Longitudinal Growth Of Long Bones Research Articles

α-parvin controls chondrocyte column formation and regulates long bone development

Endochondral ossification requires proper control of chondrocyte proliferation, differentiation, survival, and organization. Here we show that knockout of α-parvin, an integrin-associated focal adhesion protein, from murine limbs causes defects in endochondral ossification and dwarfism. The mutant long bones were shorter but wider, and the growth plates became disorganized, especially in the proliferative zone. With two-photon time-lapse imaging of bone explant culture, we provide direct evidence showing that α-parvin regulates chondrocyte rotation, a process essential for chondrocytes to form columnar structure. Furthermore, loss of α-parvin increased binucleation, elevated cell death, and caused dilation of the resting zones of mature growth plates. Single-cell RNA-seq analyses revealed alterations of transcriptome in all three zones (i.e., resting, proliferative, and hypertrophic zones) of the growth plates. Our results demonstrate a crucial role of α-parvin in long bone development and shed light on the cellular mechanism through which α-parvin regulates the longitudinal growth of long bones.

G-Protein-Coupled Estrogen Receptor-1 Positively Regulates the Growth Plate Chondrocyte Proliferation in Female Pubertal Mice.

Estrogen enhances long bone longitudinal growth during early puberty. Growth plate chondrocytes are the main cells that contribute to long bone elongation. The role of G-protein-coupled estrogen receptor-1 (GPER-1) in regulating growth plate chondrocyte function remains unclear. In the present study, we generated chondrocyte-specific GPER-1 knockout (CKO) mice to investigate the effect of GPER-1 in growth plate chondrocytes. In control mice, GPER-1 was highly expressed in the growth plates of 4- and 8-week-old mice, with a gradual decline through 12 to 16 weeks. In CKO mice, the GPER-1 expression in growth plate chondrocytes was significantly lower than that in the control mice (80% decrease). The CKO mice also showed a decrease in body length (crown–rump length), body weight, and the length of tibias and femurs at 8 weeks. More importantly, the cell number and thickness of the proliferative zone of the growth plate, as well as the thickness of primary spongiosa and length of metaphysis plus diaphysis in tibias of CKO mice, were significantly decreased compared with those of the control mice. Furthermore, there was also a considerable reduction in the number of proliferating cell nuclear antigens and Ki67-stained proliferating chondrocytes in the tibia growth plate in the CKO mice. The chondrocyte proliferation mediated by GPER-1 was further demonstrated via treatment with a GPER-1 antagonist in cultured epiphyseal cartilage. This study demonstrates that GPER-1 positively regulates chondrocyte proliferation at the growth plate during early puberty and contributes to the longitudinal growth of long bones.

NFI-C Is Required for Epiphyseal Chondrocyte Proliferation during Postnatal Cartilage Development.

Stringent regulation of the chondrocyte cell cycle is required for endochondral bone formation. During the longitudinal growth of long bones, mesenchymal stem cells condense and differentiate into chondrocytes. Epiphyseal chondrocytes sequentially differentiate to form growth-plate cartilage, which is subsequently replaced with bone. Although the importance of nuclear factor 1C (Nfic) in hard tissue formation has been extensively studied, knowledge regarding its biological roles and molecular mechanisms in this process remains insufficient. Herein, we demonstrated that Nfic deficiency affects femoral growth-plate formation. Chondrocyte proliferation was downregulated and the number of apoptotic cell was increased in the growth plates of Nfic -/- mice. Further, the expression of the cell cycle inhibitor p21 was upregulated in the primary chondrocytes of Nfic -/- mice, whereas that of cyclin D1 was downregulated. Our findings suggest that Nfic may contribute to postnatal chondrocyte proliferation by inhibiting p21 expression and by increasing the stability of cyclin D1 protein.

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.

