Polystyrene microplastic-induced endoplasmic reticulum stress contributes to growth plate endochondral ossification disorder in young rat.

BACKGROUND: Previous studies on the effects of microplastics (MPs) on bone in early development are limited. This study aimed to investigate the adverse effects of MPs on bone in young rats and the potential mechanism. METHODS: Three-week-old female rats were orally administered MPs for 28 days, and endoplasmic reticulum (ER) stress inhibitor salubrinal (SAL) and ER stress agonist tunicamycin (TM) were added to evaluate the effect of ER stress on toxicity of MPs. The indicators of growth and plasma markers of bone turnover were evaluated. Tibias were analyzed using micro-computed tomography (micro-CT). Histomorphological staining of growth plates was performed, and related gene expression of growth plate chondrocytes was tested. RESULTS: After exposure of MPs, the rats had decreased growth, shortened tibial length, and altered blood calcium and phosphorus metabolism. Trabecular bone was sparse according to micro-CT inspection. In the growth plate, the thickness of proliferative zone substantial reduced while the thickness of hypertrophic zone increased significantly, and the chondrocytes were scarce and irregularly arranged according to tibial histological staining. The transcription of the ER stress-related genes BIP, PERK, ATF4, and CHOP dramatically increased, and the transcription factors involved in chondrocyte proliferation, differentiation, apoptosis, and matrix secretion were aberrant according to RT-qPCR and western blotting. Moreover, the addition of TM showed higher percentage of chondrocyte death. Administration of SAL alleviated all of the MPs-induced symptoms. CONCLUSION: These results indicated that MPs could induce growth retardation and longitudinal bone damage in early development. The toxicity of MPs may attribute to induced ER stress and impaired essential processes of the endochondral ossification after MPs exposure.

Control of skeletal morphogenesis by the Hippo-YAP/TAZ pathway.

The Hippo-YAP/TAZ pathway is an important regulator of tissue growth, but can also control cell fate or tissue morphogenesis. Here, we investigate the function of the Hippo pathway during the development of cartilage, which forms the majority of the skeleton. Previously, YAP was proposed to inhibit skeletal size by repressing chondrocyte proliferation and differentiation. We find that, in vitro, Yap/Taz double knockout impairs murine chondrocyte proliferation, whereas constitutively nuclear nls-YAP5SA accelerates proliferation, in line with the canonical role of this pathway in most tissues. However, in vivo, cartilage-specific knockout of Yap/Taz does not prevent chondrocyte proliferation, differentiation or skeletal growth, but rather results in various skeletal deformities including cleft palate. Cartilage-specific expression of nls-YAP5SA or knockout of Lats1/2 do not increase cartilage growth, but instead lead to catastrophic malformations resembling chondrodysplasia or achondrogenesis. Physiological YAP target genes in cartilage include Ctgf, Cyr61 and several matrix remodelling enzymes. Thus, YAP/TAZ activity controls chondrocyte proliferation in vitro, possibly reflecting a regenerative response, but is dispensable for chondrocyte proliferation in vivo, and instead functions to control cartilage morphogenesis via regulation of the extracellular matrix.

Effects of acute femoral head ischemia on the growth plate and metaphysis in a piglet model of Legg-Calvé-Perthes disease.

OBJECTIVE: To determine the effects of acute (≤7 days) femoral head ischemia on the proximal femoral growth plate and metaphysis in a piglet model of Legg-Calvé-Perthes disease (LCPD). We hypothesized that qualitative and quantitative histological assessment would identify effects of ischemia on endochondral ossification. DESIGN: Unilateral femoral head ischemia was surgically induced in piglets, and femurs were collected for histological assessment at 2 (n = 7) or 7 (n = 5) days post-ischemia. Samples were assessed qualitatively, and histomorphometry of the growth plate zones and primary spongiosa was performed. In a subset of samples at 7 days, hypertrophic chondrocytes were quantitatively assessed and immunohistochemistry for TGFß1 and Indian hedgehog was performed. RESULTS: By 2 days post-ischemia, there was significant thinning of the proliferative and hypertrophic zones, by 63 µm (95% CI -103, -22) and -19 µm (95% CI -33, -5), respectively. This thinning persisted at 7 days post-ischemia. Likewise, at 7 days post-ischemia, the primary spongiosa was thinned to absent by an average of 311 µm (95% CI -542, -82) in all ischemic samples. TGFß1 expression was increased in the hypertrophic zone at 7 days post-ischemia. CONCLUSIONS: Alterations to the growth plate zones and metaphysis occurred by 2 days post-ischemia and persisted at 7 days post-ischemia. Our findings suggest that endochondral ossification may be disrupted at an earlier time point than previously reported and that growth disruption may occur in the piglet model as occurs in some children with LCPD.

