Objectives. The aim of this experimental study on New Zealand’s white rabbits was to investigate the transplantation of autogenous growth plate cells in order to treat the injured growth plate. They were assessed in terms of measurements of radiological tibial varus and histological characteristics. . Methods. An experimental model of plate growth medial partial resection of the tibia in 14 New Zealand white rabbits was created. During this surgical procedure the plate growth cells were collected and cultured. While the second surgery was being performed, the autologous cultured growth plate cells were grafted at the right tibia, whereas the left tibia was used as a control group. . Results. Histological examinations showed that the grafted right tibia presented the regular shape of the plate growth with hypertrophic maturation, chondrocyte columniation and endochondral calcification. Radiological study shows that the mean tibial deformity at the left angle was 20.29° (6.25 to 33) and 7.21° (5 to 10) in the right angle. . Conclusion. This study has demonstrated that grafting of autogenous cultured growth plate cells into a defect of the medial aspect of the proximal tibial physis can prevent bone bridge formation, growth arrest and the development of varus deformity. Cite this article: Bone Joint Res 2014;3:310–16

Objectives. The aim of this study was to examine whether asymmetric loading influences macrophage elastase (MMP12) expression in different parts of a rat tail intervertebral disc and growth plate and if MMP12 expression is correlated with the severity of the deformity. Methods. A wedge deformity between the ninth and tenth tail vertebrae was produced with an Ilizarov-type mini external fixator in 45 female Wistar rats, matched for their age and weight. Three groups were created according to the degree of deformity (10°, 30° and 50°). A total of 30 discs and vertebrae were evaluated immunohistochemically for immunolocalisation of MMP12 expression, and 15 discs were analysed by western blot and zymography in order to detect pro- and active MMP12. Results. No MMP12 expression was detected in the nucleus pulposus. Expression of MMP12 in the annulus progressively increased from group I to groups II and III, mainly at the concave side. Many growth plate chondrocytes expressed MMP12 in the control group, less in group I and rare in groups II and III. Changes in cell phenotype and reduction of cell number were observed, together with disorganisation of matrix microstructure similar to disc degeneration. ProMMP12 was detected at the area of 54 kDa and active MMP12 at 22 kDa. Conclusions. Expression of MMP12 after application of asymmetric loading in a rat tail increased in the intervertebral disc but decreased in the growth plate and correlated with the degree of the deformity and the side of the wedged disc. Cite this article: Bone Joint Res 2014;3:273–9

Sex hormones play important roles in the regulation of the proliferation, maturation and death of chondrocytes in the epiphyseal growth plate. We have investigated the effects of male castration on the cell kinetics of chondrocytes as defined by the numbers of proliferating and dying cells. The growth plates of normal rabbits and animals castrated at eight weeks of age were obtained at 10, 15, 20 and 25 weeks of age. Our study suggested that castration led to an increase in apoptosis and a decrease in the proliferation of chondrocytes in the growth plate. In addition, the number of chondrocytes in the castrated rabbits was less than that of normal animals of the same age

INTRODUCTION. Endochondral ossification in the growth plate is directly responsible for skeletal growth and its de novo bone-generating activity. Growth plates are vulnerable to disturbances that may lead to abnormal skeletal development. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used analgesics but have been reported to impair endochondral ossification-driven fracture healing. Despite the general awareness that NSAIDs affect endochondral ossification, the consequences of NSAIDs on skeletal development are unknown. We hypothesise that the NSAID celecoxib leads to impaired growth plate development and consequently impairs skeletal development. METHODS. Healthy skeletally immature (5 weeks old) C57BL/6 mice were treated for ten weeks with celecoxib (daily oral administration 10 mg/kg) or placebo (water) (institutional approval 2013–094) (n=12 per group). At 15 weeks postnatally, total growth plate thickness, the thickness of specific growth plate zones, (immuno)histological analysis of extracellular matrix composition in the growth plate, cell number and cell size, longitudinal bone growth and bone micro-architecture by micro-CT were analysed. Inhibition of COX-2 activity was confirmed by determining PGE2 levels in plasma using an ELISA. RESULTS. No significant difference in total growth plate thickness or thickness of the resting zone, proliferative or hypertrophic zone was found between groups. Staining of growth plate extracellular matrix components revealed, however, a significantly higher proteoglycan content and less collagen type II staining in the proliferative zone. In the hypertrophic zone of the growth plates of celecoxib treated mice collagen type X was hardly detectable as compared to placebo mice. In addition, a significantly decreased cell number was observed in the hypertrophic zone of the growth plate and cells were significantly smaller in the celecoxib group. Micro-CT analysis of the subchondral bone region directly beneath the growth plate showed significantly higher bone density, bone volume density and trabecular thickness following celecoxib treatment. Despite the detected differences in extracellular matrix composition of the growth plate, no difference was found in the length of the tibia in celecoxib treated mice. DISCUSSION. In summary, there are no measurable differences found in murine skeletal formation as a result of treatment with celecoxib in this study. However, there are notable phenotypic features found in the maturation of the growth plate (hypertrophic zone and subchondral bone) as a result from the celecoxib treatment, of which the potential consequences we do not yet understand. SIGNIFICANCE. When follow-up actions from the use of celecoxib on the growing individual are found this may warrant re-evaluation for the use of celecoxib in these individuals

