Hypertrophic Zone Research Articles

ADAM10 is indispensable for longitudinal bone growth in mice.

Skeletal development is a highly sophisticated process in which the expression of a variety of growth factors, signaling molecules, and extracellular matrix proteins is spatially and temporally orchestrated. In the present study, we show that ADAM10, a transmembrane protease that is critically involved in the functional regulation of various membrane-bound molecules, plays an essential role in the longitudinal growth of long bones and in skeletal development. We found that mutant mice lacking ADAM10 in osteochondroprogenitors exhibited marked growth retardation and had shorter long bones than the control mice. Histomorphometric analysis revealed that the mutant mice had a shorter hypertrophic zone and that their hypertrophic chondrocytes were smaller in size than those of the control mice. Unexpectedly, we found that the mRNA expression of the chemokine CXCL12 and its receptor CXCR4 were significantly reduced in cartilage tissues lacking ADAM10. Further, exogenous supplementation of recombinant CXCL12 rescued the defect in the ADAM10-deficient growth plate in an ex vivo culture model. Taken together, our data show a previously unknown role for ADAM10 in skeletal development that involves its regulation of the CXCL12 and CXCR4 signaling pathway.

Tissue Distribution and Receptor Activation by Somapacitan, a Long Acting Growth Hormone Derivative.

Somapacitan is a long-acting, once-weekly, albumin-binding growth hormone (GH) derivative. The reversible albumin-binding properties leads to prolonged circulation half-life. Here, we investigated and compared somapacitan with human GH on downstream receptor signaling in primary hepatocytes and hepatocellular models and using isothermal titration calorimetry to characterize receptor binding of somapacitan in the presence or absence of human serum albumin (HSA). With non-invasive fluorescence imaging we quantitatively visualize and compare the temporal distribution and examine the tissue-specific growth hormone receptor (GHR) activation at distribution sites. We found that signaling kinetics were slightly more rapid and intense for GH compared with somapacitan. Receptor binding isotherms were characterized by a high and a low affinity interaction site with or without HSA. Using in vivo optical imaging we found prolonged systemically biodistribution of somapacitan compared with GH, which correlated with plasma pharmacokinetics. Ex vivo mouse organ analysis revealed that the temporal fluorescent intensity in livers dosed with somapacitan was significantly increased compared with GH-dosed livers and correlated with the degree of downstream GHR activation. Finally, we show that fluorescent-labeled analogs distributed to the hypertrophic zone in the epiphysis of proximal tibia of hypophysectomized rats and that somapacitan and GH activate the GHR signaling in epiphyseal tissues.

Factors Affecting Outcomes of Slipped Capital Femoral Epiphysis

Slipped capital femoral epiphysis (SCFE) is a frequent cause of nontraumatic painful hip of the adolescence. It is the result of the separation of the proximal femoral growth cartilage at the level of the hypertrophic cell zone. The femoral neck metaphysis rotates externally and migrates proximally relative to the femoral head epiphysis, which is stably seated in the acetabulum; early diagnosis and in situ stabilization grants the best long term results. Numerous factors affect treatment outcomes. Not all implants have the same effect on the slipped physis. Application of the traditionally used implants, such as non-threaded pins and cannulated screws, is questioned. Modern implants are available, which stabilize the slip without accelerating physis fusion. This allows femoral head and neck growth and remodeling to limit the post-slip sequellae on hip anatomy and function. Femoroacetabular impingement (FAI) complicates almost all slips. It causes progressive labral and articular cartilage damage and leads to early hip osteoarthritis and total hip replacement, approximately ten years earlier compared to the general population. Avascular necrosis of the femoral head is a dramatic complication, seen almost exclusively in unstable slips. It develops within months after the slip and leads to immediate articular joint degeneration and the need for total hip replacement. Another serious complication of SCFE is chondrolysis, which is a rapid progressive articular cartilage degeneration leading to a narrow joint space and restriction of hip motion. Implant-related complications, such as migration and loosening, may lead to the progression of the slip. Though bilateral disease is quite frequent, there is no consensus about the need for preventive surgery on the healthy contralateral hip. Diagnosis of SCFE is frequently missed or delayed, leading to slips of higher severity. Silent slippage of the capital femoral epiphysis is highly suspected as an underlying cause of cam-type FAI and early-onset hip osteoarthritis. There is controversy, whether asymptomatic implants should be removed. Novel surgical techniques, such as the modified Dunn procedure and hip arthroscopy, seem to be effective modalities for the prevention of FAI in SCFE.

Analysis of Association between Morphometric Parameters of Growth Plate and Bone Growth of Tibia in Mice and Humans.

