Effects of low-intensity pulsed ultrasound on fracture healing and caveolin-1 gene expression in the radius defects of rabbits / 复旦学报（医学版）

Objective To explore the effect of low-intensity pulsed ultrasound (LIPUS) on long bone fracture healing and to examine caveolin-1 gene expression in the radius defects of rabbits.Methods A total of 24 New Zealand rabbits with 3-mm bone defects at lower 1/3 in both radii were randomly assigned to 4 groups (n=6).Daily LIPUS treatment was performed to the right fracture sites at a intensity of 30 mW/cm2 for 20 minutes,while the left sites received sham treatment with power off.To assess the effects of LIPUS on bone defects,X-ray imaging and hematoxylin-eosin staining were applied 7,14,21,28 days after the surgery.Additionally,the immunohistochemical staining was used to determine the subcllular localization of caveolin-1 and semi-quantify the caveolin-1 level,qPCR was performed to detect the mRNA level of caveolin-1,gene Col2a1 and Col10a1,and osteocalcin.Results On day 14,the radiological score of the right radii and mineralized callus area were significantly higher than that of the left ones,both of them were elevated with time flied.Histological examination suggested that the differentiation and apoptosis of chondrocytes along with the formation and bridging of the bone trabeculas appeared earlier in the right radius defects.The immunohistochemical staining showed that on day 7 and 14,the level of caveolin-1 increased with the proliferation and differentiation of condrocytes,and was significantly higher in callus tissues on the right sites.On day 21 and 28,the mesenchymal stem cells migrated to the surface of cartilage matrix started to differentiate into osteoblasts,the level of caveolin-1 decreased,and was significantly lower on the right sites.The result of qPCR indicated that compared with the left sites the caveolin-1 gene expression on the right sites was significantly higher on day 7,while significantly lower on day 21.The mRNA expression levels of Col2a1,Col10a1 and osteocalcin on the right sites were significantly higher on day 7 and 14,but they were significantly lower on day 21 and 28,except for Col10a1 on day 28.Conclusions Advancing endochondral ossification is considered to be a crucial mechanism during long bone fracture healing promoted by LIPUS.The caveolin-1 gene expression first increased in the chondrocytes then decreased in the mesenchymal stem cells during the process.

Progress in exogenous factors affecting the growth and remodeling of condylar process / 华西口腔医学杂志

Condyle is a critical growth region of the mandible where mandible by endochondral ossification occurs. Condylar cartilage belongs to the secondary cartilage, which is not only affected by genetic factors but also by stress, drug intake, and other local factors. To promote the growth of the mandible, various exogenous and local factors were used to alter the biological environment of the condylar cartilage to stimulate endochondral ossification. This article reviews studies on the influence of exogenous factors on condylar growth and reconstruction. This literature review will provide a reference point for the treatment of patients with mandibular retraction.

Bone regeneration with bone marrow mesenchymal stem cells, platelet rich plasma and decellularized cartilage matrices complex / 实用口腔医学杂志

Objective:To study the bone regeneration capacity of the complex of bone marrow mesenchymal stem cells(BMSCs) and platelet rich plasma (PRP) with decellularized cartilage matrix (DCM).Methods:BMSCs were isolated from young rabbit and cultured;PRP were prepared from the fresh blood of rabbit and the DCM were come from the fresh ears of rabbits and processed into a mixture of BMSCs-PRP-DCM.The mixture was injected subcutaneously into nude mice,8 weeks after injection bone regeneration was examine by HE staining and Massons staining decellularization.Results:The BMSCs show good differentiation capacity in vitro and the cartilage fragments were well decellularized.Excellent endochondral ossification ability of the complex was observed by in vivo experiment.Conclusion:The BMSCs-PRP-DCM complex has good capacity of endochondral ossification.

Effect and mechanism of total flavone of epimedium on primary callus formation in ovariectomized rats with fractures / 中国骨伤

