The effects of simulated weightlessness on bone biomechanical and biochemical properties in the maturing rat

Histomorphometric and biomechanical changes in bone resulting from hypogravity (simulated weightlessness) were examined in this study. Using a head-down hindlimb suspension model, three groups of six male rats underwent simulated weightlessness for periods of one, two and three weeks while a fourth recovery group was suspended for two weeks followed by two weeks of normal activity. Biomechanical data were collected during static and dynamic bending and torsion tests on intact femora. Histomorphometric values were determined from midshaft bone cross sections and material properties were obtained using ash and calcium assays. The experimental groups exhibited significantly lower geometric and material properties than the controls, resulting in structural hypotrophy; geometric and material changes contributed equally to the structural changes. Recovery following a return to normal activity was indicated, although full recovery may take longer than the weightlessness period. In the rat, altered maturation and reduced bone strength were the sequelae of weightlessness.     

泼尼松灌胃与肌内注射对大鼠骨密度、骨生物力学性能及骨代谢的影响

目的观察泼尼松灌胃与肌内注射两种不同给药方法对大鼠骨密度、骨生物力学及骨代谢的影响。方法将45只SPF级雄性SD大鼠随机分为3组（正常组15只、灌胃组15只、肌内注射组15只）,其中正常组大鼠作为阴性对照,予0.9%生理盐水灌胃2 m L/d;灌胃组大鼠给予泼尼松0.5 mg/（kg·d）灌胃;肌内注射组大鼠给予泼尼松0.5 mg/（kg·d）;12周后测定离体的大鼠椎体骨密度及血清β-CTX、PINP水平变化,采用三点弯曲试验测量股骨皮质骨最大载荷、弹性载荷、断裂载荷等生物力学指标。结果与正常组相比,灌胃组及肌内注射组大鼠椎骨骨密度值均显著性降低（P〈0.05）;与灌胃组相比,肌内注射组大鼠椎骨骨密度显著下降（P〈0.05）;与正常组相比,灌胃组及肌内注射组大鼠股骨的弹性载荷、最大载荷、断裂载荷均显著降低（P〈0.05）,肌内注射组与灌胃组大鼠的弹性载荷、最大载荷、断裂载荷相比差异无显著性（P〉0.05）。与正常组相比,灌胃组及肌内注射组大鼠中血清β-CTX水平均显著升高（P〈0.05）而PINP水平均显著降低（P〈0.05）,与灌胃组相比,肌内注射组大鼠血清β-CTX水平显著升高（P〈0.05）而PINP水平显著降低（P〈0.05）。骨组织切片HE染色显示：肌内注射组大鼠的骨小梁明显纤细疏松,造血组织明显减少,脂肪组织明显增多。结论泼尼松对大鼠的骨密度、骨生物力学及骨代谢指标都有影响,而肌内注射泼尼松比口服对骨密度、骨强度、骨代谢的影响更大,更易造成骨质疏松症。因此,建议临床使用泼尼松时选择口服作为给药方式更安全。     

The effects of total hip arthroplasty on the structural and biomechanical properties of adult bone

The responsiveness of bone to mechanical stimuli changes throughout life, with adaptive potential generally declining after skeletal maturity is reached. This has led some to question the importance of bone functional adaptation in the determination of the structural and material properties of the adult skeleton. A better understanding of age-specific differences in bone response to mechanical loads is essential to interpretations of long bone adaptation. The purpose of this study is to examine how the altered mechanical loading environment and cortical bone loss associated with total hip arthroplasty affects the structural and biomechanical properties of adult bone at the mid-shaft femur. Femoral cross sections from seven individuals who had undergone unilateral total hip arthroplasty were analyzed, with intact, contralateral femora serving as an approximate internal control. A comparative sample of individuals without hip prostheses was also included in the analysis. Results showed a decrease in cortical area in femora with prostheses, primarily through bone loss at the endosteal envelope; however, an increase in total cross-sectional area and maintenance of the parameters of bone strength, I(x), I(y), and J, were observed. No detectable differences were found between femora of individuals without prostheses. We interpret these findings as an adaptive response to increased strains caused by loading a bone previously diminished in mass due to insertion of femoral prosthesis. These results suggest that bone accrued through periosteal apposition may serve as an important means by which adult bone can functional adapt to changes in mechanical loading despite limitations associated with senescence.     