The purposes of this study are to evaluate which growth plate parameters are associated with bone growth in mice and to compare the mouse results with those in humans. The sagittal sections of the proximal growth plate of the mouse tibia from neonate to young adult stages were subjected to histomorphometric and functional analyses. The radiographic images of tibias of human patients until puberty were analyzed to obtain the tibia length and the proximal growth plate height. It was found that a linear correlation best modeled the relationship between the growth plate variables with the tibia growth rate and length. In mice, total height, resting zone height, combined height of the proliferation and prehypertrophic zones, proliferation activity, and the total width of tibia growth plate showed high linear correlation with tibia bone length and bone growth rate, but the hypertrophic zone height and the growth plate area did not. In both mice and humans, the total growth plate width of tibia was found to have the strongest correlation with tibia length and growth rate. The results validated that growth plate total height, the height of the resting zone and cell proliferation activity are appropriate parameters to evaluate the balance between growth plate activity and bone growth in mice, consistent with previous reports. The study also provided a new growth plate parameter candidate, growth plate width for growth plate activity evaluation in both mouse and human tibia bone.

Research advances in repair of growth plate injury

Growth plate, the developmental center of endochondral osteogenesis, can be divided morphologically and functionally into a resting zone, a proliferative zone, a prehypertrophic zone and a hypertrophic zone. Injuries to growth plate often lead to bone growth defects including limb length discrepancy and angulation deformity in children. Currently, their orthopedic corrective surgeries are invasive and limitedly effective and no effective biotherapy has been available. Previous studies on animal models of growth plate damage have investigated the related cellular and molecular events in the repair of damaged growth plates in the 4 distinct inflammatory, fibrogenic, osteogenic and remodeling phases. Related molecules involved in the regulation of the above processes, such as inflammatory cytokines tumor necrosis factor alpha, mitogenic platelet-derived growth factor and bone morphogenetic protein, are found to participate in the regulation of growth plate injury. Exploration of the mechanisms may provide new targets for biotherapy. In addition, development of cartilage tissue engineering, especially application of mesenchymal stem cells, also provides potential interventions for growth plate injury. Key words: Tissue engineering; Wounds and injuries; Bone marrow cells; Growth plate; Chondrogenesis

Growth and Bone Mineral Density Changes in Ovariectomized Rats Treated with Estrogen Receptor Alpha or Beta Agonists

BackgroundEstrogen controls the pubertal growth spurt, growth plate closure, and accretion of bone mineral density (BMD) of long bones after biding estrogen receptor (ER). There are two subtypes of ER, ERα and ERβ. If each ER subtype has different effects, we may control those actions by manipulating the estrogen binding intensity to each ER subtype and increase the final adult height without markedly reducing BMD or impairing reproductive functions. The purpose of our study was to compare these effects of ERα and ERβ on long bones in ovariectomized rats.MethodsThirty female rats were ovariectomized and randomly divided into 3 groups. The control, propylpyrazole triol (PPT), and 2,3-bis (4-hydroxyphenyl) propionitrile (DPN) groups were subcutaneously injected for 5 weeks with sesame oil, PPT as an ERα agonist, and DPN as an ERβ agonist, respectively. The crown-lump length and body weight were measured weekly. BMD, serum levels of growth hormone (GH) and estradiol were checked before and after 5 weeks of injections. Pituitary GH1 expression levels were determined with quantitative real-time polymerase chain reaction, the proximal tibias were dissected, decalcified and stained with hematoxylin-eosin, and the thicknesses of epiphyseal plates including proliferative and hypertrophic zones were measured in 20-evenly divided sites after 5 weeks of injections. Comparisons for auxological data, serum hormone and pituitary GH1 expression levels, BMD, and epiphyseal plate thicknesses among 3 groups before and after injections were conducted.ResultsThere was no significant difference in body lengths among 3 groups. The body weights were significantly lower, but, serum GH, pituitary GH1 expression levels, and BMDs were higher in PPT group than the other 2 groups after 5 weeks of injections. There was no significant difference in the thicknesses of the total epiphyseal plate, proliferative, and hypertrophic zone among 3 groups.ConclusionERα is more involved in pituitary GH secretion and bone mineral deposition than ERβ. Weight gain might be prevented with the ERα agonist.

Biochemical and histopathologic assessment of effects of acitretin on epiphyseal growth plate in rats.