Our aim was to evaluate the expression of transcription factors CCAAT/enhancer-binding protein-beta (C/EBP. β. ) and C/EBP-homologous protein (CHOP) in the growth plate. Proximal tibial epiphyseal growth plates from ten 15-day-old Wistar rats were used. Additionally, anti-proliferating cell nuclear antigen (PCNA), anti-5-bromo-2’-deoxyuridine (BrdU) immunostaining, terminal transferase dUTP nick end-labelling (TUNEL) and nucleolar organiser region-associated proteins (AgNOR) techniques were peformed. The histological morphology of the growth plate from C/EBP. β. knock-out mice was also analysed. The normal growth plate showed that C/EBP. β. and CHOP factors are expressed both in the germinative/ upper proliferative and in the lower proliferative zones. Furthermore, BdrU+ and PCNA+ cells were present exclusively in the germinative and proliferative zones, while TUNEL+ and AgNOR+ cells were seen in all three zones of the growth plate. Acellular areas, hypocellularity, the increase in cell death and anomalies in the architecture of the cell columns were observed in the growth plates of C/EBP. β. (−/ −) knockout mice. We suggest that C/EBP. β. and CHOP transcription factors may be key modulators participating in the chondrocyte differentiation process in the growth plate

Growth plates taken from five- to 20-week-old Japanese white rabbits were immunostained for c-Myc protein. This was localised both in the proliferating zone and upper hypertrophic zone at five weeks, whereas after ten weeks it was found mostly in the lower hypertrophic zone. The proliferating chondrocytes tended to show nuclear staining and the hypertrophic cells cytoplasmic staining, although the terminal hypertrophic chondrocytes sometimes expressed the protein in their nuclei. In the younger rabbits, c-Myc co-localised with proliferating cell nuclear antigen, whereas in the hypertrophic zone of older rabbits, it was present in some chondrocytes the nuclei of which also contained DNA breaks. Our study suggests that, in the rabbit growth plate, c-Myc is associated with different cellular processes, depending on the age and the developmental stage of the chondrocytes

Chondrocytes of the growth plate are generally assumed to undergo apoptosis, but the mechanisms which induce this cell death are not known. The Fas receptor is a mediator of the apoptotic signal in some systems. We studied its expression in situ in growth plates of rabbits aged from five to 20 weeks. In addition, we investigated the immunolocalisation in the growth plates of the bone proteins, osteonectin and osteocalcin, and the changes in their expression with age. The Fas-positive chondrocytes were found mostly in the hypertrophic zone, as were the osteonectin-positive and osteocalcin-positive cells. The percentage of Fas-positive cells increased with age whereas little change was found in the number of osteonectin-positive and osteocalcin-positive chondrocytes. Many of the Fas-positive chondrocytes were also TUNEL-positive. This strongly suggests that apoptosis in the growth plate is mediated through the Fas system. Double immunostaining for osteocalcin and Fas showed that not all hypertrophic chondrocytes were of the same cell type. Some chondrocytes stained for osteocalcin only, others for Fas only, while some were positive for both

The growth plates of the femoral head of Japanese white rabbits aged 5, 10, 15 and 20 weeks were stained for apoptotic and proliferating chondrocytes using the TUNEL and PCNA antibody staining techniques. Both TUNEL- and PCNA-positive chondrocytes were detected in all of the specimens. The positive ratios of both stainings were calculated for the whole plate and for the resting, proliferating and hypertrophic zones. The highest ratios in both stainings occurred in the hypertrophic zone in all age groups. With growth, the TUNEL-positive ratio increased whereas the proliferating ratio decreased. We suggest that the increase in chondrocytic death by apoptosis and the decrease in cell proliferation potential led to closure of the growth plate