<p><b>OBJECTIVE</b>To explore the effects and related mechanisms of total flavone of epimedium treatment(TFE)on primary callus for mation in ovariectomized rats.</p><p><b>METHODS</b>Forty male SD rats weighted from 209 to 246 g and aged 6 to 8 weeks were selected. Six weeks after ovariectomy a femur fracture model with middiaphyseal segment fracture was established, estimated and randomly divided into TFE group (150 mg·kg⁻¹·d⁻¹) and control group(received saline). HE staining was used to evaluate the morphologic difference of primary callus during the bone callus healing between these two groups. The relative expression of Runt-related transcription factor 2(Runx2) mRNA in the callus was identified by real-time polymerase chain reaction. Immunohistochemical technique was used to observe the Casein kinase 2-interacting protein 1(CKIP-1) protein level in the callus of the two groups. Maximum fracture load was tested by three point bend test.</p><p><b>RESULTS</b>The BMD, primary callus volume, trabecular member(Tb.N) and trabecular thickness(Tb.Th) were higher in TFE group than that in control group(<0.001). The Tb.N and Tb.Th of primary callus were higher in TFE group than control group (=0.001). The volume and bone volume/tissue volume of primary callus were in TFE group than control group(<0.01). The trabecular separation(Tb.Sp) of primary callus were in control group higher than TFE group(<0.01). The HE staining of the 6 week slices showed that the degree of cartilage ossification in callus of the TFE group was significantly higher than that in control group under high magnification. Real-time PCR revealed that the comparative expression of Runx2 mRNA in control group was higher than that in TFE group(<0.001); the positive number of CKIP-1 was less in TFE group than that in control group (<0.001). TFE could increase the maximum load of the primary callus (<0.001).</p><p><b>CONCLUSIONS</b>TFE can promote the cartiage ossification of callus in ovariectomized rats, enhancing the bone strength and bone quality in the process of fracture healing via the CKIP-1/Runx2 pathway.</p>

5-Aza-2'-deoxycytidine acts as a modulator of chondrocyte hypertrophy and maturation in chick caudal region chondrocytes in culture / 대한해부학회지

This study was carried out to explore the effect of DNA hypomethylation on chondrocytes phenotype, in particular the effect on chondrocyte hypertrophy, maturation, and apoptosis. Chondrocytes derived from caudal region of day 17 embryonic chick sterna were pretreated with hypomethylating drug 5-aza-2'-deoxycytidine for 48 hours and then maintained in the normal culture medium for up to 14 days. Histological studies showed distinct morphological changes occurred in the pretreated cultures when compared to the control cultures. The pretreated chondrocytes after 7 days in culture became bigger in size and acquired more flattened fibroblastic phenotype as well as a loss of cartilage specific extracellular matrix. Scanning electron microscopy at day 7 showed chondrocytes to have increased in cell volume and at day 14 in culture the extracellular matrix of the pretreated cultures showed regular fibrillar structure heavily embedded with matrix vesicles, which is the characteristic feature of chondrocyte hypertrophy. Transmission electron microscopic studies indicated the terminal fate of the hypertrophic cells in culture. The pretreated chondrocytes grown for 14 days in culture showed two types of cells: dark cells which had condense chromatin in dark patches and dark cytoplasm. The other light chondrocytes appeared to be heavily loaded with endoplasmic reticulum indicative of very active protein and secretory activity; their cytoplasm had large vacuoles and disintegrating cytoplasm. The biosynthetic profile showed that the pretreated cultures were actively synthesizing and secreting type X collagen and alkaline phosphatase as a major biosynthetic product.

The study of the mechanism in the endochondral ossification of bone mesenchymal stem cells in mice by continued enhanced function of fibroblast growth factor type Ⅱ receptor mutation / 重庆医学

Objective To study the role of ERK signal pathway in the endochondral ossification of bone mesenchymal stem cells ,and to explore the mechanism of ERK signal pathway in persistent enhanced FGFR2 function on development of mice BMSCs by a knock‐in mouse model with the FGFR2S252W/+ .Methods Mice with neo‐FGFR2 gain‐of‐function mutation were mated with EII‐Cre mice .The genotype of generation mice were identified by PCR and divided into wild type group and mutant type group ac‐cording to their genotype .6 week‐old mice were sacrificed to receive bone mesenchymal stem cells .The western blot was used to compare the level of P‐ERK and ERK and the RT‐PCR was applied to detect the genes of Col2 ,Col10 ,OC ,OP in chondrogenic dif‐ferentiation medium of BMSCs .Then ,treatment of cultured BMSCs with PD98059 ,compare the changes of genes and utilize the in vitro culture of long bones detect the role of ERK signal pathway in the endochondral ossification by FGFR2 mutant .Results We successfully derive BMSCs from FGFR2S252W/+ mutant mice and found the activity of ERK signal pathway of FGFR2S252W/+ was en‐hanced .After been cultured in chondrogenic differentiation medium ,the expressions of the BMSCs mRNA of Col2 ,Col10 from mu‐tant group were decreased ,while the expressions of OC ,OP were increased .Those OC ,OP genes levels showed an increased treated by PD98059 .Using in vitro culture of long bones ,we found the retardation of total length growth of long bones has been rescued by PD98059 treatment ,suggesting that ERK signal pathways was responsible for the retarded long bone development in FGFRS252W/+mice .Conclusion The results indicate these effects are mediated by the ERK signal pathway .Furthermore ,the retardation of long bones has been recued by PD98059 treatment ,suggesting that ERK signal pathway is responsible for the retarded long bone devel‐opment in FGFR2S252W/+ mice .

