Resilient anatomy and local plasticity of naive and stress haematopoiesis.

The bone marrow adjusts blood cell production to meet physiological demands in response to insults. The spatial organization of normal and stress responses are unknown owing to the lack of methods to visualize most steps of blood production. Here we develop strategies to image multipotent haematopoiesis, erythropoiesis and lymphopoiesis in mice. We combine these with imaging of myelopoiesis1 to define the anatomy of normal and stress haematopoiesis. In the steady state, across the skeleton, single stem cells and multipotent progenitors distribute through the marrow enriched near megakaryocytes. Lineage-committed progenitors are recruited to blood vessels, where they contribute to lineage-specific microanatomical structures composed of progenitors and immature cells, which function as the production sites for each major blood lineage. This overall anatomy is resilient to insults, as it was maintained after haemorrhage, systemic bacterial infection and granulocyte colony-stimulating factor (G-CSF) treatment, and during ageing. Production sites enable haematopoietic plasticity as they differentially and selectively modulate their numbers and output in response to insults. We found that stress responses are variable across the skeleton: the tibia and the sternum respond in opposite ways to G-CSF, and the skull does not increase erythropoiesis after haemorrhage. Our studies enable in situ analyses of haematopoiesis, define the anatomy of normal and stress responses, identify discrete microanatomical production sites that confer plasticity to haematopoiesis, and uncover unprecedented heterogeneity of stress responses across the skeleton.

Identification of candidate genes affecting the tibia quality in Nonghua duck.

The skeleton is a vital organ providing structural support in poultry. Weakness in bone structure can lead to deformities, osteoporosis, cage fatigue, and fractures, resulting in economic losses. Research has substantiated that genetic factors play a significant role in influencing bone quality. The discovery of genetic markers associated with bone quality holds paramount importance for enhancing genetic traits related to the skeletal system in poultry. This study analyzed nine phenotypic indicators of tibia quality in 120-day-old ducks. The phenotypic correlation revealed a high correlation among diameter, Perimeter, and weight (0.69-0.78), and a strong correlation was observed between toughness and breaking strength (0.62). Then, we conducted a genome-wide association analysis of the phenotypic indicators to elucidate the genetic basis of tibial quality in Nonghua ducks. Among the 11 candidate genes that were annotated, TAPT1, BST1, and STIM2 were related to the diameter indicator, ZNF652, IGF2BP1, CASK, and GREB1L were associated with the weight and toughness indicators. RFX8, GLP1R, and DNAAF5 were identified for ash, calcium, and phosphorus content, respectively. Finally, KEGG and GO analysis for annotated genes were performed. STIM2 and BST1 were enriched into the Calcium signalling pathway and Niacin and nicotinamide metabolic pathway, which may be key candidate genes affecting bone quality phenotypes. Gene expression analysis of the candidate genes, such as STIM2, BST1, TAPT1, and CASK showed higher expression levels in bones compared to other tissues. The obtained results can contribute to new insights into tibial quality and provide new genetic biomarkers that can be employed in duck breeding.

Reduction of the trans-cortical vessel was associated with bone loss, another underlying mechanism of osteoporosis.

RATIONALE: Numerous studies have established a robust association between bone morrow microvascular diseases and osteoporosis. This study sought to investigate the relationship between alterations in trans-cortical vessel (TCVs) and the onset of osteoporosis in various mouse models. METHODS: Aged mice, ovariectomized mice, and db/db mice, were utilized as osteoporosis models. TCVs in the tibia were detected using tissue clearing and light sheet fluorescence microscopy imaging. Femurs bone mass were analyzed using micro-CT scanning. Correlations between the number of TCVs and bone mass were analyzed using Pearson correlation analysis. RESULTS: All osteoporosis mouse models showed a significant reduction in the number of TCVs compared to the control group. Correlation analysis revealed a positive association between the number of TCVs and bone mass. TCVs were also expressed high levels of CD31 and EMCN proteins as type H vessels. CONCLUSIONS: This study underscores a consistent correlation between the number of TCVs and bone mass. Moreover, TCVs may serve as a potential biomarker for bone mass evaluation.

miR-181b-1-3p affects the proliferation and differentiation of chondrocytes in TD broilers through the WIF1/Wnt/ß-catenin pathway.

