Insulin exerts direct, IGF-1 independent actions in growth plate chondrocytes

Insufficient insulin production or action in diabetic states is associated with growth retardation and impaired bone healing, while the underling mechanisms are unknown. In this study, we sought to define the role of insulin signaling in the growth plate. Insulin treatment of embryonic metatarsal bones from wild-type mice increased chondrocyte proliferation. Mice lacking insulin receptor （IR） selectively in chondrocytes （CartIR-/-） had no discernable differences in total femoral length compared to control littermates. However, CartIR-/- mice exhibited an increase in chondrocyte numbers in the growth plate than that of the controls. Chondrocytes lacking IR had elevated insulin-like growth factor （IGF）-IR mRNA and protein levels. Subsequently, IGF-1 induced phosphorylafion of Akt and ERK was enhanced, while this action was eliminated when the cells were treated with IGF-1R inhibitor Picropodophyllin. Deletion of the IR impaired chondrogenic differentiation, and the effect could not be restored by treatment of insulin, but partially rescued by IGF-1 treatment. Intriguingly, the size of hypertrophic chondrocytes was smaller in CartIR-/- mice when compared with that of the control littermates, which was associated with upregnlation of tuberous sclerosis complex 2 （TSC2）. These results suggest that deletion of the IR in chondrocytes sensitizes IGF-1R signaling and action, IR and IGF-1R coordinate to regulate the proliferation, differentiation and hypertrophy of growth plate chondrocytes.

Fibroblast growth factor receptor 3 effects on proliferation and telomerase activity in sheep growth plate chondrocytes

Background： Fibroblast growth factor receptor 3 （FGFR3） inhibits growth-plate chondrocyte proliferation and limits bone elongation. Gain-of-function FGFR3 mutations cause dwarfism, reduced telomerase activity and shorter telomeres in growth plate chondroyctes suggesting that FGFR3 reduces proliferative capacity, inhibits telomerase, and enhances senescence. Thyroid hormone （1-3） plays a role in cellular maturation of growth plate chondrocytes and a known target of T3 is FGFR3. The present study addressed whether reduced FGFR3 expression enhanced telomerase activity, mRNA expression of telomerase reverse transcriptase （TERT） and RNA component of telomerase （TR）, and chondrocyte proliferation, and whether the stimulation of FGFR3 by T3 evoked the opposite response. Results： Sheep growth-plate proliferative zone chondrocytes were cultured and transfected with siRNA to reduce FGFR3 expression; FGFR3 siRNA reduced chondrocyte FGFR3 mRNA and protein resulting in greater proliferation and increased TERT mRNA expression and telomerase activity （p 〈 0.0.5）. Chondrocytes treated with T3 significantly enhanced FGFR3 mRNA and protein expression and reduced telomerase activity （p 〈 0.05）; TERT and TR were not significantly reduced. The action of T3 at the growth plate may be partially mediated through the FGFR3 pathway. Conclusions： The results suggest that FGFR3 inhibits chondrocyte proliferation and reducing telomerase activity indicating an important role for telomerase in capacity during bone elongation. by down-regulating TERT expression sustaining chondrocyte proliferative

AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

Mice Deficient in NF-κB p50 and p52 or RANK Have Defective Growth Plate Formation and Post-natal Dwarfism

NF-κBp50/p52 double knockout （dKO） and RANK KO mice have no osteoclasts and develop severe osteopetrosis associated with dwarfism. In contrast, Op/Op mice, which form few osteoclasts, and Src KO mice, which have osteoclasts with defective resorptive function, are osteopetrotic, but they are not dwarfed. Here, we compared the morphologic features of long bones from p50/p52 dKO, RANK KO, Op/Op and Src KO mice to attempt to explain the differences in their long bone lengths. We found that growth plates in p50/p52 dKO and RANK KO mice are significantly thicker than those in WT mice due to a 2-3-fold increase in the hypertrophic chondrocyte zone associated with normal a proliferative chondrocyte zone. This growth plate abnormality disappears when animals become older, but their dwarfism persists. Op/Op or Src KO mice have relatively normal growth plate morphology. In-situ hybridization study of long bones from pS0/ p52 dKO mice showed marked thickening of the growth plate region containing type 10 collagen-expressing chondrocytes. Treatment of micro-mass chondrocyte cultures with RANKL did not affect expression levels of type 2 collagen and Sox9, markers for proliferative chondrocytes, but RANKL reduced the number of type 10 collagen-expressing hypertrophic chondrocytes. Thus, RANK/NF-κB signaling plays a regulatory role in post-natal endochondral ossification that maintains hypertrophic conversion and prevents dwarfism in normal mice.