KIF5B modulates central spindle organization in late-stage cytokinesis in chondrocytes

BackgroundThe growth plate is a special region of the cartilage that drives longitudinal growth of long bones. Proliferating chondrocytes in the growth plate, arranged in columns, divide perpendicular to the long axis of the growth plate then intercalate to re-align with parental columns. Which molecular partners maintain growth plate columnar structures and chondrocyte cytokinesis has not been fully revealed. It is reported that kinesin family member 3A (KIF3A), a subunit of kinesin-2, plays an important role in maintaining columnar organization in growth plates via controlling primary cilia formation and cell proliferation.ResultHere we identify kinesin family member 5B (KIF5B), the heavy chain of kinesin-1, a ubiquitously expressed motor protein for anterograde intracellular transport along the microtubule network, as a key modulator of cytokinesis in chondrocytes via maintenance of central spindle organization. We show that KIF5B is concentrated in the central spindle during cytokinesis in both primary chondrocytes and chondrogenic ATDC5 cells.ConclusionThe failure of cytokinesis in KIF5B null chondrocytes leads to incomplete cell rotation, disrupting proliferation and differentiation, and results in a disorganized growth plate.

La maladie des exostoses multiples

Hereditary multiple exostosis (HEM) is one of the most common hereditary diseases. It is characterized by the proliferation of bone protuberances, especially located in the metaphysis of long bones. The disease phenotype may also associate abnormalities in the shape and length of long bones, such as the typical “Bessel Hagen” deformity. Clinically, the main complaint of patients remains pain, but the psychological and social consequences should not be minimized. The rare complication (2 to 5% of cases) but the most feared is the transformation into chondrosarcoma, which motivates regular clinical and radiological monitoring of these patients. This follow-up, although essential, remains poorly defined, including at a minimum, an annual clinical examination and radiological follow-up based on the symptoms described by the patients. The treatment remains mainly surgical, with patients being most often multi-operated during the course of the disease. Medical treatment remains secondary. In recent years, progress has been made in understanding the pathophysiology of this disease, particularly with the discovery of the mutation of EXT genes, found in 80% of HME cases. These tumor suppressor genes encode proteins that act in the synthesis of heparan sulfates (HS). The decrease in the amount of HS leads to changes in certain metabolic pathways, which explains the development of ectopic growth plaques, which are the cause of bone exostosis, but also the poor longitudinal growth of long bones. The greater knowledge of the mechanisms underlying this disease makes it possible to consider potential therapeutic targets.

Osterix regulates corticalization for longitudinal bone growth via integrin \u03b23 expression

Corticalization, coalescence of trabecular bone into the metaphyseal cortex, is important for the longitudinal growth of long bones. However, little is known about the molecular mechanisms controlling corticalization. To understand the molecular mechanisms underlying corticalization, we analyzed osteoblast-specific Osterix-knockout mice (Col-OMT). In control mice, corticalization was initiated after 7 postnatal days, and the number of osteoblasts in the peripheral spongiosa was increased compared to the number in the central spongiosa. In contrast, in Col-OMT mice, corticalization was delayed, and the number of osteoblasts in peripheral zones was unchanged compared to the central zone. Furthermore, femoral length was decreased in Col-OMT mice at 1 month. Because Col-OMT mice exhibited impaired matrix coalescence and osteoblast migration, we evaluated integrin signaling in Col-OMT mice. Osterix bound to the Itgb3 promoter and increased transcription of the Itgb3 gene in osteoblast cells. Interestingly, the inner and outer cortical bones were separated in Itgb3-null mice at postnatal day 7. In Itgb3-null mice, the number of osteoblasts in peripheral zones was not changed, and the femoral length was decreased. Taken together, these results indicate that Osterix regulates corticalization for longitudinal bone growth via the control of integrin β3 expression in osteoblasts. Our findings imply that the ability to control osteoblast function during corticalization may help in the treatment of short stature.

