Targeted Limb Heating Augments the Actions of IGF1 in the Growth Plate and Increases Bone Elongation in Growing Mice

Bone elongation disorders can lead to painful musculoskeletal disabilities in adulthood. Existing treatment options to correct left-right asymmetry in limb length include invasive surgeries and/or drug regimens. These are often only partially effective. Previous studies in weanling mice have shown that a daily application of mild heat (40°C) to limbs on one side of the body could be used to noninvasively enhance bone elongation. However, the impact of heat-treatment on bone at the cellular level remains elusive. The epiphyseal growth plate, the band of cartilage located at each end of long bones, is the main site of longitudinal growth and is regulated by local and systemic growth factors. Insulin-like growth factor 1 (IGF1) is the major regulator of growth and controls bone elongation by promoting chondrocyte proliferation and hypertrophy. The objective of this study was to build upon an established method of targeted limb heating to determine how heat-treatment influences IGF1 action in the growth plate. This study tests the hypothesis that exposure to warm temperature augments the actions of IGF1 in the growth plate and permanently increases length of the extremities. This dissertation demonstrates that differences of less than 1.5% are functionally significant measured by a nearly 20% increase in hindlimb weight bearing on heat-treated sides. Heat-enhanced bone elongation is documented in female C57BL/6 mice after 7 days of heat-treatment during the most active period of growth from 3-4 weeks of age. This increase in bone elongation is accompanied by increased chondrocyte proliferation and hypertrophy in the proximal tibial growth plate. Moreover, this study is the first to show that targeted limb-heating impacts local action of IGF1 in growth plate chondrocytes. Results suggest that heat-induced limb length is IGF1 dependent since the growth effects are attenuated when IGF1 activity is blocked. Administration of a low dose of IGF1 (2.5mg/kg) was found to augment heat enhanced bone elongation and effects were sustained to skeletal maturity (12 weeks of age). These studies help contribute to the ultimate goal of developing a noninvasive method for lengthening bones that may translate in a clinical setting to treat linear growth disorders in children

Phenotypic characterization of cartilage cells during endochondral ossification (an avian growth plate model).