IntroductionAcitretin is a commonly used retinoid in dermatology. Although there are generally known side effects, the effects on the epiphyseal plaque and bone metabolism are not clear in the literature.AimTo histopathologically investigate the effects on the epiphyseal plate and assess variations in bone metabolism caused by acitretin.Material and methodsThree groups were formed with 10 rats in each group. The 1st group (n = 10, 5 male, 5 female) were administered 10 mg/kg/day oral acitretin solution and the 2nd group (n = 10, 5 male, 5 female) were administered 3 mg/kg/day oral acitretin solution. The control group were given normal standard feed and water. Rats were sacrificed at the end of 4 weeks. The proximal tibias were excised and histopathologically and immunohistochemically assessed. Biochemical assessment was also carried out.ResultsStaining with haematoxylin-eosin found reductions in the epiphyseal plate in the 1st and 2nd group compared to the control group, though this situation was not statistically significant. Immunohistochemical studies did not encounter Type II collagen in the epiphyseal bone, proliferative zone and hypertrophic zone in the control group, low dose acitretin solution group and high dose acitretin solution group. Type II collagen was not observed in osteoids and osteoblasts. Type I collagen was not observed in the hypertrophic zone and proliferative zone of any group.ConclusionsOur data show that though acitretin caused degeneration of the epiphyseal plate, it did not cause clear thinning and we identified no significant variations in bone metabolism markers.

P363 Cardiac magnetic resonance characteristics of patients in the grey zone of hypertrophy

Abstract Funding Acknowledgements National Research, Development and Innovation Office (NKFIH) of Hungary (K 120277); Project no. NVKP_16-1-2016-0017. Supported by the ÚNKP-18-3-IV Background Differentiation between athlete’s heart and hypertrophic cardiomyopathy (HCM) may cause difficulties especially in patients in the grey zone of hypertrophy. Purpose We aimed to analyse cardiac magnetic resonance (CMR) characteristics of healthy athletes, sedentary HCM and athletic HCM patients in the grey zone of hypertrophy. Methods Male HCM patients with slightly elevated maximal end-diastolic wall thickness (EDWT 13-18 mm) and highly trained healthy athletes (n = 34, 20.4 ± 2.3 training hrs/week) were consecutively enrolled. HCM patient group was divided to sedentary (n = 35, <7 training hrs/week) and athletic HCM group (n = 13; >7 h/week, 13.2 ± 5.0 h training hrs/week).We determined conventional CMR parameters (left ventricular (LV) ejection fraction (EF),BSA-corrected end-diastolic (EDVi) and end-systolic volume (ESVi), mass (Mi)), derived parameters such as EDWT/LVEDVi, LVM/LVEDV and CMR based strain parameters such as global longitudinal (GLS), radial (GRS) and circumferential strain (GCS), standard deviation of peak LS, RS and CS (SD LS peak, SD RS peak, SD CS peak), standard deviation of time to peak LS, RS and CS (TTP LS, TTP RS, TTP CS) using feature tracking. Presence of late gadolinium enhancement (LGE) was determined on delayed contrast enhanced images. Inter-observer analysis for strain values was performed in a subgroup of 20 patients conducted by two blinded observer. Results Healthy athletes showed lower LVEF, GCS and GRS than sedentary and athletic HCM patients, respectively (57.9 ± 5.2 vs 65.3 ± 7.4, 62.7 ± 5.1; -19.7 ± 7.0 vs -24.4 ± 4.6, -22.8 ± 4.8; 61.7 ± 11.2 vs 75.4 ± 21.4, 69.3 ± 19.8). Both healthy athletes and athletes with HCM showed higher LVEDVi, LVESVi, LVSVi and LVMi than sedentary HCM patients. Although sedentary athletes showed higher SD peak strain values and more pronounced intraventricular dyssynchrony than healthy athletes (TTP LS 15.4 ± 4.9 vs 11.7 ± 4.3), none of the global strain values, SD of peak strain values or TTP strain values showed any difference between athletic HCM and healthy athletes. EDWT/LVEDVi ratio was significantly lower in healthy athletes than in sedentary and athletic HCM, respectively (0.11 ± 0.02 vs 0.19±.0.04; 0.18 ± 0.04). Inter-observer analysis showed good agreement in GLS (κ=0.77), GRS (κ=0.69) and TTP LS (κ=0.74), moderate agreement in GCS (κ=0.54), and only fair or poor agreement in SD peak strain, TTP CS and TTP RS values. None of the healthy athletes showed LGE, 62% of athletic HCM and 71% of sedentary HCM patients showed LGE in the hypertrophic segments or in the insertion points. Conclusions Based on our results CMR characteristics of sedentary and athletic HCM differs significantly, therefore establishing diagnostic parameters and cut-off values based on comparison between athletic HCM and healthy athletes would be essential. Presence of late gadolinium enhancement and derived parameters may have an important role in the differential diagnosis.