Effects of Bisphosphonate on the Expression of Matrix Enzymes during Endochondral Ossification / 체질인류학회지

Bisphosphonates have been reported to have chondroprotective activities in addition to its original functions. However, mechanisms for these just began to be elucidated. Under the hypothesis that bisphosphonates may regulate expression and activities of matrix enzymes during degradation of cartilage for bone formation, we administrated an alendronate (1 mg/kg) to newborn rats subcutaneously once a day for 4, 7, and 10 days. To identify the effects of alendronate on cartilage, thickness of cartilage layer was measured by histomorphometry on the proximal epiphysis of tibia. Immunofluorescence staining and RT-PCR were performed to investigate the expressions of matrix enzymes in both in vitro and in vivo. MTS assay revealed that at 10(-3) M in concentration, alendronate significantly reduced viability of chondrocytes. The mRNA expressions of MMP-1, MMP-9, EMMPRIN, and TIMP-3 in primary chondrocytes were decreased by the alendronate treatment. Interestingly, TIMP-1 mRNA expression was significantly increased, whereas a constitutive form, TIMP-2 was relatively unchanged by the treatment. The thickness of proliferating layer at postnatal day 7 was not significantly different, whereas thickness of hypertrophied layer was significantly thicker in the alendronate group than in the control (p<0.01). Immunofluorescence demonstrated that the expressions of MMP-9, TIMP-2 and -3 were reduced, whereas TIMP-1 expression was increased by the alendronate administration. These results suggest that the alendronate have chondroprotective properties by down-regulation of MMPs and up-regulation of TIMPs during endochondral ossification.

Representación del desarrollo de la espongiosa primaria por medio de un sistema de reacción-difusión: Una hipótesis sobre el inicio de la formación de hueso inmaduro. Parte 1: Descripción del modelo / Representation of the development of the primary spongiosa by means of a reaction-diffusion system: a hypothesis on the onset of immature bone formation. Part 1: Model description

Se presenta un modelo bioquímico que predice la formación de la arquitectura de la espongiosa primaria, a partir de la interacción de 2 factores moleculares: VEGF (factor de crecimiento endotelial vascular) y MMP13 (metaloproteinasas 13). Se supone que el MMP13 regula la degradación del cartílago y el VEGF permite la vascularización y el avance del frente de osificación mediante la presencia de osteoblastos. El acople de este conjunto de moléculas se representa mediante ecuaciones de reacción-difusión con parámetros en el espacio de Turing, y se obtiene como resultado un patrón espacio-temporal estable que da paso a la formación de las trabéculas presentes en el tejido esponjoso...

A biochemical model is presented which predicts the formation of the architecture of the primary spongiosa, based on the interaction of two molecular factors: VEGF (vascular endothelial growth factor) and MMP-13 (metalloproteinases-13). It is assumed that MMP-13 regulates cartilage degradation, and VEGF allows vascularization and the advance of the ossification front through the presence of osteoblasts. The coupling of this set of molecules is represented by means of reaction-diffusion equations with Turing space parameters, and a stable spatio-temporal pattern is obtained which leads to the formation of the trabeculae present in the spongy tissue...

Representación del desarrollo de la espongiosa primaria por medio de un sistema de reacción-difusión: Una hipótesis sobre el inicio de la formación de hueso inmaduro. Parte 2: Implementación numérica / Representation of the development of the primary spongiosa by means of a reaction-diffusion system: a hypothesis on the onset of immature bone formation. Part 2: Numerical implementation

Se presenta la implementación numérica del modelo bioquímico descrito mediante el sistema de reacción-difusión de la parte 1. De los resultados obtenidos se puede concluir que la retroalimentación química de los 2 factores moleculares a través de un sistema de reacción-difusión (RD) con parámetros en el espacio de Turing, puede explicar la aparición de los patrones espacio-temporales encontrados en la arquitectura de la espongiosa primaria. Para la solución numérica fue usado el método de los elementos finitos junto con el método de Newton-Raphson para aproximar las ecuaciones diferenciales parciales lineales. Los patrones de osificación obtenidos pueden representar la formación de la espongiosa primaria durante la osificación endocondral...