by Lee Kwong Man, Simon.Thesis (M.Phil.)--Chinese University of Hong Kong, 1990.Bibliography: leaves 88-94.ABSTRACT --- p.IACKNOWLEDGEMENTS --- p.IVTABLE OF CONTENTS --- p.VChapter CHAPTER ONE - --- INTRODUCTION --- p.1Chapter CHAPTER TWO - --- STRUCTURE OF CARTILAGEChapter 2.1 --- Characteristics of Cartilage --- p.4Chapter 2.2 --- Types of Cartilage --- p.4Chapter 2.3 --- Matrix --- p.5Chapter 2.3.1 --- Collagen --- p.5Chapter 2.3.2 --- Proteoglycan --- p.7Chapter 2.4 --- Diffusion of Solultes in Cartilage --- p.9Chapter 2.5 --- Chondroytes --- p.10Chapter CHAPTER THREE - --- PHYSIOLOGICAL CHANGES WITHIN CARTILAGEChapter 3.1 --- Endochondral Ossification --- p.12Chapter 3.2 --- Hormone Responses on Cartilage --- p.16Chapter 3.3 --- Degradative enzymatic system --- p.17Chapter 3.3.1 --- Evidence for the Role of Enzymes in osteoarthritis --- p.18Chapter 3.3.2 --- Neutral Protease Acivity --- p.20Chapter 3.3.3 --- Neutal proteases in osteoarthritis --- p.21Chapter 3.3.4 --- Collagenase activity in articular cartilage --- p.22Chapter CHAPTER FOUR - --- METHODOLOGIESChapter 4.1 --- Isolation of Chick Growth Plate and Articular Chondrocytes --- p.24Chapter 4.2 --- Countercurrent Centrifugal Elutriation --- p.25Chapter 4.3 --- Size Determination of Chondrocytes --- p.26Chapter 4.4 --- Chondrocyte Cell Culture --- p.28Chapter 4.5 --- Flow Cytometry Cell Cycle Analysis of Elutriated Chondrocytes --- p.28Chapter 4.6 --- Thymidine Incorporation Assay on Elutriated Chondrocytes --- p.29Chapter 4.7 --- Sulfur Incorporation Assay on Elutriated Chondrocytes --- p.30Chapter 4.8 --- Hyalurondiase Assay on Elutriated Chondrocytes --- p.31Chapter 4.9 --- Alkaline Phosphatase Assay on Elutriated Chondrocytes --- p.32Chapter 4.10 --- Acid Phosphatase Assay --- p.33Chapter 4.10.1 --- Total Acid Phosphatase Assay on Elutriated Chondrocytes --- p.33Chapter 4.10.2 --- Optimal pH Determination of Phosphatase in Isolated Chondrocytes --- p.33Chapter 4.10.3 --- Enzyme Kinetics of Acid Phosphatase of Isolated Growth Plate and Articular Chondrocytes --- p.34Chapter 4.10.4 --- Tartrate Inhibition Effect on Acid Phosphatase of Growth Plate and Articular Chondrocytes --- p.35Chapter 4.10.5 --- Distribution of Acid Phosphatase Isoenzymes Among Chondrocytes of Different Size --- p.35Chapter 4.11 --- Hormonal Effects on Acid and Alkaline Phosphatase Activities in Growth Plate and Articular Chondrocytes --- p.36Chapter CHAPTER FIVE - --- RESULTSChapter 5.1 --- Morphology of the Isolated Chick Chondrocytes --- p.39Chapter 5.2 --- Countercurrent Centrifugal Elutriation VI --- p.39Chapter 5.3 --- Thymidine Incorporation Assay on Elutriated Chondrocytes --- p.44Chapter 5.4 --- Flow Cytometer Cell Cycle Analysis of Elutriated Chondrocytes --- p.44Chapter 5.5 --- Sulfate Incorporation Assay on Elutriated Chondrocytes --- p.48Chapter 5.6 --- Hyaluronidase Assay on Elutriated Chondrocytes on --- p.48Chapter 5.7 --- Alkaline Phosphatase Assay on Elutriaed Chondrocytes --- p.48Chapter 5.8 --- Acid Phosphatase Assay --- p.52Chapter 5.8.1 --- pH Curve of Phosphatase in Isolated Chondrocytes --- p.52Chapter 5.8.2 --- Enzyme Kinetics of Acid Phosphatase oflsolated Growth Plate and Articular Chondrocytes --- p.52Chapter 5.8.3 --- Tartrate Inhibition Effect on Acid Phosphatae of Growth Plate and Articular Chondrocytes --- p.55Chapter 5.8.4 --- Distribution of Acid Phosphatase Isoenzymes Among Chondrocytes in Different Size --- p.57Chapter 5.9 --- Hormonal Effects on Acid and Alkaline Phosphatase Activities in Growth Plate and Articular Chondrocytes --- p.59Chapter CHAPTER SIX - --- DISCUSSIONChapter 6.1 --- Identification of Chondrocyte Subpopulations --- p.63Chapter 6.2 --- Characterization of Chondrocyte Subpopulations --- p.72Chapter 6.3 --- Characterization of Acid Phosphatase in Chick Chondrocytes --- p.74LIST OF FIGURES --- p.84LIST OF TABLES --- p.87REFERENCES --- p.88Chapter APPENDIX I --- Principle of Countercurrent Centrifugal Elutriation --- p.95Chapter APPENDIX II --- Principle of Flow Cytometry --- p.98Chapter APPENDIX III --- Reagents for Experiments --- p.10

The Effect of Muscle Paralysis on the Development of the Skeleton of the Chick Embryo