Introduction. The proteoglycan aggrecan is a major component of the cartilaginous matrices which provides resistance against compressive forces. Spontaneously occurring functional null mutations in the aggrecan gene (Acan) in various species lead to perinatal chondrodysplasia. The aim of the present study was to investigate the cellular and biomechanical properties of the cartilaginous growth plate, and the development of intervertebral disc in a novel, experimentally induced aggrecan mutant mouse strain carrying an insertion in exon 5 of the Acan gene. Methods. The novel aggrecan mutant mice were generated by inserting a loxP site into exon 5 (E5i) by homologous recombination in ES cells. Wild type and homozygous mutant (Acan-E5i/E5i) mice were analyzed by skeletal staining, histology and immunohistochemistry. Proliferation and survival were assessed by phosphorylated histone H3 immunostaining and TUNEL assay, respectively. Shape index (SI) in the proliferative zone (PZ) of the growth plate (GP) was calculated as a ratio of the long and short axes of the cells. Orientation of the PZ chondrocytes was characterized by the angle between the cell long axis and longitudinal direction of the bone growth. Imaging and stiffness measurements were performed by atomic force microscopy (AFM). Results. Acan-E5i/E5i mice are characterized by severe dwarfism, short snout, protruding tongue, cleft palate, and die at birth due to respiratory failure. On sections the cartilage of mutant mice appeared as tightly packed chondrocytes surrounded by a compressed matrix. At E18.5 and E14.5, the mutant PZ consisted of rounded (SI=1.71 at E18.5; SI=1.72 at E14.5) non-oriented chondrocytes, compared to the wild type PZ with flattened (SI=3.92 at E18.5; SI=3.90 at E14.5), columnar cells oriented with right angle to the longitudinal axis of the growth. At E13, the shape and orientation of mutant chondrocytes were similar to control. AFM at E14.5 and E18.5 demonstrated a stiffer matrix with denser collagen network in the mutant compared to wild type. The mutant cartilage had increased apoptosis and reduced proliferation rate at E18.5. The IVDs development appeared normal at E13.5-E14.5, however, the IVD was severely malformed at E18.5. Discussion. We have shown that aggrecan deficiency impairs cartilage biomechanics and results in a stiffer matrix. The altered mechanical properties might be responsible for the disorganization of mutant GP and compression of the IVD at around birth. Interestingly, the altered matrix mechanics is dispensable for early flattening and orientation of GP proliferative chondrocytes. In summary, aggrecan is essential for proper cartilage cytoarchitecture and morphogenesis by ensuring the suitable mechanical environment

Background. The cartilaginous growth plate (GP) is a zonal structure, in which chondrocytes are organized into columns and drive the longitudinal elongation of the endochondral bones. In the proliferative zone (PZ), cells exhibit high mitotic activity, are flattened and oriented along the mediolateral (ML) axis of the GP. Mitotic figures in the elongated chondrocytes lie perpendicular to the proximo-distal (PD) direction of growth, while cell divisions occur parallel to the columns followed by a gliding movement of the daughter cells. The mechanisms responsible for the geometrical anisotropy and columnar arrangement of PZ chondrocytes are poorly understood. Here, we assessed the function of the adhesive receptor β1 integrins on spindle and division geometry in chondrocytes using mouse genetics. Methods. GP slices were prepared from wild type (wt) and β1fl/fl-Col2a1cre mice. Primary rib chondrocytes were cultured on substrate-coated glass slides and fluorescently stained with anti-alpha-tubulin and anti-pericentrin antibodies, with phalloidin and DAPI. Confocal stacks were built from images acquired by confocal microscopy. Cell and spindle orientation relative to the PD axis (in vivo) or to the substrate plane (SP) in vitro were determined by geometric functions. Shape indexes (SI) were calculated as the ratio of the length and height of the cell. Results. During GP morphogenesis, aggregating mesenchymal cells (E11) were polygonal (SI=1.43) and nonoriented. Upon chondrogenic differentiation at E12, wt GP chondrocytes showed moderate flattening (SI=1.93) and tend to align perpendicular to the PD axis. At E13, PZ chondrocytes further flattened (SI=3.41) and largely organized into lines along the ML axis. At E14, the first vertical stacks formed, which were gradually elongated along the PD axis at later stages and composed of extremely flat (SI=4.91), ML-oriented chondrocytes. Early spindles were randomly oriented at all stages, whereas late spindles were aligned along the long axis of the cell. In contrast, β1 integrin null PZ chondrocytes were rounded (SI=1.37), displayed random orientation of both early and late spindles, and failed to organize into columns. On collagen II, both wt and β1-null primary chondrocytes remained rounded and displayed random spindle orientation relative to the SP. On fibronectin (FN), wt chondrocytes were flattened (SI=3.26) and their spindle was aligned parallel to SP. Mutant cells lacking the major FN receptor α5β1 integrin did not spread, were rounded (SI=1.73) and aligned their spindle randomly. On vitronectin, both cell types spread and flattened well (SI=4.11 and SI=4.20) accompanied by parallel spindle orientation to the SP. Conclusion. We propose that β1 integrins provide adhesive cues for chondrocyte geometry and orientation, which in turn orients the mitotic spindle and determines the division axis; and for chondrocyte intercalation to shape the three-dimensional structure of the GP