The influence of dexamethasone administered prenatally on cartilage of newborn spiny mouse (Acomys cahirinus) offspring

Considering the negative effects of glucocorticoid treatment, especially during fetal development it is important to investigate effectors decreasing such disadvantages. The aim of this study was to investigate the effect of prenatally administered dexamethasone (Dex), a synthetic glucocorticoid, on the histomorphometry of the femur in the offspring of spiny mice. The study was performed on 24 pregnant spiny mice. The time of the experiment included the prenatal period between the 20th day of gestation until birth (pregnancy lasts on average of 36-38 days). The mice from the experimental group received dexamethasone per os in a dose of 125 mg/kg birth weight daily. At the end, the newborns from the experimental and control group were weighted and euthanized. Maternal Dex treatment resulted in a 17% decrease in birth weight in newborns. Dex administration significantly reduced the thickness of the hypertrophy zone of the growth plate by 34% and total thickness by 8,7%. In addition, Dex decreased the number of cells in the articular cartilage by 27% and significantly decreased their diameter by 5%. Dex also affected the structure and spatial distribution of thick and thin collagen fibers, lowering the proportion of thin fibers compared with the control group. Moreover, Dex treatment considerably lowered the amount of proteoglycans in articular and growth cartilages. Exposure to glucocorticoids in pregnant spiny mice affects cartilage development by accelerating maturity of collagen fibers and growth plate, presumably along with further disruption of longitudinal growth of long bones.

Growth plate cartilage shows different strain patterns in response to static versus dynamic mechanical modulation.

Longitudinal growth of long bones and vertebrae occurs in growth plate cartilage. This process is partly regulated by mechanical forces, which are one of the underlying reasons for progression of growth deformities such as idiopathic adolescent scoliosis and early-onset scoliosis. This concept of mechanical modulation of bone growth is also exploited in the development of fusionless treatments of these deformities. However, the optimal loading condition for the mechanical modulation of growth plate remains to be identified. The objective of this study was to evaluate the effects of in vitro static versus dynamic modulation and of dynamic loading parameters, such as frequency and amplitude, on the mechanical responses and histomorphology of growth plate explants. Growth plate explants from distal ulnae of 4-week-old swines were extracted and randomly distributed among six experimental groups: baseline ([Formula: see text]), control ([Formula: see text]), static ([Formula: see text]) and dynamic ([Formula: see text]). For static and dynamic groups, mechanical modulation was performed in vitro using an Indexed CartiGen bioreactor. A stress relaxation test combined with confocal microscopy and digital image correlation was used to characterize the mechanical responses of each explant in terms of peak stress, equilibrium stress, equilibrium modulus of elasticity and strain pattern. Histomorphometrical measurements were performed on toluidine blue tissue sections using a semi-automatic custom-developed MATLAB toolbox. Results suggest that compared to dynamic modulation, static modulation changes the strain pattern of the tissue and thus is more detrimental for tissue biomechanics, while the histomorphological parameters are not affected by mechanical modulation. Also, under dynamic modulation, changing the frequency or amplitude does not affect the biomechanical response of the tissue. Results of this study will be useful in finding optimal and non-damaging parameters for the mechanical modulation of growth plate in fusionless treatments.

Impact of Long-Term Exposure to the Tyrosine Kinase Inhibitor Imatinib on the Skeleton of Growing Rats

The tyrosine kinase (TK) inhibitor imatinib provides a highly effective therapy for chronic myeloid leukemia (CML) via inhibition of the oncogenic TK BCR-ABL1. However, off-target TKs like platelet-derived growth factor receptors (PDGF-R) and colony-stimulating factor-1 receptor (c-fms), involved in bone remodeling, are also inhibited. Thus, pediatric patients with CML on imatinib exhibit altered bone metabolism, leading to linear growth failure. As TKI treatment might be necessary for a lifetime, long-term effects exerted on bone in children are of major concern. Therefore, we studied the skeletal long-term effects of continuous and intermittent imatinib exposure in a juvenile rat model.Four-weeks-old male Wistar rats were chronically exposed to imatinib via drinking water over a period of 10 weeks. Animals were exposed to a standard and high imatinib dosage continuously and to the high imatinib dose intermittently. Bone mass and strength were assessed using pQCT, micro-computed tomography (μCT), and biomechanical testing at the prepubertal, pubertal, and postpubertal age. Bone length and vertebral height as well as biochemical markers of bone turnover were analyzed.Femoral and tibial bone length were dose-dependently reduced by up to 24% (p<0.0001), femoral and tibial trabecular bone mass density (BMD) were reduced by up to 25% (p<0.01), and femoral breaking strength was lowered by up to 20% (p<0.05). Intermittent exposure mitigated these skeletal effects. Long-term exposure resulted in reduced vertebral height by 15% and lower trabecular BMD by 5%. Skeletal changes were associated with suppressed serum osteocalcin (p<0.01) and non-significantly elevated serum CTX-I and PINP levels.In conclusion, imatinib mainly impaired longitudinal growth of long bones rather than the vertebrae of growing rats. Interestingly, intermittent imatinib exposure has less skeletal side effects, which may be beneficial in pediatric patients taking imatinib.