Thiram is a plant fungicide, its excessive use has exceeded the required environmental standards. It causes tibial dyschondroplasia (TD) in broilers which is a common metabolic disease that affects the growth plate of tibia bone. It has been studied that many microRNAs (miRNAs) are involved in the differentiation of chondrocytes however, their specific roles and mechanisms have not been fully investigated. The selected features of tibial chondrocytes of broilers were studied in this experiment which included the expression of miR-181b-1-3p and the genes related to WIF1/Wnt/ß-catenin pathway in chondrocytes through qRT-PCR, western blot and immunofluorescence. The correlation between miR-181b-1-3p and WIF1 was determined by dual luciferase reporter gene assay whereas, the role of miR-181b-1-3p and WIF1/Wnt/ß-catenin in chondrocyte differentiation was determined by mimics and inhibitor transfection experiments. Results revealed that thiram exposure resulted in decreased expression of miR-181b-1-3p and increased expression of WIF1 in chondrocytes. A negative correlation was also observed between miR-181b-1-3p and WIF1. After overexpression of miR-181b-1-3p, the expression of ACAN, ß-catenin and Col2a1 increased but the expression of GSK-3ß decreased. It was observed that inhibition of WIF1 increased the expression of ALP, ß-catenin, Col2a1 and ACAN but decreased the expression of GSK-3ß. It is concluded that miR-181b-1-3p can reverse the inhibitory effect of thiram on cartilage proliferation and differentiation by inhibiting WIF1 expression and activating Wnt/ß-catenin signaling pathway. This study provides a new molecular target for the early diagnosis and possible treatment of TD in broilers.

Micellized protein transduction domain-bone morphogenetic protein-2 accelerates bone healing in a rat tibial distraction osteogenesis model.

The clinical application of growth factors such as recombinant human bone morphogenetic protein-2 (rh-BMP-2), for functional bone regeneration remains challenging due to limited in vivo efficacy and adverse effects of previous modalities. To overcome the instability and short half-life of rh-BMP-2 in vivo, we developed a novel osteogenic supplement by fusing a protein transduction domain (PTD) with BMP-2, effectively creating a prodrug of BMP-2. In this study, we first created an improved PTD-BMP-2 formulation using lipid nanoparticle (LNP) micellization, resulting in downsizing from micrometer to nanometer scale and achieving a more even distribution. The micellized PTD-BMP-2 (mPTD-BMP-2) demonstrated improved distribution and aggregation profiles. As a prodrug of BMP-2, mPTD-BMP-2 successfully activated Smad1/5/8 and induced mineralization with osteogenic gene induction in vitro. In vivo pharmacokinetic analysis revealed that mPTD-BMP-2 had a much more stable pharmacokinetic profile than rh-BMP-2, with a 7.5-fold longer half-life. The in vivo BMP-responsive element (BRE) reporter system was also successfully activated by mPTD-BMP-2. In the in vivo rat tibia distraction osteogenesis (DO) model, micro-computed tomography (micro-CT) scan findings indicated that mPTD-BMP-2 significantly increased bone volume, bone surface, axis moment of inertia (MOI), and polar MOI. Furthermore, it increased the expression of osteogenesis-related genes, and induced bone maturation histologically. Based on these findings, mPTD-BMP-2 could be a promising candidate for the next-generation osteogenesis drug to promote new bone formation in DO surgery. STATEMENT OF SIGNIFICANCE: This study introduces micellized bone morphogenetic protein-2 (mPTD-BMP-2), a next-generation osteogenic supplement that combines protein transduction domain (PTD) and nano-sized micelle formulation technique to improve transduction efficiency and stability. The use of PTD represents a novel approach, and our results demonstrate the superiority of mPTD-BMP-2 over rh-BMP-2 in terms of in vivo pharmacokinetic profile and osteogenic potential, particularly in a rat tibial model of distraction osteogenesis. These findings have significant scientific impact and potential clinical applications in the treatment of bone defects that require distraction osteogenesis. By advancing the field of osteogenic supplements, our study has the potential to contribute to the development of more effective treatments for musculoskeletal disorders.