Introduction and Objective. Neoangiogenesis drives the replacement of mineralized cartilage by trabecular bone during bone growth regulated by molecules like e.g. VEGF, OPG and RANKL and the close interaction of progenitors of osteoblasts, chondrocytes, endothelial cells and osteoclasts/chondroclasts. The Heparan sulfate proteoglycan Syndecan-1 (Sdc-1) plays a role in the interaction between osteoclasts and osteoblasts and the development of blood vessels. As the processes of osteogenesis and angiogenesis are closely related to each other in bone, we expected Sdc-1 to have an influence on vessel structure during aging. Therefore, angiogenesis at the growth plate in mice of different ages was compared and the influence of Syndecan-1 deficiency was characterized. Materials and Methods. Animals: C57BL/6 (WT) and Sdc1−/− mice were used for native bone analysis at 4, 12 and 18 month age. Femura were dissected, cryoprotected and embedded. Histology: Embedded bones were sectioned into 80um thick slices so that the 3D network of the vascularization of the bone could be visualized using an anti-Endomucin antibody and DAPI as counter staining. For semi-automatical quantification of the vessel bulbs we used a custom made software. In vitro angiogenesis: For aortic ring assay, aortic tissue was isolated from 4 month old mice, cut into 0.5mm rings and embedded in collagen type I matrix. Microvessel outgrowth was quantified after 6 days of culture. Results. We verified our custom-made software using slices of WT mice and showed that there is no variation of the number of bulbs with regard to the width of the growth plate in periphery versus center zones in all age groups which indicates a homogeneous distribution of angiogenesis throughout the interface of cartilage to newly forming bone. Furthermore, in both, WT and Sdc-1 deficient mice the number of bulbs decreased significantly with age. However, Sdc-1 knockout mice at the age of 4 and 12 month showed a highly significant decrease in angiogenesis close to the growth plate compared to WT mice, whereas in older mice these differences were gone. Quantification of microvessel outgrowth of aortic tissue revealed a significant decrease in number of vessels from rings taken from Syndecan-1 deficient mice compared to WT mice. Conclusions. Syndecan-1 has a significant impact on neoangiogenesis in vitro and in vivo during aging as demonstrated at the chondro-osseous border of the native bone, emphasizing the importance to further characterize the function of Syndecan-1 regulated processes during enchondral ossification

Introduction and Objective. Klinefelter Syndrome (KS, karyotype 47,XXY) is the most frequent chromosomal aneuploidy in males, as well as the most common cause of infertility in men. Patients suffer from a lack of testosterone, i.e. hypergonadotropic hypogonadism provoking infertility, but KS men also show an increased predisposition to osteoporosis and a higher risk of bone fracture. In a mouse model for human KS, bone analysis of adult mice revealed a decrease in bone mass that could not be rescued by testosterone replacement, suggesting a gene dosage effect originating from the supernumerary X-chromosome on bone metabolism. Usually, X chromosome inactivation (XCI) compensates for the dosage imbalance of X-chromosomal genes between sexes. Some studies suggested that expression of genes that escape silencing of the supernumerary X-chromosome (e.g. androgen receptor) has an impact on sex differences, but may also cause pathological changes in males. As a promising new such candidate for a musculoskeletal escape gene, we identified the integral membrane protein (ITM) 2a, which is encoded on the X-chromosome and related to enchondral ossification. The aim of the project was to characterize systemic bone loss in the course of aging in our KS mouse model, and whether the supernumerary X-chromosome causes differences in expression of genes related to bone development. Materials and Methods. Bone structure of 24 month (=aged) old male wild type (WT) and 41, XXY mice (B6Ei.Lt-Y) were analysed by μCT. Afterwards bones were paraffin embedded and cut. In addition, tissue of brain, liver, kidney, lung and heart were also isolated and embedded for IHC staining. Using an anti-ITM2a antibody, expression and cellular localization of ITM2a was evaluated. IHC was also performed on musculoskeletal tissue of WT embryos (E18.5) and neonatal mice to determine possible age-related differences. Results. In 24 month old mice, the analysis of the lumbar vertebrae revealed a significantly lower BV/TV, trabecular bone volume and trabecular number in the XXY- group compared to WT. Trabecular thickness appeared lower but did not reach significance, with the cortical thickness being significantly higher in the XXY- group. High expression of ITM2a was detected in bone slices of both karyotypes in the chondrocytes inside the growth plate, as well as in megakaryocytes and leucocytes as well as endothelial cells of blood vessels inside the bone marrow. Osteocytes, along with erythrocytes and erythropoetic stem cells were negative for ITM2a. Other organs that showed ITM2a positive staining were kidney (blood vessels), heart (muscle) and brain (different structures). Liver and lung tissue were negative for ITM2a. No obvious difference in the intensity of the ITM2a-expression was observed between the WT and the XXY-karyotype. Analyses of embryotic bone tissue (WT) showed high expression of ITM2a in proliferating, hypertrophic and resting chondrocytes in the growth plates of tibia and femur. In comparison, the neonatal animals (WT) did not show any protein-expression in chondrocytes. Furthermore, within the metaphysis of both, embryotic and neonatal bones, endothelial cells and osteoblasts were ITM2a-positive. Further analyses of bones and tissues from young mice (4–6 month) are ongoing. Conclusions. Bone analyses revealed a significant reduction in trabecular bone mass along with fewer and thinner trabeculae in XXY mice compared to the WT, especially in the spine. ITM2a expression was visible in different cell types inside the bone, and in addition, different expression patterns at different stages of development (embryonic/neonatal) were observed. However, we have not found a significant difference in the quantity of ITM2a between tissues of XXY-karyotypes and WT. Further analyses of X-chromosomal encoded and therefore dysregulated modulators in XXY-karyotype mice and patients may reveal new sex chromosomal effector proteins in bone metabolism

