts throughout the cranial hemisphere from 9 o'clock (posterior) to 3 o'clock (anterior). Epiphyseal extension was correlated to alpha angle measurements at the same points. RESULTS Epiphyseal extension was increased in all positions in the athletes compared with the control group. On average, athletes showed epiphyseal extension of 0.67 to 0.83 versus 0.53 to 0.71 in control subjects. In the control group epiphyseal extension was increased at all measurement points in hips after physeal closure compared with before physeal closure. In contrast, the subgroup of athletes with a closed growth plate only had increased epiphyseal extension at the 3 o'clock position compared with the athletes with an open [corrected] growth plate (0.64-0.70). We observed a correlation between an alpha angle greater than 55° and greater epiphyseal extension in the anterosuperior femoral head quadrant: the corresponding Spearman r values were 0.387 (all hips) and 0.285 (alpha angle>55°) for the aggregate anterosuperior quadrant. CONCLUSIONS These findings suggest that a cam-type abnormality in athletes is a consequence of an alteration of the growth plate rather than reactive bone formation. High-level sports activity during growth may be a new and distinct risk factor for a cam-type deformity.

Accelerated hypertrophic chondrocyte kinetics in GDF-7 deficient murine tibial growth plates

The Growth/Differentiation Factors (GDFs) are a subgroup of the Bone Morphogenetic Proteins (BMPs) well known for their role in joint formation and chondrogenesis. Mice deficient in one of these signaling molecules, GDF-5, have recently been shown to exhibit a decreased rate of endochondral bone growth in the proximal tibia due to a significantly longer hypertrophic phase duration. GDF-7 is a related family member, which exhibits a high degree of sequence identity with GDF-5. The purpose of the present study was to determine whether GDF-7 deficiency also alters the endochondral bone growth rate in mice and, if so, how this is achieved. Stereologic and cell kinetic parameters in proximal tibial growth plates from 5-week-old female GDF-7 -/- mice and wild type control littermates were examined. GDF-7 deficiency resulted in a statistically significant increase in growth rate (+26%; p = 0.0084) and rate of cell loss at the chondrosseous junction (+25%; p = 0.0217). Cells from GDF-7 deficient mice also exhibited a significantly shorter hypertrophic phase duration compared to wild type controls (-27%; p = 0.0326). These data demonstrate that, in the absence of GDF-7, the rate of endochondral bone growth is affected through the modulation of hypertrophic phase duration in growth plate chondrocytes. These findings further support a growing body of evidence implicating the GDFs in the formation, maturation, and maintenance of healthy cartilage.

Altered hypertrophic chondrocyte kinetics in GDF-5 deficient murine tibial growth plates

The growth/differentiation factors (GDFs) are a subgroup of the bone morphogenetic proteins best known for their role in joint formation and chondrogenesis. Mice deficient in one of these signaling proteins, GDF-5, exhibit numerous skeletal abnormalities, including shortened limb bones. The primary aim of this study was determine whether GDF-5 deficiency would alter the growth rate in growth plates from the long bones in mice and, if so, how this is achieved. Stereologic and cell kinetic parameters in proximal tibial growth plates from 5-week-old female GDF-5 -/- mice and control littermates were examined. GDF-5 deficiency resulted in a statistically significant reduction in growth rate (-14%, p=0.03). The effect of genotype on growth rate was associated with an altered hypertrophic phase duration, with hypertrophic cells from GDF-5 deficient mice exhibiting a significantly longer phase duration compared to control littermates (+25%, p=0.006). These data suggest that one way in which GDF-5 might modulate the rate of endochondral bone growth could be by affecting the duration of the hypertrophic phase in growth plate chondrocytes.