Effects of Selenium on Fusarium Growth and Associated Fermentation Products and the Relationship with Chondrocyte Viability

This study determined the effects of selenium on the growth of Fusorium strains and the effects of products extracted from the fungal cultures on relevant indicators of chondrocytes injury.

GROWTH DIFFERENTIATION FACTOR-5 STIMULATES THE GROWTH AND ANABOLIC METABOLISM OF ARTICULAR CHONDROCYTES

Objective To observe the effect of growth differentiation factor-5 (GDF-5) on the growth and anabolic metabolism of articular chondrocytes. Methods The articular chondrocytes isolated from rats were treated with various concentrations of rmGDF-5, and the growth of chondrocytes measured by MTT assay, the cellular cartilage matrices formation detected sulfated glycosaminoglycan by Alcian blue staining and type Ⅱcollagen by RT-PCR. Results After 7 days culture, MTT assay showed that GDF-5 enhanced the growth of chondrocytes in a dose-dependent manner, RT-PCR showed that GDF-5 clearly induced the synthesis of type Ⅱ collagen because of the col2a1 mRNA band more and more strong in a dose-dependent. Chondrocytes were cultured with GDF-5 for 14 days, the intensity of Alcian blue staining was greatly enhanced, especially, at a high concentration of 1000ng/mL, and GDF-5 enhanced the accumulation of the Alcian blue-stainable material in a concentration-dependent manner and in a does-dependent manner. Conclusion GDF-5 enhanced the growth of mature articular chondrocytes, and stimulated the cellular cartilage matrices formation in mono-layer culture.

Combined distal tibial rotational osteotomy and proximal growth plate modulation for treatment of infantile Blount's disease

Infantile Blount's disease is a condition that causes genu varum and internal tibial torsion. Treatment options include observation, orthotics, corrective osteotomy, elevation of the medial tibial plateau, resection of a physeal bar, lateral hemi-epiphysiodesis, and guided growth of the proximal tibial physis. Each of these treatment options has its disadvantages. Treating the coronal deformity alone(genu varum) will result in persistence of the internal tibial torsion(the axial deformity). In this report, we describe the combination of lateral growth modulation and distal tibial external rotation osteotomy to correct all the elements of the disease. This has not been described before for treatment of Blount's disease. Both coronal and axial deformities were corrected in this patient. We propose this combination(rather than the lateral growth modulation alone) as the method of treatment for early stages of Blount's disease as it corrects both elements of the disease and in the same time avoids the complications of proximal tibial osteotomy.

Automated Cell Detection and Morphometry on Growth Plate Images of Mouse Bone

Microscopy imaging of mouse growth plates is extensively used in biology to understand the effect of specific molecules on various stages of normal bone development and on bone disease. Until now, such image analysis has been conducted by manual detection. In fact, when existing automated detection techniques were applied, morphological variations across the growth plate and heterogeneity of image background color, including the faint presence of cells (chondrocytes) located deeper in tissue away from the image’s plane of focus, and lack of cell-specific features, interfered with identification of cells. We propose the first method of automated detection and morphometry applicable to images of cells in the growth plate of long bone. Through ad hoc sequential application of the Retinex method, anisotropic diffusion and thresholding, our new cell detection algorithm (CDA) addresses these challenges on bright-field microscopy images of mouse growth plates. Five parameters, chosen by the user in respect of image characteristics, regulate our CDA. Our results demonstrate effectiveness of the proposed numerical method relative to manual methods. Our CDA confirms previously established results regarding chondrocytes’ number, area, orientation, height and shape of normal growth plates. Our CDA also confirms differences previously found between the genetic mutated mouse Smad1/5CKO and its control mouse on fluorescence images. The CDA aims to aid biomedical research by increasing efficiency and consistency of data collection regarding arrangement and characteristics of chondrocytes. Our results suggest that automated extraction of data from microscopy imaging of growth plates can assist in unlocking information on normal and pathological development, key to the underlying biological mechanisms of bone growth.