In osteoarthritis, chondrocytes acquire a hypertrophic phenotype that contributes to matrix degradation. Inflammation is proposed as trigger for the shift to a hypertrophic phenotype. Using in vitro culture of human chondrocytes and cartilage explants we could not find evidence for a role of inflammatory signalling activation. We found, however, that tissue repair macrophages may contribute to the onset of hypertrophy (doi: 10.1177/19476035211021907) Intra-articularly injected triamcinolone acetonide to inhibit inflammation in a murine model of collagenase-induced osteoarthritis, increased synovial macrophage numbers and osteophytosis, confirming the role of macrophages in chondrocyte hypertrophy occurring in osteophyte formation (doi: 10.1111/bph.15780). In search of targets to inhibit chondrocyte hypertrophy, we combined existing microarray data of different cartilage layers of murine growth plate and murine articular cartilage after induction of collagenase-induced osteoarthritis. We identified common differentially expressed genes and selected those known to be associated to inflammation. This revealed EPHA2, a tyrosine kinase receptor, as a new target. Using in silico, in vitro and in vivo models we demonstrated that inhibition of EPHA2 might be a promising treatment for osteoarthritis. Recently, single cell RNA-seq. has revealed detailed information about different populations of chondrocytes in articular cartilage during osteoarthritis. We re-analysed a published scRNA-seq data set of healthy and osteoarthritic cartilage to obtain the differentially expressed genes in the population of hypertrophic chondrocytes compared to the other chondrocytes, applied pathway analyses and then used drug databases to search for upstream inhibitors of these pathways. This drug repurposing approach led to the selection of 6 drugs that were screened and tested using several in vitro models with human chondrocytes and cartilage explants. In this lecture I will present this sequence of studies to highlight different approaches and models that can be used in the quest for a disease modifying drug for osteoarthritis

Neoangiogenesis drives the replacement of mineralised cartilage by trabecular bone during bone growth regulated by molecules like e.g. VEGF, OPG and RANKL. The Heparan sulfate proteoglycan Syndecan-1 (Sdc1) plays a role in the interaction of osteoclasts and osteoblasts and the development of blood vessels. We expected Sdc1 to have an influence on bone structure and vessel development. Therefore, bone structure and angiogenesis at the growth plate in mice was compared and the influence of Syndecan-1 deficiency was characterised. Animals: Femura of male and female C57BL/6 WT (5♀, 6♂) and Sdc1-/- (9♀, 5♂) mice were used for native bone analysis at 4 month age. Histology: Bone structure was analysed using microCT scans with a resolution of 9µm. Vascularisation was visualised using an anti-Endomucin antibody in 80µm thick cryosections. In vitro angiogenesis: Bone marrow isolates were used to generate endothelial progenitor cells by sequential cultivation on fibronectin. Microvessel development was analysed 4h after plating on matrigel. Bone structure in male Sdc1 deficient mice was significantly reduced compare to male WT, whereas female mice of both genotypes did not differ. Sdc1 deficient mice at the age of 4 month showed a high decrease in the number of vessel bulbs at the chondro-osseous border (growth plate) compared to WT mice. However, no sex related differences were shown. Quantification of microvessel outgrowth of endothelial cells revealed a decreased amount of sprouting, but increased length of microvessels of Sdc1-/- cells compared to WT. Syndecan-1 has a significant impact on neoangiogenesis at the chondro-osseous border of the native bone, but the impact of Syndecan-1 deficiency on the loss of bone structure was significantly higher in male mice. This emphasises the importance to further characterise the function of Syndecan-1 regulated processes during enchondral ossification in a sex dependent manner