Effects of exercise on the biomechanical,biochemical and structural properties of tendons

Tendon has been shown to undergo remodeling in response to strength or endurance training, however, compared to muscle, studies of the effects of exercise on tendon are limited and the information is inconsistent. Exercise may influence the structure, chemical composition and/or mechanical properties of tendon. Studies that have examined mechanical changes of tendon in response to endurance training suggest that ultimate failure strength and stiffness increase with training. Available reports indicate that increases in tensile strength and stiffness are probably not associated with increases in collagen concentration or with tendon hypertrophy. The paucity of data renders it impossible to evaluate the response of other structural, chemical and mechanical parameters to training. Furthermore, few investigators have included discrete measures of structural, biomechanical and biochemical variables within a single study. The lack of integrative studies makes it difficult to definitively associate changes in the mechanical properties of tendon with chemical composition and structure.     

Relationship between physical,biochemical and biomechanical properties of tissue-engineered cartilage-carrier-constructs

Three-dimensional cartilage-carrier-constructs were produced according to a standard protocol from chondrocytes of an adult mini-pig. Physical parameters (height and weight) correlated very well with total DNA content (r²=0.86, re. 0.94). The relation between DNA content and glycosaminoglycan content was less but still significant. No significant relationship was found between the elasticity module and the DNA content, even if the elasticity module increased slightly at higher DNA content. With respect to later implantation, selection of a construct for implantation based on the weight, which can be determined non-invasive and under sterile conditions, seems to be justifiable.Revisions requested14 October 2004; Revisions received 7 December 2004     

Nck1 deficiency accelerates unloading‐induced bone loss

Mechanical stress is an important signal to determine the levels of bone mass. Unloading‐induced osteoporosis is a critical issue in bed‐ridden patients and astronauts. Many molecules have been suggested to be involved in sensing mechanical stress in bone, though the mechanisms involved in this phenomenon are not fully understood. Nck1 is an adaptor protein known to mediate signaling from plasma membrane‐activated receptors to cytosolic effectors regulating actin cytoskeleton remodeling. Nck1 has also been implicated in cellular responses to endoplasmic reticulum stress. In vitro, in case of cell stress the actin cytoskeleton is disrupted and in such cases Nck1 has been reported to enter the nucleus of the cells to mediate the nuclear actin polymerization. However, the role of Nck1 in vivo during the bone response to mechanical stimuli is unknown. The purpose of this study is to examine the role of Nck1 in unloading‐induced bone loss in vivo. Sciatic and femoral nerve resection was conducted. Neurectomy‐based unloading enhanced Nck1 gene expression in bone about twofold. Using the Nck1 deficient mice and control Nck1+/+, effects of neurectomy‐based unloading on bone structure were examined. Unloading reduced bone volume in wild type mice by 30% whereas the levels in bone loss were exacerbated to 50% in Nck1 deficient mice due to neurectomy after 4 weeks. These data demonstrate that Nck1 gene deficiency accelerates the mechanical unloading‐induced bone loss suggesting Nck1 to be a crucial molecule in mechanical stress mediated regulation in bone metabolism. J. Cell. Physiol. 228: 1397–1403, 2013. © 2012 Wiley Periodicals, Inc.     