The role of hedgehog signalling in skeletal health and disease.

Hedgehog ligands bind to protein patched homologue 1 (PTC), a conserved receptor that activates the GLI family of transcription factors, which are involved in development, disease and skeletal repair processes. During embryonic development, hedgehog signalling helps to pattern the limbs and plays a critical part in regulating chondrocyte differentiation and osteogenesis during the longitudinal growth of long bones. This signalling pathway also regulates mesenchymal cell differentiation during skeletal repair and regeneration. In pathologic and degenerative processes, such as osteoarthritis or cartilaginous tumour formation, hedgehog signalling is dysregulated. Several pharmacologic strategies can modulate hedgehog signalling, and targeting this pathway could lead to the development of novel therapeutic approaches. For example, by precisely regulating the level of activity of the hedgehog signalling pathway, the pace of degeneration in osteoarthritis could be slowed, bone repair could be enhanced, and cartilaginous tumour cell viability could be inhibited. As such, regulation of hedgehog signalling could be manipulated to safeguard skeletal health.

Comparative proteomic analysis of hypertrophic chondrocytes in osteoarthritis.

BackgroundOsteoarthritis (OA) is a multi-factorial disease leading progressively to loss of articular cartilage and subsequently to loss of joint function. While hypertrophy of chondrocytes is a physiological process implicated in the longitudinal growth of long bones, hypertrophy-like alterations in chondrocytes play a major role in OA. We performed a quantitative proteomic analysis in osteoarthritic and normal chondrocytes followed by functional analyses to investigate proteome changes and molecular pathways involved in OA pathogenesis.MethodsChondrocytes were isolated from articular cartilage of ten patients with primary OA undergoing knee replacement surgery and six normal donors undergoing fracture repair surgery without history of joint disease and no OA clinical manifestations. We analyzed the proteome of chondrocytes using high resolution mass spectrometry and quantified it by label-free quantification and western blot analysis. We also used WebGestalt, a web-based enrichment tool for the functional annotation and pathway analysis of the differentially synthesized proteins, using the Wikipathways database. ClueGO, a Cytoscape plug-in, is also used to compare groups of proteins and to visualize the functionally organized Gene Ontology (GO) terms and pathways in the form of dynamical network structures.ResultsThe proteomic analysis led to the identification of a total of ~2400 proteins. 269 of them showed differential synthesis levels between the two groups. Using functional annotation, we found that proteins belonging to pathways associated with regulation of the actin cytoskeleton, EGF/EGFR, TGF-β, MAPK signaling, integrin-mediated cell adhesion, and lipid metabolism were significantly enriched in the OA samples (p ≤10−5). We also observed that the proteins GSTP1, PLS3, MYOF, HSD17B12, PRDX2, APCS, PLA2G2A SERPINH1/HSP47 and MVP, show distinct synthesis levels, characteristic for OA or control chondrocytes.ConclusionIn this study we compared the quantitative changes in proteins synthesized in osteoarthritic compared to normal chondrocytes. We identified several pathways and proteins to be associated with OA chondrocytes. This study provides evidence for further testing on the molecular mechanism of the disease and also propose proteins as candidate markers of OA chondrocyte phenotype.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-015-9085-6) contains supplementary material, which is available to authorized users.