Summary Statement. The implantation of scaffold-free CTE from suspension culture into growth-plate defects resulted in a significant reduction in growth arrest of the rabbit tibia. Introduction. In childhood and adolescence, the growth plate injury can cause partial premature arrest of growth plate, which can make problems such as leg length discrepancy and angular deformity. Bone bridge resection and variable implantation materials such as fat, bone wax, silastic and craniopalst has been investigated. However, those procedures may show limitations including the control of bone growth and long term safety of implant materials in vivo. As an alternative, homogeneous or heterogeneous cartilage cells and stem cell transplants have been tried. In this method, scaffold for cell transplantation is needed. But, so far the most suitable scaffold has not been established. Recently, some authors generated a cartilage tissue equivalent (CTE) using a suspension culture with biophysical properties similar to native hyaline cartilage. Therefore we are able to transplant the CTE without scaffold to the physeal defect. The purpose of this study was to investigated the effects of a transplantation of a vitro-generated scaffold-free tissue-engineered cartilage tissue equivalent (CTE) using a suspension chondrocyte culture in a rabbit growth arrest model. Material and Method. Cartilage tissue equivalent culture. The CTE was generated by the suspension culture of chondrocytes (2 × 10. 7. /well/1 mL) which was isolated from articular cartilage of 5 weeks New Zealand white rabbit on a 24-well plate (2.4 cm. 2. /well) treated with poly HEMA (nunc, Roskide, Denmark) for up to 8 and 16 weeks. (2)Partial growth arrest animal model. An experimental model for growth arrest was created by excising the growth plate at the proximal medial side of tibia with the 4 mm in diameter and 4 mm in depth from 6-week-old New Zealand white rabbits. Two experimental groups were set to evaluate CTE implantation; group I, no implantation as controls; group II, implantation of CTE. (3) Evaluation of effect of the transplantation of CTE. Serial plain radiographs were performed at one week. The medial proximal tibial angle (MPTA) was measured for assessing the degree of angular deformity. Histologic examination using HE stain, Alcian bule and immunohistochemistry was done at 4 and 8 weeks after surgery. Results. Radiographic results: In group I, all damaged growth plates were arrested and angular deformities appeared 4 weeks later. In groups II, angular deformities were much less than in the control group. Histologic result: In group I, bone bridge formation was shown at the damaged growth plate at 4 weeks after surgery. In group II, regeneration of growth plates was recognised at 4 and 8 week after surgery. However, the thickness of regenerated growth plate at 8 weeks specimen was thinner than that of 4 weeks specimen. Discussion and Conclusion. The implantation of scaffold-free CTE from suspension culture into growth-plate defects resulted in a significant reduction in growth arrest of the rabbit tibia

The use of retrograde femoral intramedullary nails in children for deformity correction is controversial. It is unknown if the injury to the central part of the growth plate results in premature bony union, leading to limb deformities or discrepancies. The aim of this study was to assess physeal healing and bone growth after insertion of a retrograde femoral nail thorough the centre of the physis in a skeletally immature experimental porcine model. Eleven immature pigs were included in the study. One leg was randomised for operation with a retrograde femoral nail (diameter 10.7 mm), whilst the non-operated contralateral remained as control. All nails were inserted centrally in coronal and sagittal plane under fluoroscopic guidance, and the nails spanned the physis. The nails were removed at 8 weeks. Both femora in all animals underwent MRI at baseline (pre-operatively), 8 weeks (after nail removal) and 16 weeks (before euthanasia). Femoral bone length was measured at 5 sites (anterior, posterior, central, lateral and medial) using 3d T1-weighted MRI. Growth was calculated after 8 weeks (growth with nail) and 16 weeks (growth without nail). Physeal cross-sectional area and percentage violated by the nail was determined on MRI. Operated side was compared to non-operated. Corresponding 95% confidence intervals were calculated. No differences in axial growth were observed between operated and non-operated sides. Mean growth difference was 0,61 mm [−0,78;2,01] whilst the nail was inserted into the bone and 0,72 mm [−1,04;1,65] after nail removal. No signs of angular bone deformities were found when comparing operated side to non-operated side. No premature bony healing at the physis occurred. Histology confirmed fibrous healing. Mean physeal violation was 5.72% [5.51; 5.93] by the femoral nail. The insertion of a retrograde femoral nail through the centre of an open physis might be a safe procedure with no subsequent growth arrest. However, experiments assessing the long term physeal healing and growth are needed