Serum equol, bone mineral density and biomechanical bone strength differ among four mouse strains

The extent of conversion of daidzein to its metabolite, equol, by intestinal microflora may be a critical step that determines if a diet rich in daidzein protects against the deterioration of bone after estrogen withdrawal. The objective was to determine the extent that daidzein is converted to equol. In addition, bone mineral content (BMC), bone mineral density (BMD) and strength of femurs and lumbar vertebrae (LV) in four mouse strains were measured. Mice were ovariectomized and fed control diet (AIN93G) with or without daidzein (200 mg daidzein/kg diet) for 3 weeks, after which serum, femurs and LV were collected. Serum daidzein and equol were elevated in all mice fed daidzein. Among mice fed daidzein, the CD-1 and Swiss–Webster (SW) mice had higher (P<.001) serum equol than C57BL/6 (C57) and C3H mice. Differences due to mouse strain were observed for all bone outcomes. C57 mice had lower femur BMC (P<.001), BMD (P<.001) and peak load at femur midpoint (P<.001) and neck (P<.001) than other mouse strains. C57 mice also had a lower femur midpoint yield load (P<.001) and resilience (P<.001) than C3H mice. C57 mice had a lower LV1–4 BMC (P<.001) and BMD (P<.001) compared with all mouse strains and peak load of LV3 was lower than CD-1 and SW mice. Differences in serum equol, BMD and bone strength properties should be considered when selecting a mouse strain for investigating whether dietary strategies that include isoflavones preserve bone tissue after ovariectomy.     

Mangiferin attenuates osteoclastogenesis,bone resorption,and RANKL‐induced activation of NF‐κB and ERK

Osteolytic bone diseases such as osteoporosis have a common pathological feature in which osteoclastic bone resorption outstrips bone synthesis. Osteoclast formation and activation are regulated by receptor activator of nuclear factor κB ligand (RANKL). The induction of RANKL‐signaling pathways occurs following the interaction of RANKL to its cognate receptor, RANK. This specific binding drives the activation of downstream signaling pathways; which ultimately induce the formation and activation of osteoclasts. In this study, we showed that a natural immunomodulator, mangiferin, inhibits osteoclast formation and bone resorption by attenuating RANKL‐induced signaling. Mangiferin diminished the expression of osteoclast marker genes, including cathepsin K, calcitonin receptor, DC‐STAMP, and V‐ATPase d2. Mechanistic studies revealed that mangiferin inhibits RANKL‐induced activation of NF‐κB, concomitant with the inhibition of IκB‐α degradation, and p65 nuclear translocation. In addition, mangiferin also exhibited an inhibitory effect on RANKL‐induced ERK phosphorylation. Collectively, our data demonstrates that mangiferin exhibits anti‐resorptive properties, suggesting the potential application of mangiferin for the treatment and prevention of bone diseases involving excessive osteoclastic bone resorption. J. Cell. Biochem. 112: 89–97, 2011. © 2010 Wiley‐Liss, Inc.     

Arabidopsis ammonium transporters,AtAMT1;1 and AtAMT1;2, have different biochemical properties and functional roles

We have compared the biochemical properties of two different Arabidopsis ammonium transporters, AtAMT1;1 and AtAMT1;2, expressed in yeast, with the biophysical properties of ammonium transport in planta. Expression of the AtAMT1;1 gene in Arabidopsis roots increased approximately four-fold in response to nitrogen deprivation. This coincided with a similar increase in high-affinity ammonium uptake by these plants. The biophysical characteristics of this high-affinity system (K_m for ammonium and methylammonium of 8 M and 31 M, respectively) matched those of AtAMT1;1 expressed in yeast (K_m for methylammonium of 32 M and K_i for ammonium of 1–10 M). The same transport system was present, although less active, in nitrate-fed roots. Ammonium-fed plants exhibited the lowest rates of ammonium uptake and appeared to deploy a different transporter (K_m for ammonium of 46 M). Expression of AtAMT1;2 in roots was insensitive to changes in nitrogen nutrition. In contrast to AtAMT1;1, AtAMT1;2 expressed in yeast exhibited biphasic kinetics for methylammonium uptake: in addition to a high-affinity phase with a K_m of 36 M, a low-affinity phase with a K_m for methylammonium of 3.0 mM was measured. Despite the presence of a putative chloroplast transit peptide in AtAMT1;2, the protein was not imported into chloroplasts in vitro. The electrophysiological data for roots, together with the biochemical properties of AtAMT1;1 and Northern blot analysis indicate a pre-eminent role for AtAMT1;1 in ammonium uptake across the plasma membrane of nitrate-fed and nitrogen-deprived root cells.     