Physeal fusion status and lesion size are more important than patient age for healing of juvenile osteochodritis dessicans lesions of the distal femur.

PURPOSE: The purpose of the study was to evaluate the effect of skeletal age and lesion size, location, and grade on the success of nonoperative treatment for juvenile osteochondritis dissecans (OCD). It is hypothesized that skeletal maturity, including a combination of maturation phenotypes, correlates with nonoperative lesion healing. METHODS: The clinical and radiographic data on 52 patients aged 7-20 years treated for OCD of the distal femur between 2010 and 2019 were retrospectively reviewed. Knee radiographs were assessed for number of lesions present and lesion location, size, and stage. Assessments of skeletal maturation were performed on all antero-posterior knee radiographs using the Roche, Wainer, and Thissen (RWT) method. Patients were categorized as healed if they demonstrated no pain on clinical examination. The relationship between skeletal maturity and nonoperative lesion healing was determined using Spearman rank correlations on available variables. RESULTS: Neither chronological nor skeletal age was associated with surgical status (Rho = 0.03, n.s., and Rho = 0.13, n.s., respectively) or the healing status of nonoperatively treated OCD lesions (Rho = 0.44, n.s., and Rho = 0.03, n.s., respectively). Epiphyseal fusion status of the distal femoral physis was moderately correlated with nonoperative healing, but was not statistically significant (lateral femoral physis: Rho = 0.43, p = 0.05; medial femoral physis: Rho = 0.43, n.s.). Lesion length correlated with surgical status (Rho = - 0.38, p = 0.009). CONCLUSION: The extent of fusion of the distal femoral physis (multi-stage grading) may be more strongly correlated with nonoperative healing than other markers of skeletal maturity or chronological age. Clinicians can use this as an additional radiographic sign when considering nonoperative treatment for juvenile OCD lesions in the distal femur. OCD lesion length and physeal fusion status appear to be more important for healing than patient age.

Quantitative T2 and T1ρ mapping are sensitive to ischemic injury to the epiphyseal cartilage in an in vivo piglet model of Legg-Calvé-Perthes disease.

OBJECTIVE: To determine if the quantitative MRI techniques T2 and T1ρ mapping are sensitive to ischemic injury to epiphyseal cartilage in vivo in a piglet model of Legg-Calvé-Perthes disease using a clinical 3T MRI scanner. We hypothesized that T2 and T1ρ relaxation times would be increased in the epiphyseal cartilage of operated vs contralateral-control femoral heads 1 week following onset of ischemia. DESIGN: Unilateral femoral head ischemia was surgically induced in eight piglets. Piglets were imaged 1 week post-operatively in vivo at 3T MRI using a magnetization-prepared 3D fast spin echo sequence for T2 and T1ρ mapping and a 3D gradient echo sequence for cartilage segmentation. Ischemia was confirmed in all piglets using gadolinium contrast-enhanced MRI. Median T2 and T1ρ relaxation times were measured in the epiphyseal cartilage of the ischemic and control femoral heads and compared using paired t-tests. Histological assessment was performed on a subset of five piglets. RESULTS: T2 and T1ρ relaxation times were significantly increased in the epiphyseal cartilage of the operated vs control femoral heads (ΔT2 = 11.9 ± 3.7 ms, 95% CI = [8.8, 15.0] ms, P < 0.0001; ΔT1ρ = 12.8 ± 4.1 ms, 95% CI = [9.4, 16.2] ms, P < 0.0001). Histological assessment identified chondronecrosis in the hypertrophic and deep proliferative zones within ischemic epiphyseal cartilage. CONCLUSIONS: T2 and T1ρ mapping are sensitive to ischemic injury to the epiphyseal cartilage in vivo at clinical 3T MRI. These techniques may be clinically useful to assess injury and repair to the epiphyseal cartilage to better stage the extent of ischemic damage in Legg-Calvé-Perthes disease.