Differential expression of Sox9 protein and proteoglycans in the epiphyseal cartilage of bromocriptine-treated pregnant and lactating rats.

Several investigations have shown that pregnancy and lactation are able to induce elongation of long bone by altering epiphyseal cartilage function in a prolactin-dependent manner. Since the transcription factor Sox9 is of utmost importance for chondrocyte proliferation and differentiation and since bromocriptine, a dopaminergic D2 agonist widely used to suppress milk production, is known to disrupt the production and release of prolactin, we herein aimed to investigate whether pregnancy and lactation as well as bromocriptine could alter the expression of Sox9. Our immunohistochemical analysis showed that the Sox9 expression levels were markedly upregulated in the tibial proliferative zone of day 21 pregnant rats. In day 8 (early) and day 14 (mid) lactating rats, the Sox9 expression was enhanced only in the proliferative zone, but not in the resting and hypertrophic zones. There was no change in Sox9 expression in day 21 (late) lactating rats. Postweaning rats manifested a decreased Sox9 expression in the hypertrophic zone. Bromocriptine had no effect on Sox9 expression in the proliferative zone of day 21 pregnant rats; however, it completely prevented the Sox9 upregulation in those of early and mid-lactating rats. A differential response was observed in the proliferative and hypertrophic zones of late lactating rats, in which bromocriptine enhanced Sox9 expression. Further investigation of cartilaginous matrix revealed no change in proteoglycans accumulation in lactating rats. In conclusion, the upregulated Sox9 expression predominantly occurred in the proliferative zone during late pregnancy and early and mid-lactation, while the bromocriptine effects depended on the periods and epiphyseal zones.

Cell cycle progression is disrupted in murine MPS VII growth plate leading to reduced chondrocyte proliferation and transition to hypertrophy.

Endochondral bone growth is abnormal in 6 of the 11 types of mucopolysaccharidoses (MPS) disorders; resulting in short stature, reduced size of the thoracic cavity and compromised manual dexterity. Current therapies for MPS have had a limited effect on bone growth and to improve these therapies or develop adjunct approaches requires an understanding of the underlying basis of abnormal bone growth in MPS. The MPS VII mouse model replicates the reduction in long bone and vertebral length observed in human MPS. Using this model we have shown that the growth plate is elongated but contains fewer chondrocytes in the proliferative and hypertrophic zones. Endochondral bone growth is in part regulated by entry and exit from the cell cycle by growth plate chondrocytes. More MPS VII chondrocytes were positive for Ki67, a marker for active phases of the cell cycle, suggesting that more MPS VII chondrocytes were in the cell cycle. The number of cells positive for phosphorylated histone H3 was significantly reduced in MPS VII chondrocytes, suggesting fewer MPS VII chondrocytes progressed to mitotic division. While MPS VII HZ chondrocytes continued to express cyclin D1 and more cells were positive for E2F1 and phos pRb than normal, fewer MPS VII HZ chondrocytes were positive for p57kip2 a marker of terminal differentiation, suggesting fewer MPS VII chondrocytes were able to exit the cell cycle. In addition, multiple markers typical of PZ to HZ transition were not downregulated in MPS VII, in particular Sox9, Pthrpr and Wnt5a. These findings are consistent with MPS VII growth plates elongating at a slower rate than normal due to a delay in progression through the cell cycle, in particular the transition between G1 and S phases, leading to both reduced cell division and transition to the hypertrophic phenotype.

Fluoride Inhibits Longitudinal Bone Growth by Acting Directly at the Growth Plate in Cultured Neonatal Rat Metatarsal Bones.