The effect of tissue-engineered cartilage biomechanical and biochemical properties on its post-implantation mechanical behavior

The insufficient load-bearing capacity of today’s tissue-engineered (TE) cartilage limits its clinical application. Focus has been on engineering cartilage with enhanced mechanical stiffness by reproducing native biochemical compositions. More recently, depth dependency of the biochemical content and the collagen network architecture has gained interest. However, it is unknown whether the mechanical performance of TE cartilage would benefit more from higher content of biochemical compositions or from achieving an appropriate collagen organization. Furthermore, the relative synthesis rate of collagen and proteoglycans during the TE process may affect implant performance. Such insights would assist tissue engineers to focus on those aspects that are most important. The aim of the present study is therefore to elucidate the relative importance of implant ground substance stiffness, collagen content, and collagen architecture of the implant, as well as the synthesis rate of the biochemical constituents for the post-implantation mechanical behavior of the implant. We approach this by computing the post-implantation mechanical conditions using a composition-based fibril-reinforced poro-viscoelastic swelling model of the medial tibia plateau. Results show that adverse implant composition and ultrastructure may lead to post-implantation excessive mechanical loads, with collagen orientation being the most critical variable. In addition, we predict that a faster synthesis rate of proteoglycans compared to that of collagen during TE culture may result in excessive loads on collagen fibers post-implantation. This indicates that even with similar final contents, constructs may behave differently depending on their development. Considering these aspects may help to engineer TE cartilage implants with improved survival rates.     

Depth-dependent biomechanical and biochemical properties of fetal, newborn, and tissue-engineered articular cartilage

Adult articular cartilage has depth-dependent mechanical and biochemical properties which contribute to zone-specific functions. The compressive moduli of immature cartilage and tissue-engineered cartilage are known to be lower than those of adult cartilage. The objective of this study was to determine if such tissues exhibit depth-dependent compressive properties, and how these depth-varying properties were correlated with cell and matrix composition of the tissue. The compressive moduli of fetal and newborn bovine articular cartilage increased with depth (p<0.05) by a factor of 4-5 from the top 0.1 mm (28+/-13 kPa, 141+/-10 kPa, respectively) to 1 mm deep into the tissue. Likewise, the glycosaminoglycan and collagen content increased with depth (both p<0.001), and correlated with the modulus (both p<0.01). In contrast, tissue-engineered cartilage formed by either layering or mixing cells from the superficial and middle zone of articular cartilage exhibited similarly soft regions at both construct surfaces, as exemplified by large equilibrium strains. The properties of immature cartilage may provide a template for developing tissue-engineered cartilage which aims to repair cartilage defects by recapitulating the natural development and growth processes. These results suggest that while depth-dependent properties may be important to engineer into cartilage constructs, issues other than cell heterogeneity must be addressed to generate such tissues.     

The effects of quercetin on bone minerals, biomechanical behavior, and structure in streptozotocin-induced diabetic rats

This study was designed to investigate the effect of quercetin (QE) on bone minerals and biomechanics in insulin-dependent diabetic rats. Diabetes was induced by 50 mg kg(-1) intraperitoneal streptozotocin (STZ) in a single dose. The rats were randomly allotted into four experimental groups: A (control), B (non-diabetic + QE), C (diabetic), and D (diabetic + QE) each containing 10 animals. The diabetic rats received QE (15 mg kg(-1) day(-1)) for 4 weeks following 8 weeks of STZ injection. Blood samples were taken to determine glucose, insulin, calcium, and magnesium levels. The rats' femora were assessed biomechanically at femoral mid-diaphysis and neck. It was found that QE treatment increased insulin, calcium, and magnesium levels. Three-point bending of the femoral mid-diaphysis and necks showed significantly lower maximum load values (F max) in animals in the STZ group than the QE + STZ or control groups (p < 0.05). The results support the conclusion that QE treatment may decrease blood glucose and increase plasma insulin, calcium, and magnesium. QE treatment may also be effective in bone mineral metabolism, biomechanical strength, and bone structure in STZ-induced diabetic rats.     

