Influence of the Zinc and Fibre Addition in the Diet on Biomechanical Bone Properties in Weaned Piglets

The effects of the zinc and fibre source in piglets’ diet on the bone mineral content, density, and strength parameters of the femur were investigated using 24 piglets fed a diet supplemented with either lignocellulose (LC) or potato fibre (PF). Half of each group of piglets consumed a diet with ZnSO4 monohydrate or with zinc glycinate (ZnGly). The diets contained similar amounts of lysine, energy, and fibre. Bone mineral content and density were over 9% higher in pigs receiving diets with ZnGly than in animals fed diets with ZnSO4. Moreover, ZnGly strongly improved maximum and elastic strength (by 25.7 and 20.0%, respectively, p < 0.0001) and bone stiffness (by 29.4%, p < 0.0001). Only the mass of the femur was affected by the type of fibre in the diet, as the femurs of piglets fed diets with LC were over 7% (p < 0.0001) heavier than in piglets fed diets with PF. The intake of digestible zinc and the zinc content in the blood serum were positively correlated with the measured bone parameters and, depending on the parameter, “r” ranged from 0.749 to 0.866 and from 0.400 to 0.479, respectively. It can be concluded that bone parameters are affected more strongly by the organic than inorganic source of zinc.

Modeling of Bimodular Bone Specimen under Four-Point Bending Fatigue Loading

The fatigue damage behavior of bone has attracted significant attention in both the mechanical and orthopedic fields. However, due to the complex and hierarchical structure of bone, describing the damage process quantitively or qualitatively is still a significant challenge for researchers in this area. In this study, a nonlinear bi-modulus gradient model was proposed to quantify the neutral axis skewing under fatigue load in a four-point bending test. The digital image correlation technique was used to analyze the tensile and compressive strains during the fatigue process. The results showed that the compressive strain demonstrated an obvious two-stage ascending behavior, whereas the tensile strain revealed a slow upward progression during the fatigue process. Subsequently, a theoretical model was proposed to describe the degradation process of the elastic modulus and the movement of the neutral axis. The changes in the bone properties were determined using the FEM method based on the newly developed model. The results obtained from two different methods exhibited a good degree of consistency. The results obtained in this study are of help in terms of effectively exploring the damage evolution of the bone materials.

Characterization of Porous Scaffolds Fabricated by Joining Stacking Based Laser Micro-Spot Welding (JS-LMSW) for Tissue Engineering Applications

A novel manufacturing approach was used to fabricate metallic scaffolds. A calibration of the laser cutting process was performed using the kerf width compensation in the calculations of the tool trajectory. Welding defects were studied through X-ray microtomography. Penetration depth and width resulted in relative errors of 9.4%, 1.0%, respectively. Microhardness was also measured, and the microstructure was studied in the base material. The microhardness values obtained were 400 HV, 237 HV, and 215 HV for the base material, HAZ, and fusion zone, respectively. No significant difference was found between the microhardness measurement along with different height positions of the scaffold. The scaffolds’ dimensions and porosity were measured, their internal architecture was observed with micro-computed tomography. The results indicated that geometries with dimensions under 500 µm with different shapes resulted in relative errors of ~2.7%. The fabricated scaffolds presented an average compressive modulus ~13.15 GPa, which is close to cortical bone properties. The proposed methodology showed a promising future in bone tissue engineering applications.

The Effect of Design Parameters on Mechanical Characteristics of Porous CoCrMo Scaffold Manufactured by Additive Manufacturing

Metallic orthopedic implants to replace or generate lost bones caused by traumatic road traffic injuries often failed prematurely after surgery. Bone resorption caused by stress shielding of metallic implants became a main concern as it can potentially lead to bone implant failure. Metallic scaffold designed in porous structures fabricated using additive manufacturing (AM) are widely used as bone implant, since the elastic modulus of the scaffolds can easily tailored according to the bone properties, and the large surfaces are beneficial to cell in-growth. The microarchitecture of scaffold can control their mechanical and biological properties, but it is found that there is lack of systematic approach to select a cell topology with full perspective requirements of bone implant. This paper presents a systematic approach of design space mapping for two CoCrMo unit cell shapes namely square and diamond to understand the relationship between geometrical parameters with additive manufacturing limitation, mechanical and bone ingrowth requirements. The compressive response of the components was simulated by finite element analysis and the influence of design parameters on the scaffold behaviour was compared theoretically with Gibson and Ashby model. The FEA give prediction for effective elastic modulus of 3 GPa to 4.8 GPa for diamond type and range of 6 GPa to 29 GPa for square type. Experimental results showed accurate prediction of compression elastic modulus with average error of 13% for diamond type and 35% for square type respectively. The significance of the methodology and the results showed that different design parameters of the structures can play a major role in the mechanical behaviour of the metallic scaffold.