In order to study the effects of paralysis on the development of the skeletal system in the chick embryo, embryos were paralysed by periodical administration of decamethonium bromide at 6, 8 and 10 days of incubation and were compared with controls to which saline was administered. 1. Experimental embryos showed retardation of the developmental stages of Hamburger & Hamilton (1951) and were of smaller size and weight. 2. Whole mount clearing showed that experimental embryos had scoliosis, cartilaginous or even bony fusion of cervical vertebrae, the knee joint and some other joints, distortion of the pubis and scapula, and failure of chondrification of the patella and the plantar tarsal sesamoid bone. 3. Linear measurement showed reduction in length of long bones, e.g. femur, tibia, humerus, ulnar and radius and lower and upper beaks in experimental embryos. The upper beak showed a greater reduction than the lower. 4. Volumetric measurement of the tibia showed a reduction in all tissues in experimental embryos and a marked reduction in cartilage formation and resorption between the 13th and 14th days of incubation. 5. Timings of onset of invasion by the osteogenic bud, of ossification assessed histologically and of calcification assessed by alizarin staining were similar between control and experimental embryos. 6. Histological and scanning electron microscopical study of the tibia revealed no changes between the two groups until the 11th day of incubation. From 12 days onwards:- i) less periosteal bone formation occurred in the experimental embryos, ii) cartilage resorption was not progressing in the experimental embryos at the extent of their corresponding controls iii) blood vessels in the Haversian canals were larger in diameter in the experimental embryos, iv) the tibiofibular interosseous ligament and fibular crest of the tibia regressed in experimental embryos. 7. Muscle fibres were atrophied and lost their orderly arrangement in the experimental embryos

The role of suppressor of cytokine signalling-2 in endochondral bone growth

Suppressor of Cytokine Signalling-2 (SOCS2) is a negative regulator of growth hormone (GH) signalling and bone growth via inhibition of the JAK/STAT pathway. This has been classically demonstrated by the overgrowth phenotype of SOCS2-/- mice which have normal systemic IGF-1 levels. The local effects of GH on bone growth are equivocal and therefore this study aimed to understand better the SOCS2 signalling mechanisms mediating the local actions on epiphyseal chondrocytes and bone growth. SOCS2, in contrast to SOCS1 and SOCS3 expression, was increased in cultured chondrocytes following GH challenge; and gain-and-loss of function studies indicated that SOCS2 acts to negatively regulate GH stimulated chondrocyte STAT phosphorylation. This was confirmed by the observation that GH stimulates the longitudinal growth of cultured SOCS2-/- embryonic metatarsals and the proliferation of chondrocytes within. Consistent with this; bone growth rates, growth plate zone widths and chondrocyte proliferation were all increased in 6-week old SOCS2-/- mice as was the number of phosphorylated STAT-5 positive hypertrophic chondrocytes. The results of these studies indicate that the SOCS2-/- mouse represents a valid model for studying the local effects of GH and IGF-1 on bone growth. Chronic paediatric inflammatory diseases are well accepted to lead to growth retardation and this is likely due to raised inflammatory cytokine levels and reduced GH/IGF-1 signalling. Whilst SOCS2 was not found to be increased in response to inflammatory cytokines, SOCS2-/- mice were protected from LPS-induced growth retardation indicating that SOCS2 antagonists may help ameliorate the negative effects of chronic inflammation on growth

DEVELOPMENT OF HYBRID-CONSTRUCT BIOPRINTING AND SYNCHROTRON-BASED NON-INVASIVE ASSESSMENT TECHNIQUES FOR CARTILAGE TISSUE ENGINEERING