Integrin expression and extracellular matrix adhesion of septoclasts, pericytes, and endothelial cells at the chondro-osseous junction and the metaphysis of the proximal tibia in young mice.

We previously reported that septoclasts, which are uncalcified growth plate (GP) cartilage matrix-resorbing cells, are derived from pericytes surrounding capillary endothelial cells. Resorption of the GP is assumed to be regulated synchronously by septoclasts, pericytes, and endothelial cells. To reveal the contribution of the extracellular matrix (ECM) to the regulatory mechanisms of septoclastic cartilage resorption, we investigated the spatial correlation between the cells and the ECM in the GP matrix and basement membrane (BM) and investigated the expression of integrins-ECM receptors-in the cells. Septoclasts attached to the transverse septa containing collagen-II/-X at the tip of their processes and to the longitudinal septa containing collagen-II/-X at the spine-like processes extending from their bodies and processes. Collagen-IV and laminin α4 in the BM were sparsely detected between septoclasts and capillary endothelial cells at the chondro-osseous junction (COJ) and were absent in the outer surface of pericytes at the metaphysis. Integrin α1/α2, integrin α1, and integrin α2/α6 were detected in the cell membranes of septoclasts, pericytes, and endothelial cells, respectively. These results suggest that the adhesion between septoclasts and the cartilage ECM forming the scaffolds for cartilage resorption and migration is provided by integrin α2-collagen-II/-X interaction and that the adhesions between the BM and pericytes or endothelial cells are mediated by integrin α1-collagen-IV and integrin α2/α6-laminin interaction, respectively.

Dietary Zinc Glycine Supplementation Improves Tibia Quality of Meat Ducks by Modulating the Intestinal Barrier and Bone Resorption.

Leg problems characterized by gait abnormity and bone structure destruction are associated with a high risk of fractures and continuous pain in poultry. Zinc (Zn) acts a pivotal part in normal bone homeostasis and has proven to be highly effective in alleviating leg problems. Therefore, the effects of graded concentration of Zn on bone quality were evaluated in this study. A total of 512 1-d-old male ducks were fed 4 basal diets added 30 mg/kg Zn, 60 mg/kg Zn, 90 mg/kg Zn, and 120 mg/kg Zn as Zn glycine for 35 d. Tibia Zn content, ash percentage, and breaking strength linearly increased with dietary elevated Zn level (P < 0.05). Broken-line analysis revealed that the recommended level of Zn from Zn glycine was 55.13 mg/kg and 64.48 mg/kg based on tibia ash and strength, respectively. To further confirm the role of dietary Zn glycine addition on bone characteristics, data from birds fed either 60 mg/kg Zn as Zn sulfate (ZnSO4), 30 mg/kg Zn, or 60 mg/kg Zn in the form of Zn glycine indicated that birds given 60 mg/kg Zn from Zn glycine diet exhibited higher tibia ash, strength, and trabecular volume compared to those fed the 30 mg/kg Zn diet (P < 0.05). Dietary 60 mg/kg Zn as Zn glycine addition decreased intestinal permeability, upregulated the mRNA expression of tight junction protein, and increased the abundance of Lactobacillus and Bifidobacterium, which was companied by declined the level of inflammatory cytokines in both the ileum and bone marrow. Regarding bone turnover, the diet with 60 mg/kg Zn from Zn glycine induced osteoprotegerin expression and thus decreased osteoclast number and serum bone resorption biomarker levels including serum tartrate-resistant acid phosphatase activity and C-terminal cross-linked telopeptide of type I collagen level when compared to 30 mg/kg Zn diet (P < 0.05). Except for the upregulation in runt-related transcription factor 2 transcription, the experimental treatments did not apparently change the bone formation biomarker contents in serum. Additionally, Zn glycine displayed a more efficient absorption rate, evidenced by higher serum Zn level, and thus had potentially greater a protective role in the intestine barrier and tibia mass as compared to ZnSO4. Collectively, the dietary supplementation of 60 mg/kg in the form of Zn glycine could suppress bone resorption mediated by osteoclast and consequently improve tibial quality of meat ducks, in which enhanced intestinal integrity and optimized gut microbiota might be involved.