Overgrowth syndrome associated with a gain‐of‐function mutation of the natriuretic peptide receptor 2 (NPR2) gene

The signal pathway of the C-type natriuretic (CNP) and its receptor, natriuretic peptide receptor 2 (NPR2) is involved in the longitudinal growth of long bones. Loss of function mutations at NPR2 cause acromesomelic dysplasia, type Maroteaux, while overproduction of CNP by chromosomal translocation and a gain-of-function mutation at NPR2 have been reported to be responsible for an overgrowth syndrome in three cases and one family, respectively. We identified a four-generation family with an overgrowth syndrome characterized by tall stature, macrodactyly of the great toes, scoliosis, coxa valga and slipped capital femoral epiphysis, similar to those previously reported in association with CNP/NPR2 overactivity. The serum level of amino-terminal proCNP was normal in the proband. A novel missense mutation of NPR2, c.1462G>C (p.Ala488Pro) was found to co-segregate with the phenotype in this family. In vitro transfection assay of the mutant NPR2 revealed overactivity of the mutant receptor at baseline as well as with the ligand. This overgrowth syndrome caused by a gain-of-function mutation at NPR2 should be differentiated from Marfan or related syndromes, and may be categorized along with the overgrowth syndrome caused by overproduction of CNP due to its phenotypical similarity as overgrowth CNP/NPR2 signalopathy.

Impact Of The Tyrosine Kinase Inhibitors Imatinib, Dasatinib, and Bosutinib In Young Rats On The Vertebral Body

BackgroundTyrosine kinase inhibitors (TKIs) such as imatinib (IMA), dasatinib (DASA), and bosutinib (BOSU) inhibit the oncogenic BCR-ABL tyrosine kinase in patients with chronic myeloid leukemia (CML). In addition, all TKIs exhibit additional off-target effects on kinases like PDGF-R and c-FMS which are involved in the regulation of bone metabolism. In pediatric patients longitudinal growth retardation during IMA treatment represents a major side effect causing special concern. In an established juvenile growing rat model (Tauer JT et al. Blood 2011, ASH Abstract #1597) we examined skeletal effects of chronically and intermittently administered IMA, DASA, and BOSU and could mimic well the clinically observed impaired longitudinal growth of the long bones (Tauer JT et al. Haematologica 2012, EHA Abstract #67). Dose-dependently IMA and DASA revealed the highest impairment on bone length while BOSU exhibited a lesser effect (Tauer JT et al. Blood 2012, ASH Abstract #4429). Due to the fact that in contrast to long bones vertebral bodies mainly consist of trabecular structure and underlie ongoing bone remodeling (e.g. Ca-homeostasis) we additionally analyzed skeletal effects of chronically and intermittently administered IMA, DASA, and BOSU in young, growing rats on the vertebral body. MethodsIn the established model, 4 weeks old male Wistar rats (n=10 per group) were chronically exposed for 10 weeks via the drinking water to concentrations representing a TKI standard dose and a high dose (= two-fold standard dose) of IMA (1 mM / 2 mM), DASA (50 µM / 100 µM), or BOSU (50 µM / 100 µM). Based on an intermittent schedule of treatment applied to adult patients (Russo D et al. Blood 2010, ASH Abstract #3412; La Rosée P et al. Ann Hematol 2013, in press DOI: 10.1007/s00277-013-1769-2), also the high doses of all TKIs were administered intermittently (3 days “on”; 4 days “off”). Controls received water only. Three to four rats from each group were sacrificed after 2 weeks (prepubertal), after 4 weeks (pubertal), and after 10 weeks (postpubertal) of exposure, respectively. Beside investigations of the long bones also the height, trabecular and cortical bone mineral density (BMD) of the vertebral body L2 were assessed using a peripheral quantitative computer tomography device dedicated for small animals. ResultsContinuous and intermittent exposure of high dose IMA reduced significantly the height of the vertebral body L2 by approx. 15% (p<0.001) at pubertal and postpubertal age. Continuous exposure of high dose DASA revealed a non-significant reduction of the height by 9% postpubertally. BOSU exposure showed no effect at all concentrations and at all time points tested. Contrasting our findings in long bones (Tauer JT et al. Blood 2012, ASH Abstract #4429) trabecular and cortical BMD of the vertebral body were not affected under any TKI exposure during growth at all doses and all time points tested. ConclusionOur established juvenile rat model is appropriate to examine and mimic side effects on growing bones by long-term exposure to TKIs. We could nicely demonstrate the clinically described impaired longitudinal growth of long bones under IMA exposure in children. While data from children with CML under long-term exposure to 2nd generation TKIs are still missing, we dare to predict the same adverse effects for DASA, however, to a lesser extend for BOSU. Here, we now demonstrate that long-term IMA exposure also impaired the height of the vertebrae whereas DASA and BOSU revealed no effect. However, contrasting serum analysis of specific markers of bone metabolism pointing to overall impaired skeletal bone remodeling, BMD of the long bones, but not of the vertebral bodies, was reduced by IMA and DASA. Thus, TKIs like IMA and DASA mainly impair the longitudinal growth of the long bones which is based on the balanced network of i) proliferating chrondrocytes within the growth plate – essential for bone growth – and ii) the bone remodeling cycle which ensures the trabecular structure. Our data support a hypothesis that during growth the perturbation in remodeling of the trabecular structure caused by TKIs differs quantitatively in vertebrae from long bones. AcknowledgmentSupported by grant DFG SU122-3/1 to MS and DFG HO 1875/10-1 to LCH. Disclosures:No relevant conflicts of interest to declare.