The Influence of Prenatal Fumonisin Exposure on Bone Properties, as well as OPG and RANKL Expression and Immunolocalization, in Newborn Offspring Is Sex and Dose Dependent

The current study examined the effects of exposure of pregnant dams to fumonisins (FBs; FB1 and FB2), from the seventh day of pregnancy to parturition, on offspring bone metabolism and properties. The rats were randomly divided into three groups intoxicated with FBs at either 0, 60, or 90 mg/kg b.w. Body weight and bone length were affected by fumonisin exposure, irrespective of sex or dose, while the negative and harmful effects of maternal FBs’ exposure on bone mechanical resistance were sex and dose dependent. The immunolocalization of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-Β ligand (RANKL), in bone and articular cartilage, indicated that the observed bone effects resulted from the FB-induced alterations in bone metabolism, which were confirmed by the changes observed in the Western blot expression of OPG and RANKL. It was concluded that the negative effects of prenatal FB exposure on the general growth and morphometry of the offspring bones, as a result of the altered expression of proteins responsible for bone metabolism, were dose and sex dependent.

Environmental and Genetic Factors Affecting Bone Diseases and Phenotypes in Mouse Models

Bone diseases and phenotypes are affected in multiple ways. We focused on studying the effects of genetic and environmental factors, especially their impact on bone properties. Firstly, we investigated the effects of β-caryophyllene (BCP), a naturally occurring dietary cannabinoid, on protecting bone from vitamin D deficiency in mice fed on a diet lacking or supplemented with vitamin D (VD). We found that the VD-deficient diet enhanced the length of femur and tibia bones (P<0.05), and increased bone volume (BV; P<0.01) and the trabecular bone volume fraction (BV/TV; P <0.01) compared to the D+ diet. When given BCP-containing diet, mice exhibited higher BV and bone mineral density (BMD; P<0.05) than the control group. The trabecular and cortical bone were also affected by VD and BCP. In addition, the inclusion of dietary BCP improved the serum concentrations of klotho (P < 0.05). In summary, these data indicate that BCP enhances the level of klotho in the serum, leading to improved bone properties and mineralization in an experimental mouse model. Under conditions lacking UV light, the D-deficient diet could affect multiple properties of bone, including trabecular and cortical bone, in mice. The D-deficient diet can also result in weight loss in mice. My second project is to evaluate the bone properties in a mouse model with Il-1rn mutation. When knockout for IL-1rn, mice of Balb/c genomic background exhibited susceptibility to spontaneous arthritis disease (SAD), while those of a DBA/1 background were resistant to developing SAD. Our progress on the study of SAD suggested that some of the bone phenotypes, BMD, BV, tibia length, and cortical thickness, were different between wildtype and IL-1rn knockout mice both in Balb/c and DBA/1 strains. The two congenic mouse strains were also evaluated for bone properties. The results revealed that IL-1rn affected BMD differently between Balb/c and DBA/1 mouse strains. The absence of IL-1rn decreased BMD in Balb/c mice and increased BMD in DBA/1 -/- mice compared with wildtype animals. QTL in DBA.B -/- which affect arthritis in congenic strains also regulated BMD, with interferon activated gene 202b (Ifi202b) being the most favored candidate gene for BMD. Our data suggest some of the bone phenotypes are affected by the regulation of gene expression in the context of IL-1ra loss. To our knowledge, this is the first study to investigate the relationship of gene interaction in bone phenotypes with the loss of IL-1ra in an animal model. In my third study, we studied the different effects between endogenously produced and diet-supplied vitamin C on spontaneous arthritis disease susceptibility. In this study, we sought to investigate whether the source of vitamin C (endogenously produced or exogenously supplied) influences the development of inflammatory arthritis using a mouse model of SAD. SAD-susceptible Balb/c IL-1rn-/- mice were bred with vitamin C-deficient Sfx mice to produce a double mutant (SAD-susceptible, vitamin C-deficient) mouse strain. The three strains were raised, with the double mutant and Sfx mice supplied with vitamin C in drinking water, and mice’s arthritis severity scores were measured biweekly. Incidence and average severity for each strain were calculated. At four months of age, the mice were sacrificed, and body measurements and leg samples were collected. X-ray microcomputed tomography was used to scan the legs to characterize the bone profile. Femur length, tibia length, and bone volume were found to be significantly lower in double mutant mice than in Balb/c IL-1rn-/- mice. There was no significant difference in bone mineral density and femur thickness between the two arthritis-susceptible strains. The double mutant mice had an earlier onset of arthritis as well as a more severe disease than that of the Balb/c knockout (KO) strain. Our findings suggest that the source of vitamin C could affect both the susceptibility of mice to SAD and the severity of disease. In addition, we examined the sex differences in several mouse models of inflammatory arthritis. To understand the basis for these differences we conducted analysis of several mouse models of inflammatory arthritis. The study of whether there are gender and symmetry differences in experimental arthritis expression in the mouse models may be of significance to the study of human rheumatoid arthritis. Our observations and statistical analyses on the incidence of arthritis in four different animal models incorporated relatively large numbers of mice allowing for robust conclusions. Our research showed that there is a sexual dimorphism for arthritis incidence and severity of arthritis in mice harboring specific genetic modifications. For F2 population the incidence of arthritis was 57.1% in female mice and 75.6% in male mice. There was a difference in severity related to sex in two populations: B6.DR1/ B6.DR4 (P < 0.001) and F2 (P = 0.023). Among these populations, scores for the right hindlimbs were significantly higher than those for the left hindlimbs in males (P<0.05). When examining disease manifestation using the collagen induced arthritis model with DBA/1 mice, sex-dimorphism did not reach statistical significance. However, left hindlimbs showed a tendency toward greater disease expression over the right. Our results suggest that sex difference of arthritis exists in animal models not only in terms of gender, but also of left and right limbs. Using animal models, this work has laid the foundation for future research on gender differences in rheumatoid arthritis. In conclusion, our studies on genetic and environmental factors regulating bone phenotypes and diseases have significant implications. Not only have they raised the concept of sexual dimorphism in disease, but they have highlighted the influence of genetic background on bone disease. The link between different sources of vitamins and immune-mediated disease is intriguing and warrants further research, as is the bone protective effect of BCP.