Trpv6 mediates intestinal calcium absorption during calcium restriction and contributes to bone homeostasis

Energy-dependent intestinal calcium absorption is important for the maintenance of calcium and bone homeostasis, especially when dietary calcium supply is restricted. The active form of vitamin D, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is a crucial regulator of this process and increases the expression of the transient receptor potential vanilloid 6 (Trpv6) calcium channel that mediates calcium transfer across the intestinal apical membrane. Genetic inactivation of Trpv6 in mice (Trpv6(-/-)) showed, however, that TRPV6 is redundant for intestinal calcium absorption when dietary calcium content is normal/high and passive diffusion likely contributes to maintain normal serum calcium levels. On the other hand, Trpv6 inactivation impaired the increase in intestinal calcium transport following calcium restriction, however without resulting in hypocalcemia. A possible explanation is that normocalcemia is maintained at the expense of bone homeostasis, a hypothesis investigated in this study. In this study, we thoroughly analyzed the bone phenotype of Trpv6(-/-) mice receiving a normal (approximately 1%) or low (approximately 0.02%) calcium diet from weaning onwards using micro-computed tomography, histomorphometry and serum parameters. When dietary supply of calcium is normal, Trpv6 inactivation did not affect growth plate morphology, bone mass and remodeling parameters in young adult or aging mice. Restricting dietary calcium had no effect on serum calcium levels and resulted in a comparable reduction in bone mass accrual in Trpv6(+/+) and Trpv6(-/-) mice (-35% and 45% respectively). This decrease in bone mass was associated with a similar increase in bone resorption, whereas serum osteocalcin levels and the amount of unmineralized bone matrix were only significantly increased in Trpv6(-/-) mice. Taken together, our findings indicate that TRPV6 contributes to intestinal calcium transport when dietary calcium supply is limited and in this condition indirectly regulates bone formation and/or mineralization.

The structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development

OBJECTIVE: During postnatal development, mammalian articular cartilage acts as a surface growth plate for the underlying epiphyseal bone. Concomitantly, it undergoes a fundamental process of structural reorganization from an immature isotropic to a mature (adult) anisotropic architecture. However, the mechanism underlying this structural transformation is unknown. It could involve either an internal remodelling process, or complete resorption followed by tissue neoformation. The aim of this study was to establish which of these two alternative tissue reorganization mechanisms is physiologically operative. We also wished to pinpoint the articular cartilage source of the stem cells for clonal expansion and the zonal location of the chondrocyte pool with high proliferative activity. METHODS: The New Zealand white rabbit served as our animal model. The analysis was confined to the high-weight-bearing (central) areas of the medial and lateral femoral condyles. After birth, the articular cartilage layer was evaluated morphologically at monthly intervals from the first to the eighth postnatal month, when this species attains skeletal maturity. The overall height of the articular cartilage layer at each juncture was measured. The growth performance of the articular cartilage layer was assessed by calcein labelling, which permitted an estimation of the daily growth rate of the epiphyseal bone and its monthly length-gain. The slowly proliferating stem-cell pool was identified immunohistochemically (after labelling with bromodeoxyuridine), and the rapidly proliferating chondrocyte population by autoradiography (after labelling with (3)H-thymidine). RESULTS: The growth activity of the articular cartilage layer was highest 1 month after birth. It declined precipitously between the first and third months, and ceased between the third and fourth months, when the animal enters puberty. The structural maturation of the articular cartilage layer followed a corresponding temporal trend. During the first 3 months, when the articular cartilage layer is undergoing structural reorganization, the net length-gain in the epiphyseal bone exceeded the height of the articular cartilage layer. This finding indicates that the postnatal reorganization of articular cartilage from an immature isotropic to a mature anisotropic structure is not achieved by a process of internal remodelling, but by the resorption and neoformation of all zones except the most superficial (stem-cell) one. The superficial zone was found to consist of slowly dividing stem cells with bidirectional mitotic activity. In the horizontal direction, this zone furnishes new stem cells that replenish the pool and effect a lateral expansion of the articular cartilage layer. In the vertical direction, the superficial zone supplies the rapidly dividing, transit-amplifying daughter-cell pool that feeds the transitional and upper radial zones during the postnatal growth phase of the articular cartilage layer. CONCLUSIONS: During postnatal development, mammalian articular cartilage fulfils a dual function, viz., it acts not only as an articulating layer but also as a surface growth plate. In the lapine model, this growth activity ceases at puberty (3-4 months of age), whereas that of the true (metaphyseal) growth plate continues until the time of skeletal maturity (8 months). Hence, the two structures are regulated independently. The structural maturation of the articular cartilage layer coincides temporally with the cessation of its growth activity - for the radial expansion and remodelling of the epiphyseal bone - and with sexual maturation. That articular cartilage is physiologically reorganized by a process of tissue resorption and neoformation, rather than by one of internal remodelling, has important implications for the functional engineering and repair of articular cartilage tissue.