Excessive intake of fluoride inhibits bone growth in both humans and animals. It is unknown whether fluoride acts directly on the growth plate to inhibit longitudinal bone growth, and its mechanism of action has not been elucidated. In this study, we used an organ culture system and SW1353 cells to evaluate the effects of fluoride on endochondral ossification. Neonatal rat metatarsal bones were dissected and cultured with or without fluoride for 7days. The total length and width of the metatarsal rudiments and the length of the calcification zone were measured. Chondrocyte proliferation, differentiation, and apoptosis were analyzed by immunohistochemistry and TUNEL assay in sectioned bones. The apoptosis was detected by flow cytometry, and the expression of apoptosis-related proteins Bax, Bcl-2, and Caspase-3 were detected by western blotting in SW1353 cells. Linear measurements demonstrated that fluoride induced a biphasic effect on longitudinal bone growth in organ culture, with a significant growth inhibition at a high concentration (10-4M) and a stimulatory action at low concentration (10-6M) of fluoride. Histomorphometrical analysis of growth plate from fluoride-exposed metatarsal rudiments showed a significant reduction in the height of the proliferative and hypertrophic chondrocyte zones. Analysis of the Col2α1 and Col10α1 expression by immunohistochemistry revealed fluoride-suppressed metatarsal growth plate chondrocyte proliferation and differentiation. In addition, fluoride increased the number of apoptotic chondrocytes in the metatarsal growth plate. Western blotting showed an up-regulated expression of Caspase-3 and Bax and down-regulated expression of anti-apoptotic protein Bcl-2 after treatment with 5 × 10-4M fluoride in SW1353 cells. Our findings indicated that fluoride inhibited longitudinal bone growth by acting directly at the growth plate in cultured neonatal rat metatarsal bones. Such growth inhibition was mediated by suppressing proliferation and differentiation, increasing apoptosis of resting chondrocytes and causing premature cell senescence in the growth plate.

NF-κB Signaling Regulates Physiological and Pathological Chondrogenesis.

The nuclear factor-κB (NF-κB) is a transcription factor that regulates the expression of genes that control cell proliferation and apoptosis, as well as genes that respond to inflammation and immune responses. There are two means of NF-κB activation: the classical pathway, which involves the degradation of the inhibitor of κBα (IκBα), and the alternative pathway, which involves the NF-κB-inducing kinase (NIK, also known as MAP3K14). The mouse growth plate consists of the resting zone, proliferative zone, prehypertrophic zone, and hypertrophic zone. The p65 (RelA), which plays a central role in the classical pathway, is expressed throughout the cartilage layer, from the resting zone to the hypertrophic zone. Inhibiting the classical NF-κB signaling pathway blocks growth hormone (GH) or insulin-like growth factor (IGF-1) signaling, suppresses cell proliferation, and suppresses bone morphogenetic protein 2 (BMP2) expression, thereby promoting apoptosis. Since the production of autoantibodies and inflammatory cytokines, such as tumor necrosis factor-α (TNFα), interleukin (IL)-1β, IL-6, and IL-17, are regulated by the classical pathways and are increased in rheumatoid arthritis (RA), NF-κB inhibitors are used to suppress inflammation and joint destruction in RA models. In osteoarthritis (OA) models, the strength of NF-κB-activation is found to regulate the facilitation or suppression of OA. On the other hand, RelB is involved in the alternative pathway, and is expressed in the periarticular zone during the embryonic period of development. The alternative pathway is involved in the generation of chondrocytes in the proliferative zone during physiological conditions, and in the development of RA and OA during pathological conditions. Thus, NF-κB is an important molecule that controls normal development and the pathological destruction of cartilage.

Phytase and phytate interactions on broilers' diet at 21 days of age

The objective was to evaluate the effects of different levels of phytase in diets with varying amounts of phytate on live performance and bone characteristics of broiler chickens at 21 D. The treatments consisted of a 3 × 5 factorial arrangement, with 3 levels of phytate, high ( HP ), medium ( MP ), and low ( LP ), and 5 diets consisting of a positive control ( PC ); negative control ( NC , with a reduction of 0.15% of calcium [ Ca ] and 0.15% of phosphorus [ P ]), and NC diet plus 0, 500, 1,000, or 1,500 FTU kg −1 of phytase. Feed intake peaked with supplementation of 1,051 FTU kg −1 phytase to the LP diets. Bone ash of broilers receiving LP showed a maximum response at 1,101 FTU kg −1 . Birds receiving the NC diet had a larger hypertrophic cartilage zone ( P < 0.05) than those receiving the PC diet. Serum Ca and P of birds receiving the NC treatment and LP diet were lower than those in broilers fed with the MP and HP diets. Broilers in the NC plus 500 FYT kg −1 treatments had lower tibia P levels than those in the PC treatment; also, broilers receiving HP diets had a higher tibia Ca content than those receiving LP diets ( P < 0.05). In general, bone P of birds fed with diets containing HP was higher than that of birds on a LP or MP diet ( P < 0.05). The use of 1,101 FTU kg −1 resulted in better bone characteristics when fed with the lowest phytate level, and this level does not negatively affect the other parameters evaluated.

Radial Extracorporeal Shock Wave Treatment Promotes Bone Growth and Chondrogenesis in Cultured Fetal Rat Metatarsal Bones.