High resolution intravital photoacoustic microscopy reveals VEGF-induced bone regeneration in mouse tibia.

Osteogenesis and angiogenesis are essential for bone homeostasis and repair. Newly formed vessels convey osteogenic progenitors during bone regeneration. However, the lack of continuous and label-free visualization of the bone microvasculature has resulted in little understanding of the neovascular dynamics. Here, we take advantage of optical-resolution photoacoustic microscopy (ORPAM) for label-free, intravital, long-term observation of the bone vascular dynamics, including angiogenesis, remodeling and quantified angiogenic effect of locally-applied vascular endothelial growth factor (VEGF) in the murine tibial defect model. We employed ex vivo confocal microscopy and micro-computed tomography (micro-CT) imaging to verify the positive role of VEGF treatment. VEGF treatment increased the concentration of total hemoglobin, vascular branching, and vascular density, which correlated with more osteoprogenitors and increased bone formation within the defect. These data demonstrated ORPAM as a useful imaging tool that detected functional capillaries to understand hemodynamics, and revealed the effectiveness of locally delivered therapeutic agents with sufficient sensitivity, contributing to the understanding of spatiotemporal regulatory mechanisms on blood vessels during bone regeneration.

Tubiechong patching promotes tibia fracture healing in rats by regulating angiogenesis through the VEGF/ERK1/2 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: The external use of traditional Chinese medicine (TCM) to treat fractures has a long history of clinical application and theoretical basis, and is also one of the characteristic treatment methods of TCM with significant efficacy and many advantages. Among the commonly used external Chinese medicines, Tubiechong is noteworthy. AIM OF THE STUDY: To elucidate whether local patching of Tubiechong can promote fracture healing and explore its mechanism of action. MATERIALS AND METHODS: A rat tibia fracture model was constructed by the modified Einhorn modeling method. X-ray films were taken to evaluate the progress of fracture healing. Serum bone alkaline phosphatase (BALP), osteocalcin (BGP) and the C-terminal content of collagen type I (CTX-I) were analyzed by ELISA. CD31 immunohistochemistry was used to evaluate angiogenesis in the tibia segment. The effects of Tubiechong decoction (TD) on HUVEC proliferation, migration and invasion were detected by MTT assay, wound healing assay and Transwell migration assay, respectively. RNA-seq was performed to identify differentially expressed genes (DEGs). Enrichment of functions and signaling pathway analysis were performed based on the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Quantitative real time polymerase chain reaction (qRT-PCR) was used to study gene expression levels. Western blotting (WB) was used to detect the expression of relevant regulatory proteins. RESULTS: The healing time of rat tibia fractures in the three TD dose groups was shortened. The serum levels of BALP, BGP and CTX- I in the TD-treated group were higher than those in the NC group. The X-ray results showed that on the 7th day after surgery, the fracture healing degree of the high-dose TD group was significantly better than that of the NC group, and the fracture healing degrees of each TD treatment group were significantly higher than those of the NC group on the 14th, 17th, and 21st days after the operation. The CD31 immunohistochemistry results showed that the number of blood vessels and the vascular area in the TD treatment group were higher than those in the NC group. In vitro, TD promoted the proliferation, wound healing and migration of HUVECs. GO analysis of transcriptome sequencing results showed that TD significantly altered the expression of genes related to cell growth, metabolism, and motility. According to KEGG annotations, VEGFA was upregulated. Eight DEGs were enriched in the VEGFA-VEGFR2 signaling pathway, of which six were upregulated. KEGG signaling pathway analysis showed that the most abundant DEGs were in mitogen-activated protein kinase (MAPK) signaling pathway. qRT-PCR showed that VEGFA gene expression in HUVECs was 7.8 times that of the control group after 1 mg/mL TD treatment for 24 h, and WB experiments showed that its protein expression was 3 times that of the control group. WB results showed that the phosphorylated ERK gene was highly expressed, while the expression levels of phosphorylated P38 and phosphorylated JNK protein remained unchanged. CONCLUSION: Tubechong patching therapy promotes tibia fracture healing in rats by regulating angiogenesis through the VEGF/ERK1/2 signaling pathway.