Molecular and biophysical mechanisms regulating hypertrophic differentiation in chondrocytes and mesenchymal stem cells

Chondrocyte hypertrophy is one of the key physiological processes involved in the longitudinal growth of long bones, yet the regulation of hypertrophy is also becoming increasingly relevant for clinical application of mesenchymal stem cells (MSCs) and screening for drugs to treat hypertrophic osteoarthritis. The extraordinary cell volume increase during hypertrophy is accompanied by an up-regulation of collagen X, matrix metalloproteinases (MMPs), and vascular endothelial growth factor (VEGF), all which are targets of the runt-related transcription factor 2 (Runx2). Many pathways, including parathyroid hormone-related protein (PTHrP)/Indian Hedgehog, Wingless/Int (Wnt)/β-catenin, and transforming growth factor beta (TGF-β)/Sma and Mad Related Family (Smad) pathways, can regulate hypertrophy, but factors as diverse as hypoxia, co-culture, epigenetics and biomaterial composition can also potently affect Runx2 expression. Control of hypertrophic differentiation can be exploited both for cartilage repair, where a stable phenotype is desired, but also in bone regeneration, where hypertrophic cartilage could act as a template for endochondral bone formation. We hope this review will motivate the design of novel engineered microenvironments for skeletal regeneration applications.

The Effects of Low-Level Laser Therapy, 670 nm, on Epiphyseal Growth in Rats

The longitudinal growth of long bones is attributed to epiphyseal growth. However, the effects of low-level laser therapy (LLLT) in such structures has still not been studied extensively in the literature. Therefore, the aim of this study was to evaluate the use of LLLT, 670 nm, at three different doses on the epiphyseal growth of the right tibia of rats. Twenty-one Wistar rats, aged four weeks, were subjected to the application of LLLT, with dosage according to the group (G4: were submitted to the application of 4 J/cm2; G8: were submitted to the application of 8 J/cm2; G16: were submitted to the application of 16 J/cm2). After completion of protocol they were kept until they were 14 weeks of age and then submitted to a radiological examination (evaluation of limb length) and euthanised. The histological analysis of the growth plates (total thickness and hypertrophic and proliferative zones) was then performed. Comparisons were made with the untreated left tibia. No differences were observed in any of the reviews (radiological and histological), when comparing the right sides (treated) to the left (untreated). It was concluded that the treatment with LLLT within the parameters used caused changes neither in areas of the epiphyseal cartilage nor in the final length of limbs.

The effects of morphine on tissue structure of the growth plate in male rats.

