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.

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

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.

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.

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.

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 mm) 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 KIC.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.

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.

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.

Growth of Voids in a Hyperelastic Rectangular Plate

The analyses of finite deformation and stress for a hyperelastic rectangular plate with some voids under an uniaxial extension were conducted. The governing differential equations were given from the incompressibility condition of the material. The solution was approximately obtained from the minimum potential energy principle. The growth of voids was discussed. One can see that an initial central circular-cylinder void becomes an elliptic-cylinder void, but an initial non-centeral circular-cylinder void becomes an elliptic-like cylinder void and the center of void has a shift. The stress distributions along the edges of voids were given and the phenomenon of stress concentration was observed. The influences of the distribution manner and size of voids, as well as the distance between them on the growth of voids were analyzed.

育苗盘规格对烟苗生长发育及烟叶产质量的影响

为筛选出既满足宜宾烟区井窖式小苗移栽又减少基质用量的育苗盘规格,采用湿润育苗的方式,研究了不同育苗盘规格对烟苗生长发育及烟叶产质量的影响。结果表明,128孔和162孔育苗盘所育烟苗生长较快,烟苗农艺性状、物质积累和合格烟苗率较高,大田烟株农艺性状、经济性状较高,但基质用量和所需育苗面积较大,育苗投入成本明显增加;200孔育苗盘所育烟苗素质、大田烟株农艺性状、经济性状等方面与128孔育苗盘相当,但其基质用量较128孔育苗盘降低39.88%,育苗盘面积较128孔育苗盘减少30.77%,育苗投入成本明显降低;288孔育苗盘虽然基质用量和所需育苗面积最低,但其所育烟苗素质,大田烟株农艺性状和经济性状显著低于其他处理。综合而言,200孔育苗盘是宜宾烟区比较理想的选择。

Using Extended Finite Element Method for Computation of the Stress Intensity Factor, Crack Growth Simulation and Predicting Fatigue Crack Growth in a Slant-Cracked Plate of 6061-T651 Aluminum

The 6061-T651 aluminium alloy is one of the most common aluminium alloys for marine components and general structures. The stress intensity factor (SIF) is an important parameter for estimating the life of the cracked structure. In this paper, the stress intensity factors of a slant-cracked plate, which is made of 6061-T651 aluminum, have been calculated using extended finite element method (XFEM) and finite element method (FEM) in ABAQUS software and the results were compared with theoretical values. Numerical values obtained from these two methods were close to the theoretical values. In simulations of crack growth at different crack angles, the crack propagation angle values were closer to the theoretical values in XFEM method. Also, the accuracy and validity of fatigue crack growth curve were much closer to the theoretical graph in XFEM than the FEM. Therefore, in this paper the capabilities of XFEM were realized in analyzing issues such as cracks.

不同水平餐桌剩余食物对肉鸡后期生长、免疫及肉质性状的影响

【目的】本试验通过研究添加不同比例餐桌剩余食物(RPL)对肉鸡后期生长性能、屠宰性能、肉质性状、免疫器官指数及血清生化指标的影响,旨在为RPL减量替代玉米豆粕和在肉鸡养殖中的应用提供数据支持。【方法】选择1日龄白羽肉仔鸡公鸡504只,随机分为4组,每组6个重复,每个重复21只鸡。试验前期(第1~21天)饲喂相同饲粮;后期(第22~42天)设计4种饲粮,对照组饲喂玉米-豆粕基础饲粮,试验组分别饲喂添加10%、20%和30%RPL的饲粮(1/2 RPL替代玉米,1/2 RPL替代豆粕)。测定肉鸡后期生长性能,42日龄屠宰后测定屠宰性能、肉质性状、免疫器官指数及血清生化指标。【结果】与对照组相比,(1)添加10%、20%RPL对肉鸡生长性能影响不显著(P>0.05),添加30%RPL显著降低平均体重、平均日采食量、平均日增重(P<0.05),极显著提高料重比(P<0.01)。(2)添加10%、20%RPL对肉鸡屠宰率、全净膛率、胸肌率和腿肌率均无显著影响(P>0.05),但添加30%RPL显著降低胸肌率(P<0.05)。(3)饲粮中添加不同比例RPL对肉鸡免疫器官指数无显著影响(P>0.05),10%、20%RPL组肉鸡血清免疫球蛋白G(IgG)含量显著升高(P<0.05),30%RPL组血清超氧化物歧化酶(SOD)活性显著降低(P<0.05)。(4)添加不同比例RPL对肉鸡胸肌和腿肌肉色、24、48和72 h滴水损失、酸度、蒸煮损失及剪切力均无显著影响(P>0.05)。【结论】在肉鸡饲粮中添加10%~20%RPL对肉鸡后期生长性能、屠宰性能、肉质性状、免疫器官指数、血清生化指标等均无负面影响;RPL可替代常规饲粮中的玉米和豆粕在肉鸡养殖后期中应用。

