Skeletal growth is enhanced by a shared role for SOX8 and SOX9 in promoting reserve chondrocyte commitment to columnar proliferation.

SOX8 was linked in a genome-wide association study to human height heritability, but roles in chondrocytes for this close relative of the master chondrogenic transcription factor SOX9 remain unknown. We undertook here to fill this knowledge gap. High-throughput assays demonstrate expression of human SOX8 and mouse Sox8 in growth plate cartilage. In situ assays show that Sox8 is expressed at a similar level as Sox9 in reserve and early columnar chondrocytes and turned off when Sox9 expression peaks in late columnar and prehypertrophic chondrocytes. Sox8-/- mice and Sox8fl/flPrx1Cre and Sox9fl/+Prx1Cre mice (inactivation in limb skeletal cells) have a normal or near normal skeletal size. In contrast, juvenile and adult Sox8fl/flSox9fl/+Prx1Cre compound mutants exhibit a 15 to 20% shortening of long bones. Their growth plate reserve chondrocytes progress slowly toward the columnar stage, as witnessed by a delay in down-regulating Pthlh expression, in packing in columns and in elevating their proliferation rate. SOX8 or SOX9 overexpression in chondrocytes reveals not only that SOX8 can promote growth plate cell proliferation and differentiation, even upon inactivation of endogenous Sox9, but also that it is more efficient than SOX9, possibly due to greater protein stability. Altogether, these findings uncover a major role for SOX8 and SOX9 in promoting skeletal growth by stimulating commitment of growth plate reserve chondrocytes to actively proliferating columnar cells. Further, by showing that SOX8 is more chondrogenic than SOX9, they suggest that SOX8 could be preferred over SOX9 in therapies to promote cartilage formation or regeneration in developmental and degenerative cartilage diseases.

Wnt7b expressed by hypertrophic chondrocytes is a stimulatory factor for endochondral ossification that is regulated by Smad4 activity.

Endochondral ossification contributes to longitudinal skeletal growth. Osteoblasts, which are bone-forming cells, appear close to terminally differentiated hypertrophic chondrocytes during endochondral ossification. We established mice with conditional knockout (cKO) of Smad4, an essential co-activator for transforming growth factor ß family signaling. The mice showed a marked increase in bone volume in the metaphysis as a result of increased bone formation by osteoblasts, in which ß-catenin, an effector of canonical Wnt signaling, accumulated. We identified Wnt7b as a factor with increased expression in growth plate cartilage in Smad4 cKO mice. Wnt7b mRNA was expressed in differentiated chondrocytes and suppressed by BMP4 stimulation. Ablation of Wnt7b blunted the increase in bone in adult Smad4 cKO mice and reduced skeletal growth in juvenile mice. Overall, we conclude that Wnt7b is a crucial factor secreted from hypertrophic chondrocytes to initiate endochondral ossification. These results suggest that Smad4-dependent BMP signaling regulates the Wnt7b-ß-catenin axis during endochondral ossification.

Pthlha and mechanical force control early patterning of growth zones in the zebrafish craniofacial skeleton.

Secreted signals in patterning systems often induce repressive signals that shape their distributions in space and time. In developing growth plates (GPs) of endochondral long bones, Parathyroid hormone-like hormone (Pthlh) inhibits Indian hedgehog (Ihh) to form a negative-feedback loop that controls GP progression and bone size. Whether similar systems operate in other bones and how they arise during embryogenesis remain unclear. We show that Pthlha expression in the zebrafish craniofacial skeleton precedes chondrocyte differentiation and restricts where cells undergo hypertrophy, thereby initiating a future GP. Loss of Pthlha leads to an expansion of cells expressing a novel early marker of the hypertrophic zone (HZ), entpd5a, and later HZ markers, such as ihha, whereas local Pthlha misexpression induces ectopic entpd5a expression. Formation of this early pre-HZ correlates with onset of muscle contraction and requires mechanical force; paralysis leads to loss of entpd5a and ihha expression in the pre-HZ, mislocalized pthlha expression and no subsequent ossification. These results suggest that local Pthlh sources combined with force determine HZ locations, establishing the negative-feedback loop that later maintains GPs.

Developmental principles informing human pluripotent stem cell differentiation to cartilage and bone.