Substantial evidence exists to show the positive effects of radialextracorporeal shock wave therapy (ESWT) on bone formation. However, it is unknown whether rESWT can act locally at the growth plate level to stimulate linear bone growth. One way to achieve this is to stimulate chondrogenesis in the growth plate without depending on circulating systemic growth factors. We wished to see whether rESWT would stimulate metatarsal rat growth plates in the absence of vascularity and associated systemic growth factors. To study the direct effects of rESWT on growth plate chondrogenesis, we asked: (1) Does rESWT stimulate longitudinal bone growth of ex vivo cultured bones? (2) Does rESWT cause any morphological changes in the growth plate? (3) Does rESWT locally activate proteins specific to growth plate chondrogenesis? Metatarsal bones from rat fetuses were untreated (controls: n = 15) or exposed to a single application of rESWT at a low dose (500 impulses, 5 Hz, 90 mJ; n = 15), mid-dose (500 impulses, 5 Hz, 120 mJ; n = 14) or high dose (500 impulses, 10 Hz, 180 mJ; n = 34) and cultured for 14 days. Bone lengths were measured on Days 0, 4, 7, and 14. After 14 days of culturing, growth plate morphology was assessed with a histomorphometric analysis in which hypertrophic cell size (> 7 µm) and hypertrophic zone height were measured (n = 6 bones each). Immunostaining for specific regulatory proteins involved in chondrogenesis and corresponding staining were quantitated digitally by a single observer using the automated threshold method in ImageJ software (n = 6 bones per group). A p value < 0.05 indicated a significant difference. The bone length in the high-dose rESWT group was increased compared with that in untreated controls (4.46 mm ± 0.75 mm; 95% confidence interval, 3.28-3.71 and control: 3.50 mm ± 0.38 mm; 95% CI, 4.19-4.72; p = 0.01). Mechanistic studies of the growth plate's cartilage revealed that high-dose rESWT increased the number of proliferative chondrocytes compared with untreated control bones (1363 ± 393 immunopositive cells per bone and 500 ± 413 immunopositive cells per bone, respectively; p = 0.04) and increased the diameter of hypertrophic chondrocytes (18 ± 3 µm and 13 ± 3 µm, respectively; p < 0.001). This was accompanied by activation of insulin-like growth factor-1 (1015 ± 322 immunopositive cells per bone and 270 ± 121 immunopositive cells per bone, respectively; p = 0.043) and nuclear factor-kappa beta signaling (1029 ± 262 immunopositive cells per bone and 350 ± 60 immunopositive cells per bone, respectively; p = 0.01) and increased levels of the anti-apoptotic proteins B-cell lymphoma 2 (718 ± 86 immunopositive cells per bone and 35 ± 11 immunopositive cells per bone, respectively; p < 0.001) and B-cell lymphoma-extra-large (107 ± 7 immunopositive cells per bone and 34 ± 6 immunopositive cells per bone, respectively; p < 0.001). In a model of cultured fetal rat metatarsals, rESWT increased longitudinal bone growth by locally inducing chondrogenesis. To verify whether rESWT can also stimulate bone growth in the presence of systemic circulatory factors, further studies are needed. This preclinical proof-of-concept study shows that high-dose rESWT can stimulate longitudinal bone growth and growth plate chondrogenesis in cultured fetal rat metatarsal bones. A confirmatory in vivo study in skeletally immature animals must be performed before any clinical studies.

Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage.

Mandibular condylar cartilage (MCC) is a multizonal heterogeneous fibrocartilage consisting of fibrous (FZ), proliferative (PZ), mature (MZ), and hypertrophic (HZ) zones. Gross sampling of the whole tissue may conceal some important information and compromise the validity of the molecular analysis. Laser capture microdissection (LCM) technology allows isolating zonal (homogenous) cell populations and consequently generating more accurate molecular and genetic data, but the challenges during tissue preparation and microdissection procedures are to obtain acceptable tissue section morphology that allows histological identification of the desirable cell type and to minimize RNA degradation. Therefore, our aim is to optimize an LCM protocol for isolating four homogenous zone-specific cell populations from their respective MCC zones while preserving the quality of RNA recovered. MCC and FCC (femoral condylar cartilage) specimens were harvested from 5-week-old Sprague–Dawley male rats. Formalin-fixed and frozen unfixed tissue sections were prepared and compared histologically. Additional specimens were microdissected to prepare LCM samples from FCC and each MCC zone individually. Then, to evaluate LCM-RNA integrity, 3′/m ratios of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (β-Actin) using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were calculated. Both fixed and unfixed tissue sections allowed reliable identification of MCC zones. The improved morphology of the frozen sections of our protocol has extended the range of cell types to be isolated. Under the empirically set LCM parameters, four homogeneous cell populations were efficiently isolated from their respective zones. The 3′/m ratio means of GAPDH and β-Actin ranged between 1.11–1.56 and 1.41–2.12, respectively. These values are in line with the reported quality control requirements. The present study shows that the optimized LCM protocol could allow isolation of four homogenous zone-specific cell populations from MCC, meanwhile preserving RNA integrity to meet the high quality requirements for subsequent molecular analyses. Thereby, accurate molecular and genetic data could be generated.