A presentation is made of the numerical implementation of the biochemical model described by means of the reaction-diffusion system in Part 1. Based on the results obtained it may be concluded that the chemical feedback of the two molecular factors by means of a reaction-diffusion (RD) system with Turing space parameters may explain the appearance of the spatio-temporal patterns found in the architecture of the primary spongiosa. For the numerical solution, use was made of the finite element method in combination with the Newton-Raphson method to approximate the linear partial differential equations. The ossification patterns obtained may represent the formation of the primary spongiosa during endochondral ossification...

Modelamiento matemático de la enfermedad de Legg-Calvé-Perthes / Mathematical modeling of Legg-Calvé-Perthes disease

La enfermedad de Legg-Calvé-Perthes es un desorden caracterizado por la necrosis avascular de la cabeza femoral del esqueleto en desarrollo. Aunque la enfermedad fue descrita hace un siglo, aún existe gran controversia respecto a su etiología y al tratamiento que se le debe dar. Tanto la etiología como el tratamiento tienen una fuerte relación con eventos biológicos y mecánicos. Un entendimiento de dichos eventos y de su acción combinada, podría dar lugar a una mejor comprensión y manejo de la enfermedad. Este trabajo propone un acercamiento a esta comprensión mediante el uso del modelamiento computacional, el cual debe tener en cuenta, entre otros factores, la patogénesis de la enfermedad y los diferentes resultados en dependencia de la edad a la cual esta se manifieste en el niño

The Legg-CalvÚ-Perthes disease is a disorder characterized by the avascular necrosis of femoral head of developing skeleton. Although this disease was described a century ago still there is a significant controversy on its etiology and its treatment. Between etiology and treatment there is a close relation with biological and mechanical events. The knowledge of such events and of its combined action, could give rise to a better understanding and management of this disease. Present paper proposes a approach to this understanding by means of the use of a computer modeling, which must to has into account among other factors, the disease pathogenesis and the different results depending on age of appearance in the child

Role of hypoxia inducible factor-1α and VHL in murine endochondral ossification / 上海交通大学学报（医学版）

Objective To investigate the role of hypoxia inducible factor-1α (HIF-1α) and von Hippel-Lindau (VHL) in murine endochondral ossification. Methods The knockout of HIF-1α or VHL gene in murine osteoblasts was accomplished by conditional knockout technique at 4th, 8th and 12th week, and the differences between wild-type group and knock-out group in endochondral ossification were detected by HE staining, micro-CT scanning, trabecular bone area measurement, calcium content measurement, tetracycline fluorescence labeling, Real-time PCR and Western blotting. Results After knockout of HIF-1α gene in osteoblasts, the expression of vascular endothelial growth factor ( VEGF) reduced, the rate of new bone formation stepped down, the content of calcium became less, and the trabecular bone volume decreased (P <0.05) . After knockout of VHL gene in osteoblasts, the expression of VEGF increased, the rate of new bone formation stepped up, the content of calcium became more, and the trabecular bone volume was promoted (P < 0.001). Conclusion During murine endochondral ossification, VHL/HIF-1α signal pathway promotes angiogenesis through the stimulation of VEGF expression, which subsequently accelerates osteogenesis.

Effects of epidermal growth factor receptor on endochondral ossification and recruitment of osteoclasts in mice / 吉林大学学报(医学版)

Objective To determine the role of epidermal growth factor receptor(EGFR) signaling in endochondral ossification.Methods Long bones,from EGFR knockout mice(EGFR-/-),wild type(EGFR+/+) and(or) heterozygous mice(EGFR+/-) were collected and Trichrome Masson staining,in situ hybridization and tartrate-resistant acid phosphatase(TRAP) staining were used to observe endochondral ossification and recruitment of osteoclasts;Osteoclast formation was observed by addition of EGFR tyrosine kinase inhibitor AG1478 to the cultured osteoclasts from bone marrow cells.Results Due to the same phenotype of EGFR+/+ and EGFR+/-,they were regarded as EGFR+/+ in our study.EGFR deficiency caused delayed primary endochondral ossification of cartilage anlage and delayed osteoclast recruitment.The hypertrophic cartilage area in EGFR-/-mice was about 5 times longer than that in EGFR+/+ mice.There was different distribution of MMP-9 between EGFR+/+ and EGFR-/-in E16.5,but there was no difference about the quantity of MMP-9 in osteoclasts between EGFR-/-and EGFR+/+.However,inhibition of EGFR signaling with AG1478 significantly decreased osteoclast formation compared with control group with DMSO(P