On the State of Stress in the Growth Plate under Physiologic Compressive Loading

The growth plate is a thin layer of cartilage sandwiched between epiphyseal and metaphyseal bone and is the location of active bone growth during childhood. It is subjected to large compressive and shear forces while protecting its resident chondrocytes from damage. We believe that computational modeling can help us better understand how the macro-scale loads are transmitted to micro-scale stresses and strains within the growth plate cartilage. As a first step in this process we analyzed the mechanical response of compression experiments performed on bovine bone/growth plate/bone samples. We endeavored to estimate the modulus of elasticity of the growth plate itself by simulating the compression experiments of these specimens using the finite element method. It is shown that when the growth plate in the compression specimens was modeled as a flat layer, the state of stress in the cartilage was triaxial and non-uniform with the hydrostatic stress being much greater than the octahedral shear stress over most of the central region of the growth plate test samples. The computational models accounted for variations in the average cartilage thickness, the non-uniaxial, non-uniform and triaxial state of stress in the thin cartilage layer, and for the estimated extrinsic compliance resulting from compression of the variable heights of bone on either side of the growth plate cartilage. However, due to lack of information on the internal structure of each sample, the models did not account for the variations in the non-flat topography of the growth plates. The models also did not include the calcified cartilage layer. Further model development is recommendedin order to determine the degree to which accounting for the complex growth plate topography influences the predicted cartilage modulus of elasticity.

Ontogeny of rat chondrocyte proliferation:studies in embryo,adult and osteoarthritic (OA) cartilage

The aim of this work was to study the ontogeny of chondrocyte cell division using embryo, adult and osteoarthritic(OA) cartilage. We searched for mitosis phases and performed a comparative evaluation of mitotic index, basic fibro-blast growth factor b (FGFb), transforming growth factor β1 (TGF-β1) receptors, cyclin dependent kinase (CDK1)and Cyclin-B expression in fetal, neonate, 3, 5, 8 weeks old rats and experimental OA. Our results showed that mitosisphases were observed in all normal cartilage studied, although, we found a decrease in mitotic index in relation to tissuedevelopment. No mitosis was detected in OA cartilage. We also found a statistical significant reduction in cell number inOA cartilage, compared with the normal tissue. Furthermore, FGFb and TGF-β1 receptors diminished in relation totissue development, and were very scarce in experimental OA. Western blot assays showed CDK-1 expression in allcases, including human-OA cartilage. Similar results were observed for Cyclin-B, except for 8 weeks, when it was notexpressed. Our results suggest that cell division seems to be scarce, if not absent within the OA cartilage studied.Nevertheless, the existence of factors essential for cell division leaves open the question concerning chondrocyteproliferation in OA cartilage, which is likely to be present in the early stages of the disease.

Exosome-loaded extracellular matrix-mimic hydrogel with anti-inflammatory property Facilitates/promotes growth plate injury repair

Growth plate cartilage has limited self-repair ability,leading to poor bone bridge formation post-injury and ultimately limb growth defects in children.The current corrective surgeries are highly invasive,and outcomes can be unpredictable.Following growth plate injury,the direct loss of extracellular matrix(ECM)coupled with further ECM depletion due to the inhibitory effects of inflammation on the cartilage matrix protein greatly hinder chondrocyte regeneration.We designed an exosome(Exo)derived from bone marrow mesenchymal stem cells(BMSCs)loaded ECM-mimic hydrogel to promote cartilage repair by directly supplementing ECM and anti-inflammatory properties.Aldehyde-functionalized chondroitin sulfate(OCS)was introduced into gelatin methacryloyl(GM)to form GMOCS hydrogel.Our results uncovered that GMOCS hydrogel could significantly promote the synthesis of ECM due to the doping of OCS.In addition,the GMOCS-Exos hydrogel could further promote the anabolism of chondrocytes by inhibiting inflammation and ultimately promote growth plate injury repair through ECM remodeling.

Mapk7 deletion in chondrocytes causes vertebral defects by reducing MEF2C/PTEN/AKT signaling

Mutation of the MAPK7 gene was related to human scoliosis.Mapk7 regulated the development of limb bones and skulls in mice.However,the role of MAPK7 in vertebral development is still unclear.In this study,we constructed Col2a1-cre;Mapk7 f/f transgenic mouse model to delete Mapk7 in cartilage,which displayed kyphosis and osteopenia.Mechanistically,Mapk7 loss decreased MEF2C expression and thus activated PTEN to oppose PI3K/AKT signaling in vertebral growth plate chondrocytes,which impaired chondrocyte hypertrophy and attenuated vertebral ossification.In vivo,systemic pharmacological activation of AKT rescued impaired chondrocyte hypertrophy and alleviated mouse vertebral defects caused by Mapk7 deficiency.Our study firstly clarified the mechanism by which MAPK7 was involved in vertebral development,which might contribute to understanding the pathology of spinal deformity and provide a basis for the treatment of developmental disorders of the spine.