Abnormal collagen fibrils in cartilage of matrilin-1/matrilin-3-deficient mice

Matrilins are oligomeric extracellular matrix adaptor proteins mediating interactions between collagen fibrils and other matrix constituents. All four matrilins are expressed in cartilage and mutations in the human gene encoding matrilin-3 (MATN3) are associated with different forms of chondrodysplasia. Surprisingly, however, Matn3-null as well as Matn1- and Matn2-null mice do not show an overt skeletal phenotype, suggesting a dominant negative pathomechanism for the human disorders and redundancy/compensation among the family members in the knock-out situation. Here, we show that mice lacking both matrilin-1 and matrilin-3 develop an apparently normal skeleton, but exhibit biochemical and ultrastructural abnormalities of the knee joint cartilage. At the protein level, an altered SDS-PAGE band pattern and a clear up-regulation of the homotrimeric form of matrilin-4 were evident in newborn Matn1/Matn3 and Matn1 knock-out mice, but not in Matn3-null mice. The ultrastructure of the cartilage matrix after conventional chemical fixation was grossly normal; however, electron microscopy of high pressure frozen and freeze-substituted samples, revealed two consistent observations: 1) moderately increased collagen fibril diameters throughout the epiphysis and the growth plate in both single and double mutants; and 2) increased collagen volume density in Matn1(-/-)/Matn3(-/-) and Matn3(-/-) mice. Taken together, our results demonstrate that matrilin-1 and matrilin-3 modulate collagen fibrillogenesis in cartilage and provide evidence that biochemical compensation might exist between matrilins.

Surviving endoplasmic reticulum stress is coupled to altered chondrocyte differentiation and function

In protein folding and secretion disorders, activation of endoplasmic reticulum (ER) stress signaling (ERSS) protects cells, alleviating stress that would otherwise trigger apoptosis. Whether the stress-surviving cells resume normal function is not known. We studied the in vivo impact of ER stress in terminally differentiating hypertrophic chondrocytes (HCs) during endochondral bone formation. In transgenic mice expressing mutant collagen X as a consequence of a 13-base pair deletion in Col10a1 (13del), misfolded alpha1(X) chains accumulate in HCs and elicit ERSS. Histological and gene expression analyses showed that these chondrocytes survived ER stress, but terminal differentiation is interrupted, and endochondral bone formation is delayed, producing a chondrodysplasia phenotype. This altered differentiation involves cell-cycle re-entry, the re-expression of genes characteristic of a prehypertrophic-like state, and is cell-autonomous. Concomitantly, expression of Col10a1 and 13del mRNAs are reduced, and ER stress is alleviated. ERSS, abnormal chondrocyte differentiation, and altered growth plate architecture also occur in mice expressing mutant collagen II and aggrecan. Alteration of the differentiation program in chondrocytes expressing unfolded or misfolded proteins may be part of an adaptive response that facilitates survival and recovery from the ensuing ER stress. However, the altered differentiation disrupts the highly coordinated events of endochondral ossification culminating in chondrodysplasia.

Fractures in Children and Adolescents

Fractures of the growing bone require fixation techniques, which preclude any injury to the growth plate regions. This requirement is met by Elastic Stable Intramedullary Nails (ESIN) which are positioned between both metaphyseal regions. Pronounced malposition and/or shortening, open fractures and fractures with impending skin perforation are indications for clavicle nailing in adolescents. Retrograde nailing with two elastic nails, inserted from lateral, is the method of choice for stabilization of humerus fractures. In radial neck fractures with severe tilting of the radial head, a retrograde nail may reduce and fix the head. In Monteggia lesions, the ulna fracture is reduced and fixed with an antegrade nail. Forearm fractures with unacceptable axial deviation are reduced and fixed with one antegrade nail in the ulna and a retrograde nail in the radius. Ascending elastic nailing is done for femur shaft and proximal femur fractures. The medial and lateral entry sites are located above the distal physis. End caps are used to prevent shortening in spiral and multiple segment fractures. Fractures of the distal third of the femur are nailed in a descending technique. The entry sites of two nails are located on the lateral cortex below the greater trochanter. Combined tibia and fibula fractures, open fractures and unstable fracture types such as spiral and multifragmental tibia fractures are good indications for ESIN. Descending nailing is the method of choice. The nail entry points are medially and laterally distal to the apophysis of the proximal tibia. Thorough knowledge of each fracture type, fracture location and age specific healing pattern is necessary for safe and effective treatment of pediatric fractures