Human pluripotent stem cells can differentiate into any cell type given appropriate signals and hence have been used to research early human development of many tissues and diseases. Here, we review the major biological factors that regulate cartilage and bone development through the three main routes of neural crest, lateral plate mesoderm and paraxial mesoderm. We examine how these routes have been used in differentiation protocols that replicate skeletal development using human pluripotent stem cells and how these methods have been refined and improved over time. Finally, we discuss how pluripotent stem cells can be employed to understand human skeletal genetic diseases with a developmental origin and phenotype, and how developmental protocols have been applied to gain a better understanding of these conditions.

Muscle forces acting on the greater trochanter lead to a dorsal warping of the apophyseal growth plate.

The apophyseal growth plate of the greater trochanter, unlike most other growth plates of the human body, exhibits a curved morphology that results in a divergent pattern resembling an open crocodile mouth on plain antero-posterior radiographs. To quantify the angular alignment of the growth plate and to draw conclusions about the function of the muscles surrounding it, we analyzed 57 MRI images of 51 children and adolescents aged 3-17 years and of six adults aged 18-52 years. We measured the angulation of the plate relative to the horizontal plane (AY angle) and the trajectories of the muscles attaching to the greater trochanter of the proximal femur. From anterior to posterior, the AY angle shows a decrease of 33.44°. In the anterior third, the cartilage is angled at a mean of 51.64°, and in the posterior third, the mean angulation is 18.6°. This indicates that the cartilage in the anterior region of the greater trochanteric apophysis is subject to more vertically oriented force vectors compared to the posterior region, as the growth plates align perpendicular to the force vectors acting on them. Combining the measured muscle trajectories with the physiological cross-sectional areas (PCSA) available from the literature revealed that, in addition to the known internal and external lateral traction ligament systems, a third, dorsally located traction ligament system exists that may be responsible for the dorsal deformation of the AY angle.

Inadequate linear catch-up growth in children born small for gestational age: Influencing factors and underlying mechanisms.

A minority of children born small for gestational age (SGA) may experience catch-up growth failure and remain short in adulthood. However, the underlying causes and mechanisms of this phenomenon are not yet fully comprehended. We reviewed the present state of research concerning the growth hormone-insulin-like growth factor axis and growth plate in SGA children who fail to achieve catch-up growth. Additionally, we explored the factors influencing catch-up growth in SGA children and potential molecular mechanisms involved. Furthermore, we considered the potential benefits of supplementary nutrition, specific dietary patterns, probiotics and drug therapy in facilitating catch-up growth.

Regulatory Pathways in Growth Plate Chondrocytes that Are Impacted by Matrix Vesicle microRNA Identified by Targeted RISC Pulldown and Sequencing of the Resulting Transcriptome.

During endochondral bone formation, growth plate chondrocytes are differentially regulated by various factors and hormones. As the cellular phenotype changes, the composition of the extracellular matrix is altered, including the production and composition of matrix vesicles (MV) and their cargo of microRNA. The regulatory functions of these MV microRNA in the growth plate are still largely unknown. To address this question, we undertook a targeted bioinformatics approach. A subset of five MV microRNA was selected for analysis based on their specific enrichment in these extracellular vesicles compared to the parent cells (miR-1-3p, miR-22-3p, miR-30c-5p, miR-122-5p, and miR-133a-3p). Synthetic biotinylated versions of the microRNA were produced using locked nucleic acid (LNA) and were transfected into rat growth plate chondrocytes. The resulting LNA to mRNA complexes were pulled down and sequenced, and the transcriptomic data were used to run pathway analysis pipelines. Bone and musculoskeletal pathways were discovered to be regulated by the specific microRNA, notably those associated with transforming growth factor beta (TGFß) and Wnt pathways, cell differentiation and proliferation, and regulation of vesicles and calcium transport. These results can help with understanding the maturation of the growth plate and the regulatory role of microRNA in MV.

Generalizing Diffusion Tensor Imaging of the Physis and Metaphysis.