Cartilage tissue engineering has been emerging as a promising therapeutic approach, where engineered constructs or scaffolds are used as temporary supports to promote regeneration of functional cartilage tissue. Hybrid constructs fabricated from cells, hydrogels, and solid polymeric materials show the most potential for their enhanced biological and mechanical properties. However, fabrication of customized hybrid constructs with impregnated cells is still in its infancy and many issues related to their structural integrity and the cell functions need to be addressed by research. Meanwhile, it is noticed that nowadays monitoring the success of tissue engineered constructs must rely on animal models, which have to be sacrificed for subsequent examination based on histological techniques. This becomes a critical issue as tissue engineering advances from animal to human studies, thus raising a great need for non-invasive assessments of engineered constructs in situ. To address the aforementioned issues, this research is aimed to (1) develop novel fabrication processes to fabricate hybrid constructs incorporating living cells (hereafter referred as “construct biofabrication”) for cartilage tissue regeneration and (2) develop non-invasive monitoring methods based on synchrotron X-ray imaging techniques for examining cartilage tissue constructs in situ. Based on three-dimensional (3D) printing techniques, novel biofabrication processes were developed to create constructs from synthetic polycaprolactone (PCL) polymer framework and cell-impregnated alginate hydrogel, so as to provide both structural and biological properties as desired in cartilage tissue engineering. To ensure the structural integrity of the constructs, the influence of both PCL polymer and alginate was examined, thus forming a basis to prepare materials for subsequent construct biofabrication. To ensure the biological properties, three types of cells, i.e., two primary cell populations from embryonic chick sternum and an established chondrocyte cell line of ATDC5 were chosen to be incorporated in the construct biofabrication. The biological performance of the cells in the construct were examined along with the influence of the polymer melting temperature on them. The promising results of cell viability and proliferation as well as cartilage matrix production demonstrate that the developed processes are appropriate for fabricating hybrid constructs for cartilage tissue engineering. To develop non-invasive in situ assessment methods for cartilage and other soft tissue engineering applications, synchrotron phase-based X-ray imaging techniques of diffraction enhanced imaging (DEI), analyzer based imaging (ABI), and inline phase contrast imaging (PCI) were investigated, respectively, with samples prepared from pig knees implanted with low density scaffolds. The results from the computed-tomography (CT)-DEI, CT-ABI, and extended-distance CT-PCI showed the scaffold implanted in pig knee cartilage in situ with structural properties more clearly than conventional PCI and clinical MRI, thus providing information and means for tracking the success of scaffolds in tissue repair and remodeling. To optimize the methods for live animal and eventually for human patients, strategies with the aim to reduce the radiation dose during the imaging process were developed by reducing the number of CT projections, region of imaging, and imaging resolution. The results of the developed strategies illustrate that effective dose for CT-DEI, CT-ABI, and extended-distance CT-PCI could be reduced to 0.3-10 mSv, comparable to the dose for clinical X-ray scans, without compromising the image quality. Taken together, synchrotron X-ray imaging techniques were illustrated promising for developing non-invasive monitoring methods for examining cartilage tissue constructs in live animals and eventually in human patients

Whole-body biomechanical load monitoring from accelerometry in team sports

Contemporary research into training load in team sports primarily focusses on the physiological load demands, whereas the biomechanical load still remains largely unexplored. While the former refers to the work-energy relationship when the players move around the pitch, the latter refers to the external forces the players are exposed to from their movements around the pitch. Monitoring of the biomechanical load helps practitioners estimate the stresses on an athlete’s musculoskeletal structures as a consequence of the external forces acting on their body. Monitoring of the biomechanical load is currently restricted to laboratory settings, but the recent introduction of GPS devices with integrated accelerometers in team sports may enable practitioners to monitor whole-body biomechanical load during training sessions and match-play. The aim of this thesis was therefore to explore if body-worn accelerometry can be used for whole-body biomechanical load monitoring in team sports. The first study of this thesis showed that although a linear relationship exists between body-worn accelerometry (e.g. from GPS integrated accelerometers) and whole-body accelerations, the linear relationship based on Newton’s second law of motion is weak regardless of accelerometer location (trunk, pelvis or tibia). Body-worn accelerometry only measures the acceleration of the segment it is attached to and is therefore inadequate to measure the complex multi-segment dynamics of the whole body during team sports movements. The second study of this thesis did however offer a potential solution to that problem, and it was demonstrated that the complex multi-segment dynamics of the body and the associated ground reaction forces (GRF), a surrogate for whole-body biomechanical load, can be estimated with a mass-spring-damper model (MSD-model). Nonetheless, the MSD-model’s accuracy to estimate GRF slightly decreases for sharp changes of direction at high intensities, because the absorption of energy and generation of energy are decoupled. Finally, a novel approach to estimate GRF from the combination of trunk-mounted accelerometry and a MSD-model was introduced in this thesis. This approach showed that trunk accelerometry data has the potential to generate the eight model parameters required to estimate GRF from a MSD-model, though further work is required in particular towards improving the model’s ability to estimate GRF across a wide range of activities. The novel approach introduced in this thesis has the potential to give practitioners in team sports the opportunity to monitor whole-body biomechanical load due to player-ground interaction in the field, a necessity if they wish to predict the consequent musculoskeletal structural adaptations of training sessions and match-play