Growth of a Void in a Rubber Rectangular Plate under Uniaxial Extension

The finite deformation and stress analyses for a rectangular plate with a center void and made of rubber with the Yeoh elastic strain energy function under uniaxial extension were studied in this paper. An approximation solution was obtained from the minimum potential energy principle. The numerical results for the growth of the cavitation and stresses along the edge of the cavitation were discussed. In addition, the stress concentration phenomenon was considered.

Key Techniques and Applications of Adaptive Growth Method for Stiffener Layout Design of Plates and Shells

The application of the adaptive growth method is limited because several key techniques during the design process need manual intervention of designers. Key techniques of the method including the ground structure construction and seed selection are studied, so as to make it possible to improve the effectiveness and applicability of the adaptive growth method in stiffener layout design optimization of plates and shells. Three schemes of ground structures, which are comprised by different shell elements and beam elements, are proposed. It is found that the main stiffener layouts resulted from different ground structures are almost the same, but the ground structure comprised by 8-nodes shell elements and both 3-nodes and 2-nodes beam elements can result in clearest stiffener layout, and has good adaptability and low computational cost. An automatic seed selection approach is proposed, which is based on such selection rules that the seeds should be positioned on where the structural strain energy is great for the minimum compliance problem, and satisfy the dispersancy requirement. The adaptive growth method with the suggested key techniques is integrated into an ANSYS-based program, which provides a design tool for the stiffener layout design optimization of plates and shells. Typical design examples, including plate and shell structures to achieve minimum compliance and maximum bulking stability are illustrated. In addition, as a practical mechanical structural design example, the stiffener layout of an inlet structure for a large-scale electrostatic precipitator is also demonstrated. The design results show that the adaptive growth method integrated with the suggested key techniques can effectively and flexibly deal with stiffener layout design problem for plates and shells with complex geometrical shape and loading conditions to achieve various design objectives, thus it provides a new solution method for engineering structural topology design optimization.

Fracture toughness and fatigue crack growth analysis of 7050-T7451 alloy thick plate with different thicknesses

The strength, fracture toughness and fatigue crack growth resistance of 7050-T7451 aluminum alloy plate with different thicknesses （35 mm and 160 ram） were investigated by means of optical microscope, scanning electron microscope and transmission electron microscope. The results show that thicker plate has lower strength and fracture toughness but higher fatigue crack growth resistance, by comparison to the thinner plate. The drop of strength is mainly attributed to grain coarsening in the thicker plate, and the increased degree of recrystallization results in the loss of Kio However, the coarsened grains in the thicker plate make cracks deflected and closure effect enhanced due to surface roughness increased. For both of plates, in the fracture surface subjected plain strain, a transition from transgranular dimpled fracture to intergranular dimpled fracture is observed during the fracture process.

Effect of basic fibroblast growth factor and hyaluronic acid on proliferation of rabbit chondrocytes in vitro

Objective: To investigate the effect of basic fibroblast growth factor (bFGF) and hyaluronic acid (HA) on the proliferation of rabbit chondrocytes in vitro. Methods: Chondrocytes from the knee joints of New Zealand white rabbits were cultured. bFGF or HA or both were added into the culture medium respectively, and the proliferation of the chondrocytes was measured with MTT 3 (4, 5 dimethylthiazol 2 yl) 2, 5 diphenyl tetra zolium bromide. (MTT, Sigma, M2128). Results: Basic fibroblast growth factor (10 ng/ml) with low concentration of fetal bovine serum in the culture medium promoted the proliferation of chondrocytes significantly, and this effect reached its maximum when concentration of bFGF reached 50 ng/ml. HA itself had no effect on the proliferation of chondrocytes. However, when bFGF was used in combination with HA, especially when the concentration of bFGF was 50 500 ng/ml and that of HA was 10 50 ng/ml, the effect on the proliferation of chondrocytes was much more than when bFGF or HA was used alone. Conclusions: bFGF can promote the proliferation of chondrocytes. HA, which has no effect on the proliferation of the cells, can maintain a normal growth of chondrocytes. When bFGF is used in combination with HA, more proliferation is obtained.