BACKGROUND: Current methods to predict height potential are inaccurate. Predicting height by using MRI of the physeal cartilage has shown promise but the applicability of this technique in different imaging setups has not been well-evaluated. PURPOSE: To assess variability in diffusion tensor imaging of the physis and metaphysis (DTI-P/M) of the distal femur between different scanners, imaging parameters, tractography software, and resolution. STUDY TYPE: Prospective. POPULATION/SUBJECTS: Eleven healthy subjects (five males and six females ages 10-16.94). FIELD STRENGTH/SEQUENCE: 3 T; DTI single shot echo planar sequences. ASSESSMENT: Physeal DTI tract measurements of the distal femur were compared between different scanners, imaging parameters, tractography settings, interpolation correction, and tractography software. STATISTICAL TESTS: Bland-Altman, Spearman correlation, linear regression, and Shapiro-Wilk tests. Threshold for statistical significance was set at P = 0.05. RESULTS: DTI tract values consistently showed low variability with different imaging and analysis settings. Vendor to vendor comparison exhibited strong correlation (ρ = 0.93) and small but significant bias (bias -5.76, limits of agreement [LOA] -24.31 to 12.78). Strong correlation and no significant difference were seen between technical replicates of the General Electric MRI scanner (ρ = 1, bias 0.17 [LOA -1.5 to 1.2], P = 0.42) and the Siemens MRI scanner (ρ = 0.89, bias = 0.56, P = 0.71). Different voxel sizes (1 × 1 × 2 mm3 vs. 2 × 2 × 3 mm3) did not significantly affect DTI values (bias = 1.4 [LOA -5.7 to 8.4], P = 0.35) but maintained a strong correlation (ρ = 0.82). Gap size (0 mm vs. 0.6 mm) significantly affects tract volume (bias = 1.8 [LOA -5.4 to 1.8]) but maintains a strong correlation (ρ = 0.93). Comparison of tractography algorithms generated significant differences in tract number, length, and volume while maintaining correlation (ρ = 0.86, 0.99, 0.93, respectively). Comparison of interobserver variability between different tractography software also revealed significant differences while maintaining high correlation (ρ = 0.85-0.98). DATA CONCLUSION: DTI of the pediatric physis cartilage shows high reproducibility between different imaging and analytic parameters. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 1.

Depletion of b-series ganglioside prevents limb length discrepancy after growth plate injury.

INTRODUCTION: Growth plate damage in long bones often results in progressive skeletal growth imbalance and deformity, leading to significant physical problems. Gangliosides, key glycosphingolipids in cartilage, are notably abundant in articular cartilage and regulate chondrocyte homeostasis. This suggests their significant roles in regulating growth plate cartilage repair. METHODS: Chondrocytes from 3 to 5 day-old C57BL/6 mice underwent glycoblotting and mass spectrometry. Based on the results of the glycoblotting analysis, we employed GD3 synthase knockout mice (GD3-/-), which lack b-series gangliosides. In 3-week-old mice, physeal injuries were induced in the left tibiae, with right tibiae sham operated. Tibiae were analyzed at 5 weeks postoperatively for length and micro-CT for growth plate height and bone volume at injury sites. Tibial shortening ratio and bone mineral density were measured by micro-CT. RESULTS: Glycoblotting analysis indicated that b-series gangliosides were the most prevalent in physeal chondrocytes among ganglioside series. At 3 weeks, GD3-/- exhibited reduced tibial shortening (14.7 ± 0.2 mm) compared to WT (15.0 ± 0.1 mm, P = 0.03). By 5 weeks, the tibial lengths in GD3-/- (16.0 ± 0.4 mm) closely aligned with sham-operated lengths (P = 0.70). Micro-CT showed delayed physeal bridge formation in GD3-/-, with bone volume measuring 168.9 ± 5.8 HU at 3 weeks (WT: 180.2 ± 3.2 HU, P = 0.09), but normalizing by 5 weeks. CONCLUSION: This study highlights that GD3 synthase knockout mice inhibit physeal bridge formation after growth plate injury, proposing a new non-invasive approach for treating skeletal growth disorders.

Imaging of sports injuries in adolescents.

Musculoskeletal injuries in adolescents tend to occur in particular locations and have distinct characteristics, as they affect an immature skeleton. Increased engagement in sports, extended training and competition periods, and early specialization in specific sports, among other factors, have contributed significantly to the rise in musculoskeletal sports injuries in adolescents. Furthermore, females show a particularly pronounced increase in sports participation, where anatomical and hormonal factors play crucial roles in the development and increased frequency of sports-related injuries. Consequently, there is a growing demand for diagnostic imaging techniques. Musculoskeletal and pediatric radiologists require a comprehensive understanding of intrinsic and extrinsic risk factors and the successive stages of skeletal development that can influence the specific characteristics of sports injuries in adolescents. These aspects are crucial for the diagnostic, prognostic, and therapeutic management of these injuries and for mitigating chronic conditions that could compromise future sports participation. This review analyzes the primary musculoskeletal injuries in adolescent athletes and highlights the pivotal role of different imaging methods in their diagnosis and management.