In vitro and in situ porcine models for the studies on phenotypic characterization of cartilage cells during endochondral ossification.

by Lee Kwong Man, Simon.Thesis (Ph.D.)--Chinese University of Hong Kong, 1996.Includes bibliographical references (leaves 239-277).ABSTRACT --- p.iACKNOWLEDGMENT --- p.viPUBLICATIONS --- p.viiABBREVIATIONS --- p.viiiTABLE OF CONTENT --- p.xiChapter CHAPTER ONE --- General Introduction --- p.1Chapter CHAPTER TWO --- Identification and Biochemical Characterization of Various Differentiative Growth Plate Chondrocytes by Countercurrent Centrifugal Elutriation --- p.8Chapter CHAPTER THREE --- Differential Expression of Glycoconjugates during Endochondral Ossification in Porcine Growth Plate --- p.50Chapter CHAPTER FOUR --- Intra- & Extra-Cellular Free Calcium Activities of Porcine Growth Plate Chondrocytes at Various Stages of Maturation --- p.90Chapter CHAPTER FIVE --- A New In Situ Model for Electrophysiological Characterization of Ionic Channels in Growth Plate Chondrocytes --- p.144Chapter CHAPTER SIX --- Effects of Quinolones on Growth Plate Chondrocytes --- p.201Chapter CHAPTER SEVEN --- Summary and Conclusion --- p.226BIBLIOGRAPHY --- p.23