Reactivation of Vertebral Growth Plate Function in Vertebral Body Tethering in an Animal Model.

Flexible spine tethering is a relatively novel fusionless surgical technique that aims to correct scoliosis based on growth modulation due to the pressure exerted on the vertebral body epiphyseal growth plate. The correction occurs in two phases: immediate intraoperative and postoperative with growth. The aim of this study was to evaluate the reactivation of vertebral growth plate function after applying corrective forces. The rat tail model was used. Asymmetric compression and distraction of caudal growth plates were performed using a modified external fixation apparatus. Radiological and histopathological data were analysed. After three weeks of correction, the activity of the structures increased across the entire growth plate width, and the plate was thickened. The height of the hypertrophic layer and chondrocytes on the concave side doubled in height. The height of chondrocytes and the cartilage thickness on the concave and central sides after the correction did not differ statistically significantly from the control group. Initiation of the correction of scoliosis in the growing spine, with relief of the pressure on the growth plate, allows the return of the physiological activity of the growth cartilage and restoration of the deformed vertebral body.

Cellular and Molecular Alterations Underlying Abnormal Bone Growth in X-Linked Hypophosphatemia.

X-linked hypophosphatemia (XLH), the most common form of hereditary hypophosphatemic rickets, is caused by inactivating mutations of the phosphate-regulating endopeptidase gene (PHEX). XLH is mainly characterized by short stature, bone deformities and rickets, while in hypophosphatemia, normal or low vitamin D levels and low renal phosphate reabsorption are the principal biochemical aspects. The cause of growth impairment in patients with XLH is not completely understood yet, thus making the study of the growth plate (GP) alterations necessary. New treatment strategies targeting FGF23 have shown promising results in normalizing the growth velocity and improving the skeletal effects of XLH patients. However, further studies are necessary to evaluate how this treatment affects the GP as well as its long-term effects and the impact on adult height.

DDB1 promotes the proliferation and hypertrophy of chondrocytes during mouse skeleton development.

The proliferation and hypertrophy of chondrocytes play important roles in endochondral ossification, which is tightly regulated during skeleton development. However, the regulation mechanism remains largely unknown. Here we show that DDB1 (Damaged DNA Binding Protein 1) has a critical function in the development of growth plates. Using chondrocyte-specific DDB1 knockout mice, we found that DDB1 deletion in chondrocytes results in dwarfism due to the aberrant skeleton development. The structure of growth plate in tibia becomes disordered at P21, not in femur. But at P70, the changes are severer in femur than tibia. Chondrocyte proliferation and differentiation are attenuated and asynchronous in both tibia and femur at P7 and P21. Furthermore, DDB1 deficiency induces p27 upregulation and subsequent cell cycle arrest in primary chondrocytes. Therefore, our data reveal that DDB1 is essential for the skeleton development by controlling chondrocyte proliferation and differentiation.

GR/Sp3/HDAC1/UGDH signaling participated in the maternal dexamethasone-induced dysplasia of the rat fetal growth plate.