Stressed or fractured: MRI differentiating indicators of physeal injury.

OBJECTIVE: To identify MRI findings that can indicate chronic physeal stress injury and differentiate it from acute Salter-Harris (SH) fracture of the pediatric knee or wrist. METHODS: IRB-approved retrospective study of consecutively selected knee and wrist MRIs from 32 athletes with chronic physeal stress injury and 30 children with acute SH fracture. MRI characteristics (physeal patency, physeal thickening, physeal signal intensity (SI), continuity of the zone of provisional calcification (ZPC), integrity of the periosteum and/or perichondrium, pattern of periphyseal and soft tissue edema signal, and joint effusion) were compared. RESULTS: Forty-eight chronic physeal stress injuries (mean age 13.1 years [8.2-17.5 years]) and 35 SH fractures (mean age 13.3 years [5.1-16.0 years]) were included. Any physeal thickening was more common with chronic stress injury (98% vs 77%, p = 0.003). Abnormal physeal SI was more common with SH fractures (91% vs 67%, p = 0.008). ZPC discontinuity strongly suggested chronic stress injury (79% vs 49%, p < 0.004). Periosteal and/or perichondrial elevation or rupture and soft tissue edema characterized most of the acute SH fractures (p < 0.001) and were seen only in 1 chronic stress injury (< 2%). While periphyseal edema was not significantly different in the two groups (p = 0.890), a joint effusion was associated with acute SH fracture (p < 0.001). CONCLUSION: Chronic physeal stress injury of the pediatric knee and wrist shows higher incidence of ZPC discontinuity and focal physeal thickening compared to SH fracture, reflecting disruption in normal endochondral ossification. However, these findings can overlap in the 2 groups. Periosteal and/or perichondrial injury, soft tissue edema signal, and joint effusion strongly suggest SH fracture and are rarely present with chronic stress injury.

Youth soccer players: patterns of injury involving the primary growth plates of epiphyses.

Youth soccer (football) is immensely popular internationally. Earlier participation, sport sub-specialization, and year-around practice have led to an increased incidence of injury from both acute trauma and repetitive overuse. The growth plates (physes) of the immature skeleton are particularly vulnerable to injury and delayed diagnosis can lead to future growth disturbance and long-term morbidity. Familiarity with the various components of the growth plate complex necessary for ensuring normal endochondral ossification is fundamental in understanding the various patterns of imaging findings following injury. This review discusses the zonal columnar arrangement of the growth plate proper and the contrasting function of the vasculature within the subjacent epiphysis and metaphysis. This is followed by an evidence-based discussion of the common patterns of injury involving the epiphyseal primary growth plate observed among youth soccer players: subcategorized into physeal fractures (direct injury) and physeal stress injuries (indirect insult to subjacent metaphysis). In this section, the role of imaging and characteristic imaging features will be discussed. While the normal physiologic and pathophysiologic mechanisms can be applied to other growth plates, such as primary growth plates underlying the apophyses and secondary growth plates surrounding the secondary ossificiation centers, which also undergo endochondral ossification, the current review is focused on injuries involving the primary growth plates underlying epiphyses.

The regenerative treatment of costal osteochondral graft implantation for partial growth arrest using a rabbit model.

PURPOSE: To investigate the feasibility of cylindrical costal osteochondral graft transplantation as a novel regenerative treatment in growth arrest. METHODS: The medial portion of the proximal tibial growth plate of 6-week-old male New Zealand White rabbits was resected to establish an experimental model of partial growth plate injury. The rabbits were divided into four groups: no-treatment, bone wax transplantation, costal chondral graft, and costal osteochondral graft groups. Radiographic and micro-computed tomography scan results were analyzed to evaluate angular deformity of the tibia and bony bridge formation at the injury site. In addition, repair of the injured growth plate cartilage was assessed histologically at 4, 8, and 12 weeks postoperatively. RESULTS: Radiographic examination revealed that bone wax transplantation continuously decreased the medial proximal tibial angle (MPTA) while the costal chondral graft implantation reduced the decrease of MPTA at 12 weeks postoperatively. The costal osteochondral graft implantation recovered the MPTA, close to the normal. Histologically, the costal osteochondral grafts retained the MPTA in the injured site compared to costal chondral grafts. Additionally, hypertrophic chondrocytes were observed at the graft site in the costal osteochondral graft group at 12 weeks, suggesting that endochondral ossification may occur at the graft site similar to normal ossification. The fluorescence in situ hybridization analysis of osteochondral grafts transplanted from male to female rabbits indicated that they were replaced by cells of host origin. CONCLUSION: The costal osteochondral graft can achieve regeneration without bony bridge formation in partial growth plate injury.