The Epiphyseal Cartilage And Growth Of Long Bones In Rana Catesbeiana

The structure of the epiphyseal cartilage of the bullfrog Rana catesbeiana and its role in the growth of long bones were examined. The epiphyseal cartilage was inserted into the end of a tubular bone shaft, defining three regions: articular cartilage, lateral articular cartilage and growth cartilage. Joining the lateral cartilage to the bone was a fibrous layer of periosteum, rich in blood vessels. Osteoblasts with alkaline phosphatase activity were found on the surface of the periosteal bone, which presented a fibrous non-mineralised tip. The growth cartilage was inside the bone. The proliferative chondrocytes presented perpendicular separation of daughter cells and there was no columnar arrangement of the cells. Furthermore, chondrocyte hypertrophy was not associated with either calcification or endochondral ossification, in apparent contrast to the avian and mammalian models. Finally, there was no reinforcement system capable of directing cell volume increase into longitudinal growth. Since bone extension depends on the intramembranous ossification of the periosteum, the growth cartilage is inside and not at the end of the bone and the cells in the growth cartilage show no columnar arrangement and separate in a direction perpendicular to the long bone axis, we conclude that the growth cartilage mainly contributes to the radial expansion of the bone.313301307Aceitero, J., Gaytan, F., Ranz, F.B., Ribes, R., Heterogeneity of the cartilage-marrow interface during uncalcified cartilage resorption in the chick embryo tibia (1988) J. Anat., 160, pp. 39-50Anderson, H.C., Mechanism of mineral formation in bone (1989) Lab. Invest., 60, pp. 1513-1520Bancroft, J. D. 1982Barreto, C., Albrecht, R.M., Bjorling, D.E., Horner, J.R., Wilsman, N.J., Evidence of the growth plate and the growth of long bones in juvenile dinosaurs (1993) Science, 262, pp. 2020-2023Bianco, P., Cancedda, F.D., Riminucci, M., Cancedda, R., Bone formation via cartilage models: The 'bordeline chondrocytes' (1998) Matrix Biol., 17, pp. 185-192Breur, G.J., Van Enkevort, B.A., Farnum, C.E., Wilsman, N.J., Linear relationship between the volume of hypertrophic chondrocytes and the rate of longitudinal bone growth in growth plates (1991) J. Orthop. Res., 9, pp. 348-359Breur, G.J., Farnum, C.E., Padgett, G.A., Wilsman, N.J., Cellular basis of decreased rate of longitudinal growth of bone in pseudoachondroplastic dogs (1992) J. Bone Jt. Surg., 74, pp. 516-528Breur, G.J., Lapierre, M.D., Kazmierczak, K., Stechuchak, K.M., McCabe, G.P., The domain of hypertrophic chondrocytes in growth plates growing at different rates (1997) Calcif. Tissue Int., 61, pp. 418-425Cancedda, R., Cancedda, F.D., Castagnola, P., Chondrocyte differentiation (1995) Int. Rev. Cytol., 159, pp. 265-359Carvalho, H.F., Understanding the biomechanics of tendon fibrocartilages (1995) J. Theor. Biol., 172, pp. 293-297Carvalho, H.F., Vidal, B.C., Structure and histochemistry of a pressure-bearing tendon of the frog (1994) Ann. Anat., 176, pp. 161-170Carvalho, H.F., Vidal, B.C., The unique fibrillar arrangement of the bullfrog pressure-bearing tendon as an indicative of great functional deformability (1994) Biol. Cell., 82, pp. 59-65Cole, A.A., Wezeman, F.H., Perivascular cells in cartilage canals of the developing mouse epiphysis (1985) Am. J. Anat., 174, pp. 119-129Cole, A.A., Wezeman, F.H., Cytochemical localization of tartrate-resistant acid phosphatase, alkaline phosphatase, and nonspecific esterase in perivascular cells of cartilage canals in the developing mouse epiphysis (1987) Am. J. Anat., 180, pp. 237-242Cotta-Pereira, G., Rodrigo, F.G., David-Ferreira, J.F., The use of tannic acid-glutaraldehyde in the study of elastic related fibers (1976) Stain Technol., 51, pp. 7-11Dell'Orbo, C., Gioglio, L., Quacci, D., Morphology of epiphyseal apparatus of a ranid frog (1992) Histol. Histopathol., 7, pp. 267-273Dickson, R.G., Ultrastruture of growth cartilage in the proximal femur of the frog, Rana temporaria (1982) J. Anat., 135, pp. 549-564Eggli, P.S., Herrmann, W., Hunziker, E.B., Schenk, R.K., Matrix compartments in the growth plate of the proximal tibia of rats (1985) Anat. Rec., 211, pp. 246-257Erlebacher, A., Filvaroff, E.H., Giltelman, S.E., Derynck, R., Toward a molecular understanding of skeletal development (1995) Cell, 80, pp. 371-378Farnum, C.E., Wilsman, N.J., Determination of proliferative characteristics of growth plate chondrocytes by labeling with bromodeoxyuridine (1993) Calcif. Tissue Int., 52, pp. 110-119Gardner, E., Gray, D.J., The prenatal development of the human femur (1970) Am. J. Anat., 129, pp. 121-140Haines, R.W., The evolution of epiphyses and of endochondral bone (1942) Biol. Rev., 17, pp. 267-292Hunziker, E.B., Schenk, R.K., Physiological mechanisms adopted by chondrocytes in regulating longitudinal bone growth in rats (1989) J. Physiol., 414, pp. 55-71Hunziker, E.B., Schenk, R.K., Cruz-Orive, L.M., Quantitation of chondrocyte performance in growth-plate cartilage during longitudinal bone growth (1987) J. Bone Jt. Surg., 69, pp. 162-173Lee, E.R., Lamplugh, L., Sherpard, N.L., Mort, J.S., The Septoclast, a cathepsin B-rich cell involved in the resorption of growth plate cartilage (1995) J. Histochem. Cytochem., 43, pp. 525-536Lewinson, D., Silbermann, M., Chondroclasts and endothelial cells collaborate in the process of cartilage resorption (1992) Anat. Rec., 233, pp. 504-514Mühlhauser, J., Resorption of the unmineralized proximal part of Meckel's cartilage in the rat (1986) A Light and Electron Microscopic Study. J. Submicrosc. Cytol. Pathol., 18, pp. 717-724Miyake, T., Cameron, A.M., Hall, B.K., Stage-specific expression patterns of alkaline phosphatase during development of the first arch skeleton in inbred C57BL/6 mouse embryos (1997) J. Anat., 190, pp. 239-260Rhodin, A.G., Comparative chondro-osseous development and growth of marine turtles (1985) Copeia, 3, pp. 752-771Schenk, R.K., Spiro, D., Wiener, J., Cartilage resorption in the tibial epiphyseal plate of growing rats (1967) J. Cell Biol., 34, pp. 275-291Shibata, S., Suzuki, S., Yamashita, Y., An ultrastructural study of cartilage resorption at the site of initial endochondral bone formation in the fetal mouse mandibular condyle (1997) J. Anat., 191, pp. 65-76Silvestrini, G., Ricordi, M.E., Bonucci, E., Resorption of uncalcified cartilage in the diaphysis of the chick embryo tibia (1979) Cell Tissue Res., 196, pp. 221-235Sorrell, J.M., Weiss, L., A light and electron microscopic study of the region of cartilage resorption in the embryonic chick (1980) Anat. Rec., 198, pp. 513-530Sorrell, J.M., Weiss, L., The cellular organization of fibroblastic cells and macrophages at regions of uncalcified cartilage resorption in the embryonic chick femur as revealed by alkaline and acid phosphatase histochemistry (1982) Anat. Rec., 202, pp. 491-499Speer, D.P., Collagenous architecture of the growth plate and perichondrial ossification groove (1982) J. Bone Jt. Surg., 64, pp. 399-407Thorp, B.H., Absence of cartilage canals in the long bone extremities of four species of skeletally immature marsupials (1990) Anat. Rec., 226, pp. 440-446Wilsman, N.J., Farnum, C.E., Lieferman, E.M., Fry, M., Barreto, C., Differential growth by growth plates as a function of multiple parameters of chondrocytic kinetics (1996) J. Orthop. Res., 14, pp. 927-93