Low concentration of lipopolysaccharide acts on MC3T3‐E1 osteoblasts and induces proliferation via the COX‐2‐independent NFκB pathway

The translocations of lipopolysaccharide (LPS) from the gut and its effects on bone healing are usually of clinical interest during bone fracture. As already widely stuided, Cyclooxygenase‐2 (COX‐2) is a key enzyme for prostaglandin E2 (PGE₂) production, which induces the nuclear factor kappa B (NFκB) activation and is beneficial to fracture healing. In order to know their roles in skeletal regeneration, mouse MC3T3‐E1 osteoblasts were treated with NFκB inhibitor BAY 11‐7082 and sc791 (a selective COX‐2 inhibitor), in the presence of LPS. Interestingly, LPS could induce osteoblasts proliferation through increasing NFκB activation and translocation. This induction was not related to COX‐2 expression, suggesting that LPS‐induced NFκB activiation is independent of COX‐2. It is possible that low concentration of LPS can act as a stimulating factor of the NFκB pathway in nonstimulated cells such as osteoblasts. COX‐2 is not necessary for the NFκB pathway during LPS‐induced proliferation of osteoblasts since sc791 had no effects on this induction. These studies provide insight into a potential mechanism by which LPS can affect bone tissue repair in the initial phase of inflammation. Copyright © 2009 John Wiley & Sons, Ltd.     

Physical, biomechanical, histologic, and biochemical effects of rapid versus conventional tissue expansion

Clinical tissue expansion has been quite successful but takes 2 to 3 months. This study compares the effects of a conventional tissue expansion regimen of 6 weeks with an accelerated regimen of 2 weeks in the dog model, which is biomechanically similar to the human. In 22 dogs, the skin expanded 34.4 percent in the 2-week and 35.8 percent in the 6-week protocol, excluding stretch and recruitment. There was thinning of the panniculus carnosus in the 6-week group and otherwise no significant decrease in dermal thickness in either group. The biomechanical properties of elasticity and creep did not differ in expanded skin from both groups, while stress/relaxation mildly decreased from a control value of 53.5 percent to 48.8 percent in the 6-week group (p less than 0.05). Collagen activity was increased in both the 6-week and the 2-week groups (p less than 0.001) over nonexpanded skin, and immunohistochemical staining with a monoclonal procollagen antibody demonstrated collagen synthesis by dermal fibroblasts in both groups. We conclude that rapid tissue expansion did not demonstrate any deleterious effects when compared with a conventional regimen.     

Inhibitory effects of biochanin A on titanium particle‐induced osteoclast activation and inflammatory bone resorption via NF‐κB and MAPK pathways

Revision operations have become a new issue after successful artificial joint replacements, and periprosthetic osteolysis leading to prosthetic loosening is the main cause of why the overactivation of osteoclasts (OCs) plays an important role. The effect of biochanin A (BCA) has been examined in osteoporosis, but no study on the role of BCA in prosthetic loosening osteolysis has been conducted yet. In this study, we utilised enzyme‐linked immunosorbent assay, computed tomography imaging, and histological analysis. Results showed that BCA downregulated the secretion levels of tumor necrosis factor‐α, interleukin‐1α (IL‐1α), and IL‐1β to suppress inflammatory responses. The secretion levels of receptor‐activated nuclear factor‐κB ligand, CTX‐1, and osteoclast‐associated receptor as well as Ti‐induced osteolysis were also reduced. BCA effectively inhibited osteoclastogenesis and suppressed hydroxyapatite resorption by downregulating OC‐related genes in vitro. Analysis of mechanisms indicated that BCA inhibited the signalling pathways of mitogen‐activated protein kinase (P38, extracellular signal‐regulated kinase, and c‐JUN N‐terminal kinase) and nuclear factor‐κB (inhibitor κB‐α and P65), thereby downregulating the expression of nuclear factor of activated T cell 1 and c‐Fos. In conclusion, BCA may be an alternative choice for the prevention of prosthetic loosening caused by OCs.