Ellis-van Creveld Syndrome in Grey Alpine Cattle: Morphologic, Immunophenotypic, and Molecular Characterization

Ellis-van Creveld (EvC) syndrome is a human autosomal recessive disorder caused by a mutation in either the EVC or EVC2 gene, and presents with short limbs, polydactyly, and ectodermal and heart defects. The aim of this study was to understand the pathologic basis by which deletions in the EVC2 gene lead to chondrodysplastic dwarfism and to describe the morphologic, immunohistochemical, and molecular hallmarks of EvC syndrome in cattle. Five Grey Alpine calves, with a known mutation in the EVC2 gene, were autopsied. Immunohistochemistry was performed on bone using antibodies to collagen II, collagen X, sonic hedgehog, fibroblast growth factor 2, and Ki67. Reverse transcription polymerase chain reaction was performed to analyze EVC1 and EVC2 gene expression. Autopsy revealed long bones that were severely reduced in length, as well as genital and heart defects. Collagen II was detected in control calves in the resting, proliferative, and hypertrophic zones and in the primary and secondary spongiosa, with a loss of labeling in the resting zone of 2 dwarfs. Collagen X was expressed in hypertrophic zone in the controls but was absent in the EvC cases. In affected calves and controls, sonic hedgehog labeled hypertrophic chondrocytes and primary and secondary spongiosa similarly. FGF2 was expressed in chondrocytes of all growth plate zones in the control calves but was lost in most EvC cases. The Ki67 index was lower in cases compared with controls. EVC and EVC2 transcripts were detected. Our data suggest that EvC syndrome of Grey Alpine cattle is a disorder of chondrocyte differentiation, with accelerated differentiation and premature hypertrophy of chondrocytes, and could be a spontaneous model for the equivalent human disease.

Comparative release of growth factors from PRP, PRF, and advanced-PRF

OBJECTIVES The use of platelet concentrates has gained increasing awareness in recent years for regenerative procedures in modern dentistry. The aim of the present study was to compare growth factor release over time from platelet-rich plasma (PRP), platelet-rich fibrin (PRF), and a modernized protocol for PRF, advanced-PRF (A-PRF). MATERIALS AND METHODS Eighteen blood samples were collected from six donors (3 samples each for PRP, PRF, and A-PRF). Following preparation, samples were incubated in a plate shaker and assessed for growth factor release at 15 min, 60 min, 8 h, 1 day, 3 days, and 10 days. Thereafter, growth factor release of PDGF-AA, PDGF-AB, PDGF-BB, TGFB1, VEGF, EGF, and IGF was quantified using ELISA. RESULTS The highest reported growth factor released from platelet concentrates was PDGF-AA followed by PDGF-BB, TGFB1, VEGF, and PDGF-AB. In general, following 15-60 min incubation, PRP released significantly higher growth factors when compared to PRF and A-PRF. At later time points up to 10 days, it was routinely found that A-PRF released the highest total growth factors. Furthermore, A-PRF released significantly higher total protein accumulated over a 10-day period when compared to PRP or PRF. CONCLUSION The results from the present study indicate that the various platelet concentrates have quite different release kinetics. The advantage of PRP is the release of significantly higher proteins at earlier time points whereas PRF displayed a continual and steady release of growth factors over a 10-day period. Furthermore, in general, it was observed that the new formulation of PRF (A-PRF) released significantly higher total quantities of growth factors when compared to traditional PRF. CLINICAL RELEVANCE Based on these findings, PRP can be recommended for fast delivery of growth factors whereas A-PRF is better-suited for long-term release.