Maternal dexamethasone decreases the body length of the newborn. However, whether dexamethasone inhibits the development of the growth plate of the fetal long bone is still unknown. Here, we found that lengths of fetal femur and growth plate were both shorter in the fetuses with maternal dexamethasone (0.2 mg/kg.d from gestation day 9 to 20), with a decreased proteoglycan content of the growth plate in the fetal rat. Notable decreases in both the gene expression and H3K9 acetylation of UDP-glucose dehydrogenase (Ugdh) gene, which codes a key enzyme in the proteoglycan biosynthesis in the chondrocyte, were also observed. Meanwhile, up-regulation of glucocorticoid receptor (GR), specific protein 3 (Sp3), and histone deacetylase 1 (Hdac1) gene expression were detected in the fetal growth plate. Similar changes were also observed in the chondrogenic rat bone marrow stromal cells (BMSCs) with excessive exogenous dexamethasone. However, antagonizing GR with RU486 and silencing Hdac1 or Sp3 with specific siRNAs could all stimulate the H3K9 acetylation and gene expression of Ugdh previously inhibited by dexamethasone. Meanwhile, dexamethasone also induced the nuclear translocation of GR, which further directly bound to the Ugdh promoter and interacted with HDAC1 and Sp3, respectively. Collectively, our study revealed that maternal dexamethasone induced the direct binding of GR to the Ugdh promoter of the chondrocyte in the rat fetal growth plate, which recruited HDAC1 and Sp3, induced deacetylation of the H3K9, and subsequently inhibited Ugdh gene expression. Such changes further led to attenuated proteoglycan synthesis in the developing chondrocyte and therefore disrupted the development of growth plate and fetal long bone.

miR-143 is implicated in growth plate injury by targeting IHH in precartilaginous stem cells.

Precartilaginous stem cells (PCSCs) are able to initiate chondrocyte and bone development. The present study aimed to investigate the role of miR-143 and the underlying mechanisms involved in PCSC proliferation. In a rat growth plate injury model, tissue from the injury site was collected and the expression of miR-143 and its potential targets was determined. PCSCs were isolated from the rabbits' distal epiphyseal growth plate. Cell viability, DNA synthesis, and apoptosis were determined with MTT, BrdU, and flow cytometric analysis, respectively. Real time PCR and western blot were performed to detect the mRNA and protein expression of the indicated genes. Indian hedgehog (IHH) was identified as a target gene for miR-143 with luciferase reporter assay. Decreased expression of miR-143 and increased expression of IHH gene were observed in the growth plate after injury. miR-143 mimics decreased cell viability and DNA synthesis and promoted apoptosis of PCSCs. Conversely, siRNA-mediated inhibition of miR-143 led to increased growth and suppressed apoptosis of PCSCs. Transfection of miR-143 decreased luciferase activity of wild-type IHH but had no effect when the 3'-UTR of IHH was mutated. Furthermore, the effect of miR-143 overexpression was neutralized by overexpression of IHH. Our study showed that miR-143 is involved in growth plate behavior and regulates PCSC growth by targeting IHH, suggesting that miR-143 may serve as a novel target for PCSC-related diseases.

Cluster of differentiation 147 (CD147) expression is linked with thiram induced chondrocyte's apoptosis via Bcl-2/Bax/Caspase-3 signalling in tibial growth plate under chlorogenic acid repercussion.

Tibial dyschondroplasia (TD) is a metabolic disease of young poultry that affects bone andcartilage's growth. It mostly occurs in broilers due to thiram toxicity in the feed. In this disease, tibial cartilage is not yet ripe for ossification, but it also results in lameness, death, and moral convictions of commercial poultry due to numerous apoptotic changes on cell level. These changes serve a cardinal role in this situation. Many potential problems indicate that chlorogenic acid (CGA) performs an extensive role in controlling apoptosis's perception. However, the actual role of CGA in TD affected chondrocytes in-vitro is still unidentified. The current study investigates the imperceptible insight of CGA on chondrocyte's apoptosis via B-cell lymphoma 2 (Bcl-2), Bcl-2 associated x-protein (Bax), and Caspase-3 with CD147 signalling. The expression of these markers was investigated by Immunofluorescence, western blot analysis, and reverse transcription-quantitative polymerase chain (RT-qPCR). Chondrocytes from the growth plate of tibia were isolated, cultured, and processed. A sub-lethal thiram (2.5 µg/mL) was used to induce cytotoxicity and then treated with an optimum dose (40 µg/ mL) of CGA. According to the results, thiram distorted chondrocyte cells with enhanced apoptotic rate. But, in case of CGA, high expression of CD147 enhanced cell viability of chondrocytes, accompanied by downregulation of Bax/Caspase-3 signalling with the upregulation of Bcl-2. The first possibility has ruled out in the present study by the observation that the cells apoptosis marker, Caspase-3 showed a significant change in CD147 overexpressing cells. Conversely, immunodepletion of CD147 with enhanced cleavage of Caspase-3, indicating the activation of apoptosis in chondrocytes cells. Therefore, these findings suggest a novel insight about CD147 in thiram induced TD about the regulation of Bcl-2/Bax/Caspase-3 apoptosis-signalling axis.