Molecular Identification of Spatially Distinct Anabolic Responses to Mechanical Loading in Murine Cortical Bone.

Osteoporosis affects over 200 million women worldwide, one-third of whom are predicted to suffer from an osteoporotic fracture in their lifetime. The most promising anabolic drugs involve administration of expensive antibodies. Because mechanical loading stimulates bone formation, our current data, using a mouse model, replicates the anabolic effects of loading in humans and may identify novel pathways amenable to oral treatment. Murine tibial compression produces axially varying deformations along the cortical bone, inducing highest strains at the mid-diaphysis and lowest at the metaphyseal shell. To test the hypothesis that load-induced transcriptomic responses at different axial locations of cortical bone would vary as a function of strain magnitude, we loaded the left tibias of 10-week-old female C57Bl/6 mice in vivo in compression, with contralateral limbs as controls. Animals were euthanized at 1, 3, or 24 hours post-loading or loaded for 1 week (n = 4-5/group). Bone marrow and cancellous bone were removed, cortical bone was segmented into the metaphyseal shell, proximal diaphysis, and mid-diaphysis, and load-induced differential gene expression and enriched biological processes were examined for the three segments. At each time point, the mid-diaphysis (highest strain) had the greatest transcriptomic response. Similarly, biological processes regulating bone formation and turnover increased earlier and to the greatest extent at the mid-diaphysis. Higher strain induced greater levels of osteoblast and osteocyte genes, whereas expression was lower in osteoclasts. Among the top differentially expressed genes at 24-hours post-loading, 17 had known functions in bone biology, of which 12 were present only in osteoblasts, 3 exclusively in osteoclasts, and 2 were present in both cell types. Based on these results, we conclude that murine tibial loading induces spatially unique transcriptomic responses correlating with strain magnitude in cortical bone. © 2022 American Society for Bone and Mineral Research (ASBMR).

HIF-1α upregulation exerts the antagonistic effect against angiogenesis inhibition in manganese deficiency-induced tibial dyschondroplasia of broiler chicks.

Manganese (Mn) is an essential microelement for broiler breeding and its deficiency causes tibial dyschondroplasia (TD). Tibial growth plate (TGP) development and metaphyseal vascularization are crucial for tibia growth in fast-growing broiler chickens, but their roles in Mn deficiency-induced TD in chicks remain unclear. This study was designed to clarify this issue. A total of 36 one-day-old broilers were divided into the control group and Mn-deficiency (Mn-D) group, which were fed with a standard diet (60 mg Mn/kg) and Mn deficiency diet (22 mg Mn/kg) for 42 days, respectively. TGP and proximal tibial metaphysis were collected to perform the related assays. This study found that Mn deficiency decreased the tibia length and TGP thickness in the TD model. Also, Mn deficiency increased the irregular and white tibial dyschondroplasia lesions (TDL) region under the TGP, and reduced the expression levels of vascular endothelial growth factor (VEGF) and macrophage migration inhibitory factor (MIF). Combined with histological assessment, it was suggested that Manganese deficiency inhibited angiogenesis in the proximal tibial metaphysis. Meanwhile, Mn deficiency enhanced the expression levels of hypoxia-inducible factor-1 α (HIF-1α), autophagy-related protein 5 (ATG5), and microtubule-associated protein 1 light chain 3 ß (LC3-II) in TGP, but decreased the expression level of SQSTM1 (P62), which suggested that autophagy was activated during this process. Collectively, these data indicate that HIF-1α up-regulation and concurrent autophagy activation exert a protective effect against Mn deficiency-induced angiogenesis inhibition, which may provide useful guidance to prevent TD in broilers.