Transphyseal arthroscopic anterior cruciate ligament reconstruction in children under 12 years.

BACKGROUND: The incidence of anterior cruciate ligament (ACL) injuries in children is on the rise. Despite this trend, the optimal management of these injuries remains a matter of ongoing debate. In this light, our study seeks to assess the clinical, radiological, and functional outcomes of transphyseal ACL reconstruction in preadolescent patients in the medium-term. METHODS: This prospective study included preadolescent patients aged up to 12 years who underwent ACL transphyseal reconstruction between 2010 and 2020 and had a minimum follow-up of 2 years. Clinical assessments encompassed joint stability and range of motion. Furthermore, leg length discrepancy (LLD) and femorotibial alignment were evaluated both clinically and radiologically using full-length lower limb standing radiographs. Pre- and postoperative functional outcomes were assessed using the International Knee Documentation Committee (IKDC) and Lysholm scales, and the return to normal sports activity was evaluated using the ACL-Return to Sport after Injury (ACL-RSI) scale. Complications and relevant follow-up data were also recorded. Statistical analyses were conducted to evaluate these outcomes. RESULTS: A total of 35 preadolescent patients, consisting of 24 males and 11 females, with a mean age at surgery of 11.2 ± 0.7 years (8.7-12), were included in the study. The mean follow-up was 52.3 ± 20.7 months (24.1-95.9). No significant growth disturbances or clinically relevant LLD were evidenced. All patients demonstrated clinically stable knees with full range of motion at the 2-year follow-up. There were statistically significant improvements in pre- and postoperative IKDC (39.3 ± 13.5 vs. 99.7 ± 0.8, p < 0.005) and Lysholm scores (48.2 ± 15.1 vs. 99.6 ± 1.4, p < 0.005). All but two patients were able to return to their pre-injury level of sports activity, with a mean ACL-RSI score of 93.5 ± 1.3. The analysis revealed an 8.6% rerupture rate and an 11.4% rate of contralateral ACL injuries, with 5-year survival rates of 92.3% and 88.8%, respectively. Subgroup analyses based on age, gender, surgical delay, or associated meniscal lesions did not reveal any significant differences in functional outcomes. Additionally, there was no discernible relationship between age or timing of ACL reconstruction and the risk of meniscal injuries. CONCLUSIONS: Our study reinforces the value of ACL reconstruction in skeletally immature preadolescent patients, with transphyseal technique proven to be a safe, effective, and technically simpler option, even for children under the age of 12. The findings indicate excellent functional outcomes, a high rate of successful return to sporting activities, and minimal to no incidence of growth-related complications in the medium-term. LEVEL OF EVIDENCE: Level II, prospective comparative cohort study, before and after intervention.

Comparison of Toxicities among Different Bumped Kinase Inhibitor Analogs for Treatment of Cryptosporidiosis.

Recent advances on the development of bumped kinase inhibitors for treatment of cryptosporidiosis have focused on the 5-aminopyrazole-4-carboxamide scaffold, due to analogs that have less hERG inhibition, superior efficacy, and strong in vitro safety profiles. Three compounds, BKI-1770, -1841, and -1708, showed strong efficacy in C. parvum infected mice. Both BKI-1770 and BKI-1841 had efficacy in the C. parvum newborn calf model, reducing diarrhea and oocyst excretion. However, both compounds caused hyperflexion of the limbs seen as dropped pasterns. Toxicity experiments in rats and calves dosed with BKI-1770 showed enlargement of the epiphyseal growth plate at doses only slightly higher than the efficacious dose. Mice were used as a screen to check for bone toxicity, by changes to the tibia epiphyseal growth plate, or neurological causes, by use of a locomotor activity box. These results showed neurological effects from both BKI-1770 and BKI-1841 and bone toxicity in mice from BKI-1770, indicating one or both effects may be contributing to toxicity. However, BKI-1708 remains a viable treatment candidate for further evaluation as it showed no signs of bone toxicity or neurological effects in mice.