Normal bone growth requires optimal estrogen levels: negative effects of both high and low dose estrogen on the number of growth plate chondrocytes

Endochondral bone formation at epiphyseal growth plate consists of the synchronized processes of chondrogenesis and cartilage ossification. Estrogen, the major female sex hormone, plays an important role in this process, particularly during the pubertal growth spurt. However, its effects on the growth plate are not completely understood. The aims of this study were to clarify the effects of estrogen on the kinetics of chondrocytes in the growth plates of 10- to 25-week-old female rabbits by studying the effects of ovariectomy or high-dose administration of estrogen on the balance between cell proliferation and death. Forty-eight Japanese white rabbits were divided into three groups: sham operated, ovariectomized, or ovariectomized with subsequent weekly injection of high dose estrogen from 10 weeks. The chondrocyte kinetics was investigated by histomorphometry and immunohistochemistry, using antibodies for caspase-3, a marker of apoptosis, and for proliferating cell nuclear antigen. Both ovariectomized and estrogen-injected rabbits showed a declination of the chondrocyte number although the latter animals indicated a more dramatic effect. Estrogen-injected rabbits showed a decrease in the cell proliferating ability together with an increase in chondrocytes undergoing apoptosis while ovariectomy mainly reduced the cell proliferating ability. Given the known importance of estrogen for bone growth, one would expect that ovariectomy and high-dose administration of estrogen would have opposite effects. However, the present study indicated that both low and high concentration had a similar effect: a decrease in the chondrocyte number compared with control, suggesting that estrogen has to be maintained within a narrow range for optimal bone growth