Mineralization pathways in the active murine epiphyseal growth plate

Endochondral ossification in the growth plate of long bones involves cartilage mineralization, bone formation and the budding vasculature. Many of these processes take place in a complex and dynamic zone, the provisional ossification zone, of the growth plate. Here we investigate aspects of mineralization in 2D and 3D in the provisional ossification zone at different length scales using samples preserved under cryogenic or fully hydrated conditions. We use confocal light microscopy, cryo-SEM and micro-CT in the phase contrast mode. We show in 9 week old BALB/c mice the presence of vesicles containing mineral particles in the blood serum, as well as mineral particles without membranes integrated with the blood vessel walls. We also observe labeled mineral particles within cells associated with bone formation, but not in the hypertrophic cartilage cells that are involved with cartilage mineralization. High resolution micro-CT images of fresh hydrated tibiae, show that there are open continuous pathways between the blood vessel extremities and the hypertrophic chondrocyte zone. As the blood vessel extremities, the mineralizing cartilage and the forming bone are all closely associated within this narrow zone, we raise the possibility that in addition to ion transport, mineral necessary for both cartilage and bone formation is also transported through the vasculature.

P4429Cardiac magnetic resonance characteristics of professional athletes and hypertrophic cardiomyopathy patients in the grey zone of hypertrophy

Abstract Differentiation between athlete's heart and hypertrophic cardiomyopathy (HCM) may cause difficulties especially in patients in the grey zone of hypertrophy. We aimed to determine conventional cardiac magnetic resonance (CMR) parameters such as left ventricular (LV) ejection fraction (EF), BSA-corrected end-diastolic (EDVi), end-systolic and stroke volume (SVi), mass (Mi), derived CMR parameters such as maximal end-diastolic wall thickness to LVEDVi ratio (EDWT/LVEDVi), LVM to LVEDV ratio (LVM/LVEDV), and CMR based strain values (global longitudinal (GLS), radial (GRS) and circumferential strain (GCS)) in male HCM patients and athletes. We consecutively enrolled male HCM patients with only slightly elevated EDWT (13–18 mm) and highly trained healthy athletes (n=30, 18.7±1.2 training hrs/week) with marked LV hypertrophy. HCM patient group was divided into sedentary (n=30, &lt;7 training hrs/week) and athletic HCM group (n=10; &gt;7 h/week, 12.7±7.3 h training hrs/week). Both sedentary and athletic HCM patients showed higher LVEF, lower LVEDVi and LVESVi and higher EDWT compared to the healthy athletes. LVMi of both healthy athletes and athletic HCM patients was significantly higher than in sedentary HCM patients, respectively (98.9±11.4; 94.5±7.8 vs 78.1±14.4 /m2). EDWT/LVEDVi ratio was higher in both sedentary and athletic HCM patients compared to healthy athletes, respectively (0.19±0.04; 0.17±0.04 vs 0.11±0.02). LVM/LVEDV also showed significant difference between HCM patients and healthy athletes. GLS and GRS showed no significant difference between the three groups, GCS was higher in athletic HCM compared to healthy athletes (−20.7±2.2 vs −17.8±2.3%). Figure 1. Feature tracking analysis of a healthy athlete and a patient with HCM. CMR characteristics of athletic and sedentary HCM may fundamentally alter. Our preliminary data suggest that besides conventional CMR parameters, derived parameters such as EDWT/LVEDVi and LVM/LVEDV ratios and deformation imaging may also help the differentiation between pathological and physiological hypertrophy. Acknowledgement/Funding National Research, Development and Innovation Office (NKFIH) of Hungary (K 120277). Project no. NVKP_16-1-2016-0017

Immunohistochemical study on the postnatal growth changes of the spheno-occipital synchondrosis and tibial cartilage