Osteochondral Allograft Transplantation of the Knee in Adolescent Patients and the Effect of Physeal Closure.

PURPOSE: The primary aim was to compare osteochondral allograft (OCA) transplantation outcomes between adolescent patients aged 16 years or younger and those older than 16 years. A secondary aim was to analyze the association between physeal closure status and outcomes. METHODS: Consecutive patients aged 18 years or younger who underwent OCA transplantation with a minimum 2-year follow-up were identified from a prospectively collected database. Patients were divided into 2 groups: those aged 16 years or younger (group 1) and those aged 17 to 18 years (group 2). Outcomes included patient-reported outcomes (PROs), complications, reoperations, and cartilage revision surgery. Outcomes were compared between groups, and physeal status was analyzed as a prognostic indicator. RESULTS: A total of 36 patients met the inclusion criteria: 18 in group 1 and 18 in group 2. There were no significant differences between the groups in terms of demographic characteristics, prior surgical procedures, and surgical details, including concomitant procedures. The mean overall follow-up period was 4.6 ± 2.5 years (range, 2-10.3 years), with no significant difference between the groups (P = .21). There were 10 reoperations (28.8%), 4 in group 1 and 6 in group 2 (P = .47). The overall time to reoperation was 2.8 years and did not significantly differ between groups (P = .75). The failure rate was 5.6%, with 1 patient in each group undergoing either graft debridement or revision OCA transplantation. All PROs were significantly improved postoperatively (P < .05), except for the Western Ontario and McMaster Universities Arthritis Index stiffness score (P = .28) and the Short Form 12 mental score (P = .19). There were no significant between-group differences in terms of PROs. Patients with closed physes had a significantly greater increase in most PROs compared with patients with open physes (P < .05). CONCLUSIONS: OCA transplantation in adolescents results in significant PRO score improvement and a low failure rate, albeit reoperations are not uncommon. Patients with closed physes show greater PRO score improvement than those with open physes. LEVEL OF EVIDENCE: Level III, retrospective comparative study.

Anterior and distal tunnel orientation for anatomic reconstruction of the medial patellofemoral ligament is safer in patients with open growth plates.

PURPOSE: In patients with open growth plates, the direction of tunneling that avoids distal femoral physis (DFP) damage in anatomic reconstructions of the medial patellofemoral ligament (MPFL) has been a topic of discussion. The objective of this study was to determine the ideal orientation for anatomic reconstructions of MPFL tunneling that minimized DFP damage while avoiding breaching the intercondylar notch. METHODS: Eighty magnetic resonance images of patients aged 10 through 17 were obtained, randomly sampled from the institutional database. A de novo software was developed to obtain 3D models of the distal femur and DFP. In each model, the anatomical insertion point of the MPFL was determined as defined by Stephen. A 20-mm-depth drilling was simulated, starting from the insertion point at every possible angle within a 90° cone using 5-, 6- and 7-mm drills. Physeal damage for each pair of angles and each drill size was determined. Damage was expressed as a percentage of total physis volume. Statistical analysis was conducted using Student's t test and one-way ANOVA. RESULTS: Maximum physeal damage (5.35% [4.47-6.24]) was obtained with the 7-mm drill when drilling 3° cephalic and 15° posterior from insertion without differences between sexes (n.s.). Minimal physeal damage (0.22% [0.07-0.37]) was obtained using the 5-mm drill aimed 45° distal and 0° anteroposterior, not affected by sex (n.s.). Considering intra-articular drilling avoidance, the safest zone was obtained when aiming 30°-40° distal and 5°-35° anterior, regardless of sex. CONCLUSION: Ideal femoral tunnel orientation, avoiding physeal damage and breaching of the intercondylar notch, was obtained when aiming 30°-40° distal and 5°-35° anterior, regardless of sex. This area is a safe zone that allows anatomic MPFL reconstruction of patients with an open physis.