Delayed Endochondral Ossification in Hsp70 Knock-out Mice Fetuses due to Maternal Hyperthermia / 대한해부학회지

To investigate the effects of maternal hyperthermia on the endochondral ossification, pregnant Hsp70 knock-out and wild type mice at gestational day (GD)8 were immersed in 43 degree C water bath until their body core temperature reached at 43 degree C. Thereafter, pregnant mice were given more 5 minutes hyperthermic exposure. Fetuses were collected at GD 15 and photographed for external appearance analysis. In addition, heat treated Hsp70 fetuses with external congenital anomalies and heat untreated wild type fetuses were used as experimental and control animals, respectively. Developing humeri at GD 17 were stained with alizarin red S and alcian bue according to the method of Kimmel and Trammell (1981).Developing upper limbs were immunostained for FGF-8, and observed with light and transmission electron microscope. 1.The proportion of average length of the humerus in heat untreated group to heat treated group was 1 :0.58.The proportion of average length of calcified part to average whole length was 1 :0.46 in control and 1 :0.18 in experimental group, respectively. 2.Apical ectodermal ridge was positively reacted to FGF-8 immunohistochemistry in the control group, but not in the experimental group. The humerus of experimental group showed more delayed endochondrial ossification than that of control group. The chondrocytes in the proliferating zone did not react in the experimental group. 3.Collagen fibers were loosely arranged in the experimental group. Mitochondria possessed early staged mitochondrial cristae, i.e.vesicular type in the experimental group. The results of this study suggest that maternal hyperthermia may inhibit the expression of FGF-8 in the developing upper limb, resulting in delayed endochondral bone growth of the humerus.

Expression of fibroblast growth factor receptor-3 during fracture healing / 第三军医大学学报

Objective To observe the spatial and temporal expression patterns of FGFR3 gene and analyze the potential roles of FGFR3 during fracture healing and explore the technique of in situ hybridization of paraffin sections of bone tissue. Methods The fracture model of mouse tibia was established by the standard methods. The expression of FGFR3 mRNA was studied by in situ hybridization during the various stages of bone repair. Results There was faint FGFR3 positive-signal in the cytoplasm of prehypertrophic chondrocytes in soft callus on day 5. FGFR3 mRNA was strongly expressed in prehypertrophic chondrocytes and hypertrophic chondrocytes in soft callus on day 7, but disappeared on day 14, 28 because chondrocytes was replaced by osteoblasts. Conclusion FGFR3 mRNA was strongly expressed in prehypertrophic chondrocytes and hypertrophic chondrocytes in the fracture callus, which shows that FGFR3, a gene that regulates the proliferation of chondrocytes during bone development, may participate in the regulation of differentiation of chondrocytes during fracture healing.

Cellular Proliferation and Apoptosis during Endochondral Fracture Healing / 대한정형외과학회잡지

PURPOSE: The authors investigated the process of endochondral ossification quantitatively and objectively in respect to proliferation and apoptosis. MATERIALS AND METHODS: Fractures were made on the left tibiae of 72 male Sprague-Dawley rats. The fracture callus was harvested at the 5th, 7th, 9th, 11th, 14th, and the 21st day after fracture. Cellular DNA content was analyzed with image cytometry, and proliferative index was determined from the data. The Ki-67 antigen expression was semiquantitatively measured by the immunohisto-chemical method. TUNEL was used for in situ localization of apoptotic cells. The expression of cell cycle inhibitors, P21 and P27, was investigated with Northern blotting. RESULTS: The proliferation index was highest on the 5th day, then gradually decreased until the 11th day. The expression of Ki-67 antigen gradually decreased with time. Apoptotic cells increased in accordance with enhanced bone formation within chondroid callus. The expression of p21 and p27 was highest on the 11th and the 14th day. CONCLUSIONS: These findings show that proliferative activity decreased with the reduction of mesenchymal tissue and the appearance of mature chondroid tissue. The apoptosis of hypertrophic chondrocytes occurred in accordance with enhanced bone formation. P21 and P27 had a certain role in the differentiation of chondrocytes.