Introduction. NF-κB transcription factors regulate a number of genes that are activated under stress conditions. Blockage of the the canonical NF-κB pathway has been emerged as a possible strategy to cure osteoarthritis and rheumatoid arthritis. However, the roles of κNF-B in normal skeletal physiology are largely unknown owing to the lack of suitable animal models. Here, we investigated the function of canonical κNF-B pathway in the cartilaginous skeleton by ablating Nemo (NF-κB essential modulator) in chondrocytes using the Col2a1 transgene. Methods. Mice were analyzed by skeletal staining, histology, proliferation and apoptosis assays at various stages. Histochemistry, GAG assay and immunohistochemistry were utilized to assess the impact of NEMO-deficiency in cytokine-induced cartilage degradation of hip explants. To identify genes regulated through the canonical NF-κB pathway in response to injury, an ex vivo hip avulsion model was applied. 24 genes known to be induced early following cartilage injury were assessed in wildtype and mutant hips by RT-PCR. Time lapse photography was used to investigate chondrocyte migration in vitro. Atomic force microscopy (AFM) was applied to assess biomechanical properties of the cartilage. Pathological changes of articular cartilage were scored in aged joints. Results. Mutant mice exhibited moderate dwarfism postnatally characterized by disorganized growth plate, abnormal chondrocyte proliferation, apoptosis and migration. AFM indentation experiments showed no changes in biomechanical properties of the mutant growth plate compared with control. Exposure of aggrecan degradation neoepitopes and release of GAGs were less pronounced in mutant hip explants stimulated by cytokines. Of the 24 genes regulated 4h following injury in wildtype hips, only Arginase-1 was suppressed in the mutant hips, while the expression levels of most other inflammatory response genes e.g. TSG-6, NOS2, COX2, IL6 and IL1b were unaffected. A small number of genes, IL-18, MMP-3 and Has-2 were further upregulated upon injury in Nemo-deficient compared with wildtype hips. Aging mutant mice showed signs of osteoarthritis comparable to wildtype. Conclusion. Nemo-deficient mice have demonstrated an important role for canonical NF-κB signaling in skeletal growth by modulating chondrocyte behavior. Even though the catabolic effects of pro-inflammatory cytokines in cartilage could be partially eased by blocking the canonical NF-κB pathway, canonical NF-κB signaling seems to play only a minor role in injury-induced inflammatory gene expression and the development of spontaneous OA

Introduction. Human Mesenchymal stem cells (hMSCs) are a promising source for articular cartilage repair. Unfortunately, under in vitro conditions, chondrogenically differentiated hMSCs have the tendency to undergo hypertrophy similar to growth plate chondrocytes. Retinoic acid (RA) signalling plays a key role in growth plate hypertrophy. Whilst RA agonists block chondrogenesis and foster hypertrophy during later stages, RAR inverse agonists (IA) enhance chondrogenesis when applied early in culture. Therefore, we hypothesized that treatment with RAR IA will attenuate hypertrophy in chondrogenically differentiated hMSCs. To test this hypothesis, we analysed early (initial chondrogenic differentiation) and late treatment (hypertrophy stage) of hMSCs with an RAR IA. Methods. Pellets of passage 2 hMSCs were formed in V-bottom well plates by centrifugation and pre-differentiated in a chemically defined medium containing 10ng/mL TGFß (CM+) for 14 days. Thereafter, pellets were cultured for an additional 14 days under 6 conditions: CM+, CM- (w/out TGFß), and hypertrophic medium (CM- with 25 ng/ml BMP 4, w/out dexamethasone). Each of these first three conditions was additionally supplemented with the RA receptor (RAR) inverse agonist BMS493 (BMS) at 2μM after 14 days of chondrogenic pre-differentiation. One additional BMP4 group was supplemented with BMS from the beginning of chondrogenic differentiation until day 14. The pellets were assessed for gene expression (Col 2, Col 10, Col 1 and MMP13) and histologically using dimethyl methylene blue (DMMB), alkaline phosphatase staining (ALP) and collagen II and X immunohistochemistry. Results. Hypertrophy was reduced by addition of BMS at day 14 and further reduced by addition from the beginning. BMS treatment resulted in smaller cells under hypertrophic conditions, higher collagen II content in chondrogenic groups and reduction in collagen X production and ALP activity in every condition. Gene expression data for hypertrophic markers, collagen X and MMP13, were upregulated under the influence of BMP4 but a distinct downregulation in MMP13 expression was shown upon addition of BMS during the late stage differentiation and further reduced upon addition during early stage chondrogenesis. Furthermore, Collagen X expression was reduced by early BMS treatment. Discussion. The treatment with the RAR IA, BMS, attenuated hypertrophic changes in chondrogenically differentiated hMSCs as demonstrated by histology, immunohistochemistry and PCR. These findings suggest an additional approach to attenuate hypertrophy in chondrogenically differentiated hMSCs. Current studies are exploring the timing and dose of BMS to most efficaciously prevent hypertrophy