Comparison of histomorphometric and radiographic effects of growth guidance with tension-band devices (eight-Plate and FlexTack) in a pig model.

Background and purpose - Temporary hemiepiphysiodesis for growth modulation in skeletally immature patients is a long-known technique. Recently the use of tension-band devices has become popular. This study compares 2 tension-band implants (eight-Plate and FlexTack) regarding their effects on the growth plate.Animals and methods - 12 pigs in 2 equally sized groups (A and B) were investigated. The right proximal medial tibia was treated with either eight-Plate or FlexTack. The left tibia of the same pig was treated with the opposite implant. After 9 weeks all implants were removed. Animals in group B were then hosted for another 5 weeks. Histomorphometric analysis of the growth plate was carried out after 9 and 14 weeks, respectively. Radiographs were taken at implantation, removal, and after 14 weeks.Results - Both tension-band devices achieved a statistically significant and clinically relevant growth inhibition, whereas the effect appeared to be more distinct after the use of FlexTack. Implant-related complications or physeal damage was not observed. After implant removal, rebound phenomenon was radiologically observed in all cases. The growth plates treated with eight-Plate showed a paradox reversal of the zonal distributions, with an increase of the proliferative zones at the previously arrested medial aspect of the physis and a decrease laterally.Interpretation - Both eight-Plate and FlexTack proved to be appropriate devices for growth-guiding treatment. The radiographic evaluation showed a change in angular axes after treatment with each implant, while the correction appeared to be faster with FlexTack. The paradox cartilaginous reaction observed after removal of the eight-Plate might be a histopathological correlate for rebound phenomenon.

An essential role for IGF2 in cartilage development and glucose metabolism during postnatal long bone growth.

Postnatal bone growth involves a dramatic increase in length and girth. Intriguingly, this period of growth is independent of growth hormone and the underlying mechanism is poorly understood. Recently, an IGF2 mutation was identified in humans with early postnatal growth restriction. Here, we show that IGF2 is essential for longitudinal and appositional murine postnatal bone development, which involves proper timing of chondrocyte maturation and perichondrial cell differentiation and survival. Importantly, the Igf2 null mouse model does not represent a simple delay of growth but instead uncoordinated growth plate development. Furthermore, biochemical and two-photon imaging analyses identified elevated and imbalanced glucose metabolism in the Igf2 null mouse. Attenuation of glycolysis rescued the mutant phenotype of premature cartilage maturation, thereby indicating that IGF2 controls bone growth by regulating glucose metabolism in chondrocytes. This work links glucose metabolism with cartilage development and provides insight into the fundamental understanding of human growth abnormalities.

Effects of mild hyperbaric oxygen on osteoporosis induced by hindlimb unloading in rats.

INTRODUCTION: Disuse-induced bone loss is caused by a suppression of osteoblastic bone formation and an increase in osteoclastic bone resorption. There are few data available for the effects of environmental conditions, i.e., atmospheric pressure and/or oxygen concentration, on osteoporosis. This study examined the effects of mild hyperbaric oxygen at 1317 hPa with 40% oxygen on unloading-induced osteoporosis. MATERIALS AND METHODS: Eighteen 8-week old male Wistar rats were randomly divided into three groups: the control for 21 days without unloading and mild hyperbaric oxygen (NOR, n = 6), the unloading for 21 days and recovery for 10 days without mild hyperbaric oxygen (HU + NOR, n = 6), and the unloading for 21 days and recovery for 10 days with mild hyperbaric oxygen (HU + MHO, n = 6). RESULTS: The cortical thickness and trabecular bone surface area were decreased in the HU + NOR group compared to the NOR group. There were no differences between the NOR and HU + MHO groups. Osteoclast surface area and Sclerostin (Sost) mRNA expression levels were decreased in the HU + MHO group compared to the HU + NOR group. These results suggested that the loss of the cortical and trabecular bone is inhibited by mild hyperbaric oxygen, because of an inhibition of osteoclasts and enhancement of bone formation with decreased Sost expression. CONCLUSIONS: We conclude that exposure to mild hyperbaric oxygen partially protects from the osteoporosis induced by hindlimb unloading.