Non-invasively Imageable Tibia-tumor-fragment Implantation Experimental-bone-metastasis Mouse Model of GFP-expressing Prostate Cancer.

BACKGROUND/AIM: Although the 5-year survival rate for localized prostate cancer is nearly 100%, prognosis for patients with metastases, of which the bone is the most common site, is poor. In order to evaluate efficacy of treatments against metastatic prostate cancer, experimental tibia-bone-metastasis mouse models of prostate cancer have been previously established. In the present study, we used a novel procedure for establishment of an experimental tibiabone metastasis mouse model, with human PC-3 prostate cancer expressing green fluorescent protein (GFP), that more closely matches prostate cancer growing in the bone. MATERIALS AND METHODS: PC-3 human prostate cancer cells, labeled with GFP, were initially subcutaneously injected into the flank of five male nude mice to obtain tumor tissues. Once the tumor tissue grew larger than 10 mm in diameter, the tumor tissue was harvested and minced into fragments of 1 mm3 A 1-mm hole was made in the proximal left tibia of eight male nude mice, using the tip of a 5-mm blade, and a tumor fragment was implanted into the hole for an exact fit. Tumor size was measured once a week, by non-invasive imaging of GFP fluorescence. The mice were sacrificed four weeks after tumor implantation. RESULTS: Tumors grew in 8 out of 8 mice (100%). All tumors were non-invasively detectable with GFP fluorescence, through the skin. Increased tumor growth in the tibia was observed every week. CONCLUSION: The establishment in the tibia of the novel experimental bone-metastatic mouse model of human prostate cancer enables facile screening, in a clinically-relevant system, of improved therapeutics for this recalcitrant disease.

Effect and Mechanism of Endothelin Receptor A Inhibitor BQ-123 Combined with Electroacupuncture on Tibia Cancer Pain in Rats.

Objective: To research the impact and mechanism of endothelin receptor A inhibitor BQ-123 combined with electroacupuncture on tibia cancer pain in rats. Methods: Sprague-Dawley (SD) rats were randomly divided into sham group (SHAM group) and bone cancer pain model group (BCP group). The behavior of SD rats was measured. The histology of the right tibia was observed by hematoxylin-eosin (HE) staining. The remaining rats were randomly divided into model, BQ-123, electroacupuncture, and BQ-123+ electroacupuncture group. Behavioral tests were performed, and mechanical pain threshold (MWT) and thermal pain threshold (TWL) were measured. The expressions of α-smooth muscle actin (αSMA), ETAR (endothelin A receptor), ETB (End of Transmission Block), P-Phosphatidylinositol 3-kinase (PI3K), and P-Protein kinase B (Akt) were detected by real-time fluorescence quantitative PCR and western blot. Results: In the BCP group, bone structure was severely damaged, local tissue swelling was obvious, bone trabecula was missing, and bone cortex was discontinuous. The optical density of Glial fibrillary acidic protein (GFAP) and CD11b immunoreactive signal in BCP group was significantly increased, and most of the ETAR of endothelin receptor was comapped with NeuN, and a small part of GFAP was comapped with CD11b, but no comapped with CD11b. The AS score of BQ-123+ electroacupuncture group was significantly lower than that of BQ-123 group and electroacupuncture group (P < 0.05), whereas the MWT and TWL values were significantly higher than that of the BQ-123 group and electroacupuncture group (P < 0.05). The mRNA expression of α-SMA and ETAR in BQ-123+ electroacupuncture group was lower than that in BQ-123 and electroacupuncture group, and the protein expression of P-PI3K and P-Akt in BQ-123+ electroacupuncture group was lower as well. Conclusion: BQ-123 may inhibit the activation of PI3K/Akt signal path combined with electroacupuncture to alleviate the effects of tibia cancer pain in rats.

Effects of a novel consensus bacterial 6-phytase variant on the apparent ileal digestibility of amino acids, total tract phosphorus retention, and tibia ash in young broilers.