基于支架和无支架策略在生长板软骨再生治疗中的应用

背景:组织工程是一种理想的生长板再生治疗方式,然而目前大多数的再生组织工程研究都是建立在传统支架策略的基础之上,随着传统支架的局限性逐渐显露,研究的方向也逐渐多样化。目的:总结基于支架和无支架策略在生长板软骨再生治疗中的应用以及各自的优势和不足。方法:检索PubMed、Wiley、Elsevier数据库收录的相关文献,英文检索词为“growth plate injury,regeneration,tissue engineering,scaffold,scaffold-free,biomimetic,cartilage”。检索时限为1990-2023年,最终纳入104篇文献进行综述。结果与结论:仿生支架策略是通过模拟生长板独特的组织结构最大程度上还原每个区域的细胞组成、生物信号和独特力学性能,进而构建能促进组织再生的仿生微环境,因此,仿生支架的设计是尽可能地从成分、结构和力学性能上模拟原生生长板,虽然取得一定的成效,但仍存在再生效果不稳定的问题。无支架策略认为支架的局限性会对再生治疗产生不利影响,因此,无支架构建物的设计是尽可能地依赖于细胞自身产生和维持细胞外基质的能力,不干扰细胞-细胞间的信号,不引入外源性物质,但存在稳定性较差、机械强度低,操作难度较大等问题。仿生策略和无支架策略的出发点不同且均存在各自的优缺点,但它们对于生长板软骨再生均能产生积极的作用。因此,后续的研究不论是采取仿生策略或无支架策略,都将聚焦于对现有技术的不断优化,以期实现有效的生长板软骨再生治疗。

葡萄籽提取物抑制大鼠生长板软骨细胞凋亡及促进胫骨的生长

背景:已有文献报道,葡萄籽提取物在抑制雄激素依赖型肿瘤的生长(如乳腺癌等)方面具有一定的作用,因此理论上葡萄籽提取物可以抑制青少年后期由于雌激素产生的骨骺闭合现象。目的:观察葡萄籽提取物对大鼠生长板软骨细胞凋亡及骨骺闭合的影响。方法:①体外实验:取对数生长期的大鼠胫骨及股骨生长板软骨细胞,分组培养:正常对照组、模型对照组(加入17β雌二醇诱导细胞凋亡)、阳性对照组(加入来曲唑与17β雌二醇)、葡萄籽提取物组(加入17β雌二醇和10μg/mL葡萄籽提取物)、Caspase-9抑制剂组(加入17β雌二醇和Caspase-9抑制剂)、Caspase-9激动剂组(加入17β雌二醇和Caspase-9激动剂),培养48 h后,采用流式细胞术检测细胞凋亡。②体内实验:将30只3月龄SD大鼠随机分为模型对照组、阳性对照组及药物低、中、高剂量组,每组5只,均皮下注射17β雌二醇(3次/周)建立骨骺闭合模型,阳性对照组灌胃给予来曲唑,药物低、中、高剂量组分别灌胃给予0.05,0.2,0.8 g/kg的葡萄籽提取物,1次/d,直至模型对照组大鼠胫骨平台骨骺闭合2/3以上停药,观察各组大鼠胫骨长度。将18只SD大鼠随机分为模型对照组、阳性对照组、药物中剂量组,每组6只,按照如上方法处理,连续给药1.5个月后,Western blot检测大鼠胫骨生长板软骨Caspase-9蛋白表达。结果与结论:①体外实验:17β雌二醇可诱发生长板软骨细胞的凋亡,而来曲唑、葡萄籽提取物与Caspase-9抑制剂均可抑制生长板软骨细胞的凋亡;②体内实验:当模型对照组大鼠胫骨平台骨骺闭合2/3以上时,阳性对照组、药物中剂量组骨骺闭合数量少于模型对照组(P<0.05),且胫骨长度大于模型对照组(P<0.05),胫骨生长板软骨Caspase-9蛋白表达低于模型对照组(P<0.05);③结果表明:适宜浓度的葡萄籽提取物可有效抑制生长板软骨细胞的凋亡并延迟骨骺闭合,具有促进骨骼生长的潜能。

软骨细胞外基质基因调控与遗传缺陷研究新进展

生长板软骨的生长发育是一个精密的生物学过程,受到细胞外基质、细胞内信号、旁分泌信号、内分泌信号等综合调控。细胞外基质在软骨细胞的结构支持中起着关键作用,作为传递生长因子与软骨发育相关信号分子的介质,调节软骨细胞的生长发育。文章对软骨细胞外基质的结构、功能,编码软骨细胞外基质基因的致病性变异影响骨骼发育的机制,以及致病性变异与表型的相关性等方面进行综述,旨在为临床软骨细胞外基质变异造成的遗传缺陷性疾病的诊断和治疗提供理论参考。