Elevated hypertrophy, growth plate maturation, glycosaminoglycan deposition, and exostosis formation in the Hspg2 exon 3 null mouse intervertebral disc.

Heparan sulfate (HS) regulates diverse cell signalling events in intervertebral disc development and homeostasis. The aim of the present study was to investigate the effect of ablation of perlecan HS/CS on murine intervertebral disc development. Genetic models carrying mutations in genes encoding HS biosynthetic enzymes have identified multiple roles for HS in tissue homeostasis. In the present study, we utilised an Hspg2 exon 3 null HS/CS-deficient mouse to assess the role of perlecan HS in disc cell regulation. HS makes many important contributions to growth factor sequestration, stabilisation/delivery, and activation of receptors directing cellular proliferation, differentiation, and assembly of extracellular matrix. Perlecan HS/CS-mediated interactions promote extracellular matrix assembly/stabilisation and tissue functional properties, and thus, removal of perlecan HS/CS should affect extracellular matrix function and homeostasis. Hspg2 exon 3 null intervertebral discs accumulated significantly greater glycosaminoglycan in the nucleus pulposus, annulus fibrosus, and vertebral growth plates than C57BL/6 wild-type (WT) I intervertebral discs. Proliferation of intervertebral disc progenitor cells was significantly higher in Hspg2 exon 3 null intervertebral discs, and these cells became hypertrophic by 12 weeks of age and were prominent in the vertebral growth plates but had a disorganised organisation. C57BL/6 WT vertebral growth plates contained regular columnar growth plate chondrocytes. Exostosis-like, ectopic bone formation occurred in Hspg2 exon 3 null intervertebral discs, and differences were evident in disc cell maturation and in matrix deposition in this genotype, indicating that perlecan HS/CS chains had cell and matrix interactive properties which repressively maintained tissue homeostasis in the adult intervertebral disc.

Unsuspected osteochondroma-like outgrowths in the cranial base of Hereditary Multiple Exostoses patients and modeling and treatment with a BMP antagonist in mice.

Hereditary Multiple Exostoses (HME) is a rare pediatric disorder caused by loss-of-function mutations in the genes encoding the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. HME is characterized by formation of cartilaginous outgrowths-called osteochondromas- next to the growth plates of many axial and appendicular skeletal elements. Surprisingly, it is not known whether such tumors also form in endochondral elements of the craniofacial skeleton. Here, we carried out a retrospective analysis of cervical spine MRI and CT scans from 50 consecutive HME patients that included cranial skeletal images. Interestingly, nearly half of the patients displayed moderate defects or osteochondroma-like outgrowths in the cranial base and specifically in the clivus. In good correlation, osteochondromas developed in the cranial base of mutant Ext1f/f;Col2-CreER or Ext1f/f;Aggrecan-CreER mouse models of HME along the synchondrosis growth plates. Osteochondroma formation was preceded by phenotypic alteration of cells at the chondro-perichondrial boundary and was accompanied by ectopic expression of major cartilage matrix genes -collagen 2 and collagen X- within the growing ectopic masses. Because chondrogenesis requires bone morphogenetic protein (BMP) signaling, we asked whether osteochondroma formation could be blocked by a BMP signaling antagonist. Systemic administration with LDN-193189 effectively inhibited osteochondroma growth in conditional Ext1-mutant mice. In vitro studies with mouse embryo chondrogenic cells clarified the mechanisms of LDN-193189 action that turned out to include decreases in canonical BMP signaling pSMAD1/5/8 effectors but interestingly, concurrent increases in such anti-chondrogenic mechanisms as pERK1/2 and Chordin, Fgf9 and Fgf18 expression. Our study is the first to reveal that the cranial base can be affected in patients with HME and that osteochondroma formation is amenable to therapeutic drug intervention.

Risk Factors for Contralateral Slipped Capital Femoral Epiphysis: A Meta-analysis of Cohort and Case-control Studies.