The effect of a novel consensus bacterial 6-phytase variant (PhyG) on apparent ileal digestibility (AID) of amino acids (AA) and phosphorus (P) utilization in young broilers when added to diets with high phytate-P (PP) content without added inorganic phosphate (Pi) and deficient in digestible (dig) AA and metabolizable energy (ME) was investigated. A total of 256 Ross 308 male broilers were assigned to 4 treatments (8 birds/cage, 8 cages/treatment) in a completely randomized design. Treatments comprised a positive control (PC, 2,975 kcal/kg ME, 3.7 g/kg dig P, 2.83 g/kg PP, 8.4 g/kg Ca, 10.6 g/kg dig lysine), a negative control (NC) without added Pi (ME -68 kcal/kg, crude protein -10 g/kg, dig AA -0.1 to -0.4 g/kg, Ca -2.0 g/kg, dig P -2.2 g/kg, Na -0.4 g/kg vs. PC), and NC plus 500 or 1,000 FTU/kg of PhyG. Test diets were corn/soy/rapeseed-meal/rice-bran-based and fed from 5 to 15 d of age. Ileal digesta and tibias were collected on day 15. Excreta was collected during days 12 to 15 to determine P retention. The NC (vs. PC) reduced (P < 0.05) P retention (-10.4% units), tibia ash (-14.3% units), weight gain (-109 g), feed intake (-82 g) and increased FCR (from 1.199 to 1.504), confirming that the NC was extremely deficient in nutrients and energy. Phytase addition to the NC linearly (P < 0.001) improved performance, but did not fully recover it to the level of the PC due to the severe nutrients/energy reduction in NC. Phytase linearly increased P retention (P < 0.001), tibia ash (P < 0.001), AID of dry matter (P < 0.05), nitrogen (P < 0.01), gross energy (P < 0.05), and all 17 individual AA (P < 0.01). At 1,000 FTU/kg, phytase increased (P < 0.05) P retention vs. PC and NC (+14.5 and +24.9% units, respectively) and increased tibia ash vs. NC (+13.8% units), equivalent to PC. The NC decreased AID of Cys, Gly, Thr, and Met vs. PC (P < 0.05). At 1,000 FTU/kg, phytase increased AID of all 17 AA vs. NC (P < 0.01), equivalent to PC. At 1,000 FTU/kg, AID AA responses (above NC) ranged from +4.5% (Met) to +15.0% (Cys), being maximal for essential Thr (+10.4%) and Val (+8.2%) and non-essential Cys (+15.0%) and Gly (+10.4%). The results highlight the efficacy of PhyG at a dose level of 500 to 1,000 FTU/kg in young broilers for improving the ileal digestibility of nitrogen, AA, and energy alongside P retention and tibia ash. The performance data emphasize the need to consider digestible nutrient intake as a response variable in exogenous enzyme studies.

Microbial phytase is widely used in commercial broiler diets to improve digestion of phosphorus (P) and reduce its excretion into the environment. Phytase improves the digestion of phosphorus and other nutrients including amino acids (AA). This study evaluated the effect of a novel consensus bacterial 6-phytase variant (PhyG) added to a nutrient-reduced diet without any added inorganic P on the digestibility of nutrients including P and AA in the ileum of young broilers. Effects on P retention and bone mineralization were also assessed. Compared to an unsupplemented negative control diet, PhyG improved growth performance, P retention, bone mineralization (tibia ash), digestibility of dry matter, nitrogen, gross energy, and all 17 individual AA during 5 to 15 d post-hatch, in a dose-dependent manner (dose range 0 to 1,000 phytase units [FTU] per kilogram of feed). For some AA, the increases in digestibility with PhyG at 1,000 FTU/kg were substantial (cysteine: +15.0%, threonine:+10.4%), and for all AA were equivalent to the responses produced by a nutritionally adequate positive control (unsupplemented) diet. The results demonstrate the efficacy of PhyG to improve AA digestibility alongside growth performance, P retention, and bone mineralization in young broilers.