Introduction: The synchondrosis contributes to the growth of overall skull. The growth plate of the tibia is an analogous structure to the synchondrosis. The fibroblast growth factor receptors (FGFRs) are known to play an essential role in the proliferation and differentiation of cartilaginous cells. Methods: This study was purposed to examine the histological features of spheno-occipital synchondrosis (SOS) and tibial cartilage (TC) and the expression pattern of FGFR-1, -2 and proliferating cell nuclei antigen (PCNA) in SOS and TC of the postnatal mouse using an immunohistochemical method. Results: The width of SOS and TC reduced with age. The width of the SOS decreased, and then maintained, while the width of TC decreased gradually. Expression pattern of FGFRs indicated that they were involved in the postnatal bone growth and development. In SOS, FGFR-1 expression increased until the 14th day, and then, it showed a notable decrease. Comparing the level of expression, TC showed a stronger level than SOS at most stages. FGFR-2 showed in the resting and proliferating zones at an earlier stage of differentiation. With age, FGFR-2 expression reduced in previous zone and increased in the hypertrophic zone at both tissues. In PCNA study, cell proliferation was active in the resting and proliferating zone at an early stage. As mouse matured, the PCNA-positive cells usually expressed in the newly formed endosteal bone surface. Conclusions: This approach revealed a temporal and spatial change of FGFR-1, -2 expressions. The expression pattern of PCNA indicated that the chondrocytes of TC had more active metabolism than SOS at most of the stages. We can also speculate that FGFR-1 is a negative regulator of chondrogenesis, and FGFR-2 is a positive regulator of chondrogenesis.

Mir-374-5p, mir-379-5p, and mir-503-5p Regulate Proliferation and Hypertrophic Differentiation of Growth Plate Chondrocytes in Male Rats

Growth plate chondrocytes undergo sequential differentiation to form the resting zone, the proliferative zone (PZ), and the hypertrophic zone (HZ). The important role of microRNAs (miRNAs) in the growth plate was previously revealed by cartilage-specific ablation of Dicer, an enzyme essential for biogenesis of many miRNAs. To identify specific miRNAs that regulate differentiation of PZ chondrocytes to HZ chondrocytes, we microdissected individual growth plate zones from juvenile rats and performed miRNA profiling using a solution hybridization method and miRNA sequencing. Thirty-four miRNAs were differentially expressed between the PZ and the HZ, and we hypothesized that some of the miRNAs that are preferentially expressed in the PZ may promote proliferation and inhibit hypertrophic differentiation. Consistent with this hypothesis, transfection of inhibitors for four of these miRNAs (mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p) decreased proliferation in primary epiphyseal chondrocytes. The inhibitors for three of these miRNAs (mir-374-5p, mir-379-5p, and mir-503-5p) also increased expression of multiple genes that are associated with chondrocyte hypertrophic differentiation. We next hypothesized that preferential expression of these miRNAs in the PZ is driven by the parathyroid hormone-related protein (PTHrP) concentration gradient across the growth plate. Consistent with this hypothesis, treatment of primary chondrocytes with a parathyroid hormone (PTH)/PTHrP receptor agonist, PTH1-34, increased expression of mir-374-5p, mir-379-5p, and mir-503-5p. Taken together, our findings suggest that the PTHrP concentration gradient across the growth plate induces differential expression of mir-374-5p, mir-379-5p, and mir-503-5p between the PZ and the HZ. In the PZ, the higher expression levels of these miRNAs promote proliferation and inhibit hypertrophic differentiation. In the HZ, downregulation of these miRNAs inhibits proliferation and promotes hypertrophic differentiation.

High Impact Exercise Improves Bone Microstructure and Strength in Growing Rats

Physical activity is beneficial for skeletal development. However, impact sports during adolescence, leading to bone growth retardation and/or bone quality improvement, remains unexplained. This study investigated the effects of in vivo low (LI), medium (MI), and high (HI) impact loadings applied during puberty on bone growth, morphometry and biomechanics using a rat model. 4-week old rats (n = 30) were divided into control, sham, LI, MI, and HI groups. The impact was applied on the right tibiae, 5 days/week for 8 weeks mimicking walking (450 µε), uphill running (850 µε) and jumping (1250 µε) conditions. Trabecular and cortical parameters were determined by micro-CT, bone growth rate by calcein labeling and toluidine blue staining followed by histomorphometry. Bio-mechanical properties were evaluated from bending tests. HI group reduced rat body weight and food consumption compared to shams. Bone growth rate also decreased in MI and HI groups despite developing thicker hypertrophic and proliferative zone heights. HI group showed significant increment in bone mineral density, trabecular thickness, cortical and total surface area. Ultimate load and stiffness were also increased in MI and HI groups. We conclude that impact loading during adolescence reduces bone growth moderately but improves bone quality and biomechanics at the end of the growing period.