BACKGROUND: Slipped capital femoral epiphysis (SCFE) is an important cause of hip pain and disability in pediatric patients. SCFE occurs bilaterally in 12% to 80% of cases, and the risk of contralateral SCFE is noted to be 2335 times higher than the index SCFE. Several studies have reported risk factors for contralateral SCFE; however, these studies have not been systematically analyzed. The purpose of this systematic review and meta-analysis was to review and analyze risk factors for subsequent contralateral SCFE and identify the strongest risk factors for a subsequent slip. METHODS: A systematic review was performed of all observational studies focusing on risk factors for subsequent contralateral SCFE indexed in Medline, Embase, and Cochrane databases. Data extraction was performed and summarized using descriptive statistics. Meta-analysis was performed for risk factors with sufficient constituent study data. Quality assessment was performed using the Newcastle-Ottawa Scale, and funnel plots were generated to assess publication bias. RESULTS: The initial search strategy identified 226 references, and after exclusions, 20 studies were included in this analysis. Demographic risk factors included age, sex, weight, body mass index, ethnicity, and urban/rural residence; clinical risk factors included endocrine abnormality, duration of symptoms, slip stability, and slip chronicity; and radiographic risk factors included slip angle, triradiate cartilage, alpha angle, posterior sloping angle (PSA), physeal sloping angle, modified Oxford score, and bone age. Younger patient age, body mass index≥95th percentile, presence of an endocrine abnormality, higher PSA of the unaffected hip, and lower modified Oxford score have been noted to be significant risk factors for contralateral SCFE. Meta-analysis showed that younger age (-0.9; confidence interval, -1.1, -0.6), and higher PSA (4.7 degrees; 95% confidence interval, 3.3-6.2 degrees) of the unaffected hip were predictive of subsequent contralateral SCFE. The majority of studies were of good quality. CONCLUSION: There are several risk factors for subsequent contralateral SCFE. On the basis of the available data, younger patients with a high PSA of the unaffected hip would most likely benefit from prophylactic fixation of the unaffected hip. LEVEL OF EVIDENCE: Level II.

A Comparison of Transphyseal Neck-Head Tunneling and Multiple Epiphyseal Drilling on Femoral Head Healing Following Ischemic Osteonecrosis: An Experimental Investigation in Immature Pigs.

BACKGROUND: Two operative procedures are currently advocated to stimulate the necrotic femoral head healing in children with Legg-Calve-Perthes disease: transphyseal neck-head tunneling (TNHT) and multiple epiphyseal drilling (MED). The purpose of this study was to compare the bone healing and physeal function after treatment with TNHT or MED in a piglet model of ischemic osteonecrosis. METHODS: Eighteen piglets were induced with osteonecrosis by surgically placing a ligature tightly around the right femoral neck. One week later, the piglets were assigned to 1 of 3 treatment groups (n=6/group): (1) local nonweight bearing only (NWB), (2) TNHT plus NWB, or (3) MED plus NWB. The unoperated left femoral heads were used as normal controls. The animals were euthanized at 8 weeks after osteonecrosis induction. Histologic, histomorphometric, radiographic, microcomputed tomography (CT), and calcein-labeling assessments were performed. Statistical analysis included a 1-way ANOVA. RESULTS: Micro-CT analyses showed higher femoral head bone volume in the MED group compared with the TNHT and the NWB groups (P<0.01). The MED group had a higher mean trabecular number (P<0.001) and new bone formation (P=0.001) based on calcein-labeling parameters compared with the TNHT and the NWB groups. In addition, the osteoclast number per bone surface was lower in the MED group compared with the NWB group (P=0.001). Histologic and micro-CT assessments of the proximal femoral physis revealed a larger physeal disruption at the site of physeal drilling in the TNHT group compared with the MED group. However, no significant differences in physeal elongation (P=0.61) and femoral neck length (P=0.31) were observed between the treatment groups. CONCLUSIONS: MED produced a higher bone volume and stimulated greater bone formation than the TNHT or the NWB alone. Both procedures did not produce a significant physeal growth disturbance during the study period. CLINICAL RELEVANCE: This preclinical study provides evidence that MED produces more favorable bone healing than the TNHT in a large animal model of Legg-Calve-Perthes disease.