Bone Phenotyping Approaches in Human, Mice and Zebrafish – Expert Overview of the EU Cost Action GEMSTONE (“GEnomics of MusculoSkeletal traits TranslatiOnal NEtwork”)

A synoptic overview of scientific methods applied in bone and associated research fields across species has yet to be published. Experts from the EU Cost Action GEMSTONE (“GEnomics of MusculoSkeletal Traits translational Network”) Working Group 2 present an overview of the routine techniques as well as clinical and research approaches employed to characterize bone phenotypes in humans and selected animal models (mice and zebrafish) of health and disease. The goal is consolidation of knowledge and a map for future research. This expert paper provides a comprehensive overview of state-of-the-art technologies to investigate bone properties in humans and animals – including their strengths and weaknesses. New research methodologies are outlined and future strategies are discussed to combine phenotypic with rapidly developing –omics data in order to advance musculoskeletal research and move towards “personalised medicine”.

The effect of light/dark cycles on performance and welfare in broiler

The purpose of this study was to compare a continuous lighting programme (23 hours of lighting (L) / 1 hour of darkness (D)) with intermittent lighting programmes (16L: 8D) and also to investigate the effects of the length of the dark cycle in the intermittent programme on the performance, carcass characteristics, water consumption, uniformity, metabolic parameters, and ammonia burns of chickens. Thus, five hundred Ross-308 male chicks were used. The 23L:1D was applied to the chicks for 7 days. On day 7, they were divided into four groups by balancing their live weight; group I: continuous 23L: 1D; group II: intermittent 4x (4L: 2D); group III: intermittent 2x (8L: 4D); group IV: continuous 16L: 8D. The study took place between days 7 and 42. At the end of the study, 10 chickens from each group were slaughtered, their carcass, blood, and bone properties were analysed. Body temperatures and ammonia burns were assessed for all broiler chickens. The mean live weight of group IV was the lowest. The difference among the groups in terms of live weight gains, feed intakes, feed conversion ratios, and survival rates was non-significant. Long-period darkness in group IV significantly dropped the water consumption. On day 21, group III’s best uniformity was calculated; but on day 42, the difference was non-significant. The highest breast ratio and the lowest wing ratio beloged to chicks in group I. Their free T4, glucose, and uric acid levels were lower; whilst their testosterone levels were higher. Body temperature and tibia ash levels were similar across all of the groups. The intermittent lighting programmes increased the number of ammonia burns. Consequently, the long-term darkness negatively affected both the chickens’ performance and well-being.

Subchondral Bone Microarchitectural and Mineral Properties and Expression of Key Degradative Proteinases by Chondrocytes in Human Hip Osteoarthritis

Background: The purpose of this study was to investigate the relationship between the expression of key degradative enzymes by chondrocytes and the microarchitectural and mineral properties of subchondral bone across different stages of cartilage degradation in human hip osteoarthritis (OA). Methods: Osteochondral samples at different stages of cartilage degradation were collected from 16 femoral heads with OA. Osteochondral samples with normal cartilage were collected from seven femoral heads with osteoporosis. Microcomputed tomography was used for the investigation of subchondral bone microarchitecture and mineral densities. Immunohistochemistry was used to study the expression and distribution of MMP13 and ADAMTS4 in cartilage. Results: The microarchitecture and mineral properties of the subchondral plate and trabecular bone in OA varied with the severity of the degradation of the overlying cartilage. Chondrocytes expressing MMP13 and ADAMTS4 are mainly located in the upper zone(s) of cartilage regardless of the histopathological grades. The zonal expression of these enzymes in OA (i.e., the percentage of positive cells in the superficial, middle, and deep zones), rather than their overall expression (the percentage of positive cells in the full thickness of the cartilage), exhibited significant variation in relation to the severity of cartilage degradation. The associations between the subchondral bone properties and zonal and overall expression of these enzymes in the cartilage were generally weak or nonsignificant. Conclusions: Phenotypic changes in chondrocytes and remodelling of subchondral bone proceed at different rates throughout the process of cartilage degradation. Biological influences are more important for cartilage degradation at early stages, while biomechanical damage to the compromised tissue may outrun the phenotypic change of chondrocytes and is critical in the advanced stages.