Alendronate disturbs femoral growth due to changes during immunolocalization of transforming growth factor-β1 and bone morphogenetic protein-2 in epiphyseal plate

BACKGROUND The epiphyseal growth plate is an important anatomical segment localized on the ends of a long bone.Despite the abovementioned atractive reasons for alendronate’s use,few data on the effect of alendronate during epiphyseal growth exist.AIM Verify the effect of alendronate on the growth epiphyseal plate,and compare its effect with the size of the femur during the double-staining of the immunolocalization of transforming growth factor-β1(TGF-β1)and bone morphogenetic protein-2(BMP2)in endochondral ossifing in specimens that have received alendronate.METHODS Forty newborn rats were randomly divided into two groups:a control group(were given applications of 1 mg/kg physiologic saline)and a group that received Alendronate(a dose of 2.5 mg/kg).These groups were then divided into two subgroups for euthanasia in two and 12 d of life.After euthanasia,the femurs were removed,and the femoral bones were measured linearly between the apex of the greater trochanter until the lower intercondylar midlle face to verify the probable bone growth between 3 and 12 d in control and alednroanto treated rats.Posteriorly,the surgical pieces were also sent to the histopathology laboratory to produce histological slides.The obtained slides were stained with hematoxylin and eosin to measure each of the cartilage zones in endochondral development.and other slides were immunohistochemically tested for anti-TGF-β1 and BMP-2 antibodies to investigate the immunolocalization of these proteins in the epiphyseal plaque area.RESULTS On the third day,some diferences between the control group and specimens treated with alendronate were verified.Macroscopiccaly,we found similarities in size between the femoral bones when we compared the control group with the specimens that received alendronate.On the 12^th day,the bone size of the mice receiving the drug was significantly smaller than those of the control group.These results coincide with changes in the TGF-β1 and BMP-2 expression.In the specimens that received alendronate,the TGF-β1 was expressed in some sites of trabecular bone that was neoformed,peripherally to the bone marrow area.The BMP-2 was also positive in proliferative chondrocytes and hypertrofic chondrocytes.On the 12^th day,all layers of chondrocytes exhibited positivity for BMP-2 in the specimens that received alendronate.In the interface between the trabecular bone and cartilage,an area of disorganized bone deposition was evident.Neoformed bone also appeared to be different at 12 d.In the control group,BMP-2 was positive in an intense area of bone trabeculae,whereas the alendronate-treated group showed TGF-β1 positive trabeculae and a greater bone area.CONCLUSION Alendronate alters the immunolocalization of TGF-β1 and BMP-2 simultaneously,a condition that changes the usual histological aspects of the cartilage zone and impairs epiphysis growth and femur growth.

THE GROWTH OF THE VOID IN A HYPERELASTIC RECTANOULAR PLATE UNDERA UNIAXIAL EXTENSION

In the presenl paper. the finite deformation and stress analysis for a hyperelasticrectangular plate with a center void under a uniaxial extension is studied. In order toconsider the effect of the exijtence of the void on the deformation and stress of theplate, the problem is reduced to the deformation and stress analysis for a hyperelusticannular plate and its approximate solution is obtained from the minimum potentialenergy principle. The growth of the cavitation iS also nunterically computed andanalysed.

NUMERICAL ANALYSIS OF DELAMINATION GROWTH FOR STIFFENED COMPOSITE LAMINATED PLATES

A study of postbuckling and delamination propagation behavior in delaminated stiffened composite plates was presented. A methodology was proposed for simulating the multi-failure responses, such as initial and postbuckling, delamination onset and propagation, etc. A finite element analysis was conducted on the basis of the Mindlin first order shear effect theory and the von-Krmn nonlinear deformation assumption. The total energy release rate used as the criteria of delamination growth was estimated with virtual crack closure technique (VCCT). A self-adaptive grid moving technology was adopted to model the delamination growth process. Moreover, the contact effect along delamination front was also considered during the numerical simulation process. By some numerical examples, the influence of distribution and location of stiffener, configuration and size of the delamination, boundary condition and contact effect upon the delamination growth behavior of the stiffened composite plates were investigated. The method and numerical conclusion provided should be of great value to engineers dealing with composite structures.