Objective. Early cell loss of up to 50% is common to in vitro chondrogenesis of mesenchymal stromal cells (MSC) and stimulation of cell proliferation could compensate for this unwanted effect and improve efficacy and tissue yield for cartilage tissue engineering. We recently demonstrated that proliferation is an essential requirement for successful chondrogenesis of MSC, however, how it is regulated is still completely unknown. We therefore aimed to identify signaling pathways involved in the regulation of proliferation during in vitro chondrogenesis and investigated, whether activation of relevant pathways could stimulate proliferation. Design. Human MSC were subjected to in vitro chondrogenesis for up to 42 days under standard conditions in the presence of 10 ng/ml TGF-β. Cells were or were not additionally treated with inhibitors of bone morphogenetic protein (BMP), insulin-like growth factor (IGF) IGF/PI3K, fibroblast growth factor (FGF) or indian hedgehog (IHH) pathways for two or four weeks. To investigate the stimulation of proliferation by exogenous factors, cells were treated with BMP-4, IGF-1, FGF-18 or purmorphamine (small molecule hedgehog agonist). Proliferation was determined by [3H]-thymidine incorporation. Results and Discussion. Quantitative assessment of proliferation revealed that proliferation arrest occurred during condensation up to day 3 and cell division was re-initiated thereafter with a peak on day 28. To test which pathways are relevant for the restart of proliferation, BMP, IGF/PI3K, FGF or IHH signaling was inhibited up to day 14. All treatments significantly reduced proliferation > 50% and, thus, seemed to participate in the re-entry into the cell cycle. To study whether the same pathways are relevant to maintain cells in a proliferative state later on, inhibitors were supplemented from day 14–28. This resulted in a significant decrease of proliferation in the groups treated with inhibitors of BMP (67% decrease), FGF (70%) and IHH (30%) signaling, while inhibition of IGF/PI3K did not influence late proliferation. Although BMP-4, IGF-1 or FGF-18 are known mitogenic factors in the growth plate, stimulation of cells by exogenous addition of these factors did not enhance proliferation in any differentiation phase. In contrast, stimulation of IHH signaling from day 14–28 significantly increased proliferation by 44%. This is in line with the documented strong mitogenic activity of hedgehog signaling in the proliferative zone of the growth plate. Thus, our data demonstrated that BMP, IGF/PI3K, FGF and IHH essentially participate in the regulation of proliferation during in vitro chondrogenesis. Early or late activation of single pathways by exogenous factors was, however, not sufficient to increase proliferation significantly with the exception of late activation of hedgehog signaling. Optimization of stimulation of the hedgehog pathway with a focus on increased tissue yield will now be the next step

Abstract. Objective. To assess the prevalence of acetabular retroversion in patients presenting with Slipped Upper Femoral Epiphysis using both validated radiological signs and CT-angle measurements. Methods. A retrospective review of all cases involving surgical management for acute SUFE presenting to the Royal Children's Hospital, Melbourne were assessed from 2012–2018. Pre-operative plain radiographs were assessed for slip angle, validated radiological signs of retroversion (post wall/crossover/ischial spine sign) and standardised post-operative CT Scans were used to assess cranial and mid-acetabular version. Results. 116 SUFEs presented in 107 patients who underwent surgical intervention; 47 females and 60 boys, with an average age of 12.7 years (range 7.5–16.6 years). Complete radiological data was available for 91 patients (99 hips) with adequate axial CT imaging of both hips. 82% patients underwent pinning in situ (PIS) with subcapital realignment surgery (SRS) performed in 18% (slip angles >75°). Contralateral prophylactic hip PIS was performed in 72 patients (87%). On the slip side, 68% of patients had 1 or more radiological signs of retroversion in the slipped hip, with 60% on the contralateral side. The mean cranial and mid-acetabular version measurements were −8°(range −30 – 8°) and 10.5°(range −10 – 25°), respectively. Conclusions. Acetabular retroversion is rare in the normal population with studies reports ranging from 0–7%. This study showed an increased prevalence of 68% in SUFE patients, which is likely to be a primary anatomical abnormality, subsequently increasing the shear forces across the proximal femoral growth plate due to superior over-coverage. The resulting CAM lesion from SUFE in combination with the pincer lesion due to retroversion can lead to premature hip impingement and degeneration. Further larger studies are required to assess if acetabular retroversion is a true risk factor, and its role in helping guide management including prophylactic pinning. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project