Dysregulation of insulin-like growth factor-1 signaling in postnatal bone elongation.

Insulin-like growth factor-1 (IGF-1) is a critical modulator of cell growth and survival, making it a central part of maintaining essentially every biological system in the body. Knowledge of the intricate mechanisms involved in activating IGF-1 signaling is not only key to understanding basic processes of growth and development, but also for addressing diseases, such as cancer and diabetes. This brief review explores how dysregulation of normal IGF-1 signaling can impact growth by examining its role in postnatal bone elongation. IGF-1 actions are dysregulated in autoimmune diseases, such as juvenile idiopathic arthritis and chronic kidney disease, which results in growth stunting. Conversely, childhood obesity results in growth acceleration, premature growth cessation, and ultimately, diminished bone quality, while systemic IGF-1 levels remain normal. Understanding the role of IGF-1 signaling in normal and dysregulated growth can add to other studies that address how this system regulates chronic diseases.

Skeletal stem cells: insights into maintaining and regenerating the skeleton.

Skeletal stem cells (SSCs) generate the progenitors needed for growth, maintenance and repair of the skeleton. Historically, SSCs have been defined as bone marrow-derived cells with inconsistent characteristics. However, recent in vivo tracking experiments have revealed the presence of SSCs not only within the bone marrow but also within the periosteum and growth plate reserve zone. These studies show that SSCs are highly heterogeneous with regard to lineage potential. It has also been revealed that, during digit tip regeneration and in some non-mammalian vertebrates, the dedifferentiation of osteoblasts may contribute to skeletal regeneration. Here, we examine how these research findings have furthered our understanding of the diversity and plasticity of SSCs that mediate skeletal maintenance and repair.

Magnetic resonance and diffusion tensor imaging of the adolescent rabbit growth plate of the knee.

PURPOSE: To assess the ability of MRI-DTI to evaluate growth plate morphology and activity compared with that of histomorphometry and micro-CT in rabbits. METHODS: The hind limbs of female rabbits aged 16, 20, and 24 wk (n = 4 per age group) were studied using a 9.4T MRI scanner with a multi-gradient echo 3D sequence and DTI in 14 directions (b-value = 984 s/mm2 ). After MRI, the right and left hind limb were processed for histological analysis and micro-CT, respectively. The Wilcoxon signed-rank test was used to evaluate the height and volume of the growth plate. Intraclass correlation and Pearson correlation coefficient were used to evaluate the association between DTI metrics and age. RESULTS: The growth plate height and volume were similar for all modalities at each time point and age. Age was correlated with all tractography and DTI metrics in both the femur and tibia. A correlation was also observed between all the metrics at both sites. Tract number and volume declined with age; however, tract length did not show any changes. The fractional anisotropy color map showed lateral diffusion centrally in the growth plate and perpendicular diffusion in the hypertrophic zone, as verified by histology and micro-CT. CONCLUSION: MRI-DTI may be useful for evaluating the growth plates.

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.

Investigating the interfaces of the epiphyseal plate: An integrated approach of histochemistry, microtomography and SEM.

We investigated the interfaces of the epiphyseal plate with over- and underlying bone segments using an integrated approach of histochemistry, microtomography and scanning electron microscopy (SEM) to overcome the inherent limitations of sections-based techniques. Microtomography was able to provide an unobstructed, frontal view of large expanses of the two bone surfaces facing the growth plate, while SEM observation after removal of the soft matrix granted an equally unhindered access with a higher resolution. The two interfaces appeared widely dissimilar. On the diaphyseal side the hypertrophic chondrocytes were arranged in tall columns packed in a sort of compact palisade; the interposed extracellular matrix was actively calcifying into a thick mineralized crust growing towards the epiphysis. Behind the mineralization front, histochemical data revealed a number of surviving cartilage islets which were being slowly remodelled into bone. In contrast, the epiphyseal side of the cartilage consisted of a relatively quiescent reserve zone whose mineralization was marginal in amount and discontinuous in extension; the epiphyseal bone consisted of a loose trabecular meshwork, with ample vascular spaces opening directly into the non-mineralized cartilage. On both sides the calcification process took place through the formation of spheroidal bodies 1-2 µm wide which gradually grew by apposition and coalesced into a solid mass, in a way distinctly different from that of bone and other calcified tissues.