Studies have shown that morphine, in addition to its analgesic properties, has several effects on cell proliferation and apoptosis. There is also evidence that opioid receptors are present on chondrocytes. Our main objective in the present study was to investigate the effects of morphine on rat femur growth cartilage. This research was carried out on 18 4-week-old male rats. Animals were divided into four groups: groups 1 (n= 3) and 2 (n= 4) were non morphine-dependent and groups 3 (n= 6) and 4 (n= 5) were morphine-dependent. Groups 1 and 3 were followed up for 4 weeks and the others for 7 weeks. We prepared femur bone biopsies, fixed the samples in 10% formalin and 10% nitric acid and stained the samples with haematoxylin and eosin. The thickness of the growth cartilage and its proliferative zone (PZ) cell number were studied. In addition, the existence of necrosis, inflammation, fibrosis, and hyalinisation were evaluated. There were no signs of inflammation, fibrosis, necrosis or hyalinisation in the growth cartilages of all rats. The morphine-dependent groups had a statistically significant difference (P< 0.001) in the number of cells in the proliferative zone and thickness of the growth cartilage compared to other groups using ANOVA analysis. It seems that morphine reduces the number of cell in the proliferative zone and decreases the thickness of the growth cartilage which may alter longitudinal growth of long bones.

Tierexperimentelle Studie zur Stimulation des Extremitätenwachstums durch elektrischen Gleichstrom*

Direct current under certain conditions will be show a positive effect on the longitudinal growth of long bones during growing-age. Examinations were made in 8 to 10 week old rabbits in a right to left attempt. The electrodes were implanted in the midshaft of tibia. The stimulation was performed by direct current of 20 and 30 microamperes. The quantitative evaluation of 12 oxytetracyclinelabled pairs of bone showed a statistically significant increase of longitudinal growth on the electrically treated side as compared with control. Possible reasons leading to bone growth by electrical stimulation are discussed.

Wnt gene expression in the post-natal growth plate: Regulation with chondrocyte differentiation

Longitudinal growth of long bones occurs at the growth plate by endochondral ossification. In the embryonic mouse, this process is regulated by Wnt signaling. Little is known about which members of the Wnt family of secreted signaling proteins might be involved in the regulation of the postnatal growth plate. We used microdissection and real-time PCR to study mRNA expression of Wnt genes in the mouse growth plate. Of the 19 known members of the Wnt family, only six were expressed in postnatal growth plate. Of these, Wnts -2b, -4, and -10b signal through the canonical β-catenin pathway and Wnts -5a, -5b, and -11 signal through the noncanonical calcium pathway. The spatial expression for these six Wnts was remarkably similar, showing low mRNA expression in the resting zone, increasing expression as the chondrocytes differentiated into the proliferative and prehypertrophic state and then (except Wnt-2b) decreasing expression as the chondrocytes underwent hypertrophic differentiation. This overall pattern is broadly consistent with previous studies of embryonic mouse growth cartilage suggesting that Wnt signaling modulates chondrocyte proliferation and hypertrophic differentiation. We also found that mRNA expression of these Wnt genes persisted at similar levels at 4 weeks, when longitudinal bone growth is waning. In conclusion, we have identified for the first time the specific Wnt genes that are expressed in the postnatal mammalian growth plate. The six identified Wnt genes showed a similar pattern of expression during chondrocyte differentiation, suggesting overlapping or interacting roles in postnatal endochondral bone formation.

Cortical bone development under the growth plate is regulated by mechanical load transfer

Longitudinal growth of long bones takes place at the growth plates. The growth plate produces new bone trabeculae, which are later resorbed or merged into the cortical shell. This process implies transition of trabecular metaphyseal sections into diaphyseal sections. We hypothesize that the development of cortical bone is governed by mechanical stimuli. We also hypothesize that trabecular and cortical bone share the same regulatory mechanisms for adaptation to mechanical loads. To test these hypotheses, we monitored the development of the tibial cortex in growing pigs, using micro-computer tomography and histology. We then tested the concept that regulatory mechanisms for trabecular bone adaptation can also explain cortical bone development using our mechanical stimulation theory, which could explain trabecular bone (re)modelling. The main results showed that, from the growth plate towards the diaphysis, the pores of the trabecular structure were gradually filled in with bone, which resulted in increased density and cortical bone. The computer model largely predicted this morphological development. We conclude that merging of metaphyseal trabeculae into cortex is likely to be governed by mechanical stimuli. Furthermore, cortex development of growing long bones can be explained as a form of trabecular bone adaptation, without the need for different regulatory mechanisms for cortical and trabecular bone.