Lycopene Improves Bone Quality and Regulates AGE/RAGE/NF-кB Signaling Pathway in High-Fat Diet-Induced Obese Mice.

OBJECTIVE: This study was aimed at examining the effects of lycopene on bone metabolism in high-fat diet (HFD)- induced obese mice and to identify the potential underlying mechanisms. METHODS: Mice were fed a HFD for 12 weeks and then continue with or without lycopene intervention (15 mg/kg) for additional 10 weeks. The effects of lycopene on blood glucose and lipid metabolism, as well as serum levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and malondialdehyde (MDA) were determined by biochemical assays. Bone histomorphological features and osteoclast activity were assessed by hematoxylin/eosin and tartrate-resistant acid phosphatase staining. Bone microstructure at the proximal tibial metaphysis and diaphysis was determined by microcomputed tomography. Tibial biomechanical strength and material profiles were measured by a three-point bending assay and Fourier transform infrared spectroscopy. Protein expressions involved in the AGE/RAGE/NF-кB signaling pathway were determined by western blot and/or immunohistochemical staining. RESULTS: Lycopene consumption reduced body weight gain and improved blood glucose and lipid metabolism in HFD-induced obese mice. In addition, lycopene treatment preserved bone biomechanical strength, material profiles, and microarchitecture in obese mice. Moreover, these alterations were associated with an increase in serum levels of T-AOC and SOD, and a decline in serum levels of MDA, as well as a reduction of AGEs, RAGE, cathepsin K, and p-NF-кBp65 and NF-кBp65 expressions in the femurs and tibias of obese mice. CONCLUSION: Lycopene may improve bone quality through its antioxidant properties, which may be linked with the regulation of the AGE/RAGE/NF-кB signaling pathway in obese mice. These results suggest that lycopene consumption may be beneficial for the management of obesity-induced osteoporosis.

Moxifloxacin Concentrations in the Knee Joint, Tibial Bone, and Soft Tissue When Combined with Rifampicin: A Randomized Porcine Microdialysis Study.

BACKGROUND: Peri and postoperative antibiotics are key adjuvant treatment tools in the management of periprosthetic joint infection (PJI). The aim of this study was to evaluate the effect of rifampicin on the area under the moxifloxacin concentration-time curve from 0 to 24 hours (AUC0-24) in the synovial fluid of the knee joint, tibial bone, and adjacent subcutaneous tissue under steady-state conditions using microdialysis in a porcine model. METHODS: Twenty female pigs were randomized to receive oral treatment with moxifloxacin monotherapy (Group A, n = 10) of 400 mg once daily for 3 days or a combination therapy (Group B, n = 10) of 400 mg of moxifloxacin once daily for 3 days and 450 mg of rifampicin twice daily for 7 days. Microdialysis was used for sampling the synovial fluid of the knee joint, tibial cancellous and cortical bone, and adjacent subcutaneous tissues. Plasma samples were taken as a reference. Measurements were obtained for 24 hours. RESULTS: Coadministration of moxifloxacin and rifampicin resulted in reductions of the moxifloxacin AUC0-24 in all targeted tissue compartments by 67% to 85% (p < 0.05). The corresponding change in plasma was 20% (p = 0.49). For both groups, the tissue penetration (the ratio of tissue free fraction AUC0-24 to plasma free fraction AUC0-24 [fAUCtissue/fAUCplasma]) was incomplete in all investigated compartments. The highest moxifloxacin tissue penetration was in the knee joint synovial fluid: 0.59 (Group A) and 0.24 (Group B). The lowest tissue penetration was in the cortical bone: 0.17 (Group A) and 0.03 (Group B). CONCLUSIONS: We found a significant reduction of the moxifloxacin concentration, expressed as the AUC0-24, in tissues relevant to acute PJI treatment when coadministered with rifampicin. CLINICAL RELEVANCE: The concentrations within the targeted tissue compartments were reduced significantly more than the concentrations in plasma, which may be particularly important as plasma concentrations are used in clinical practice to assess moxifloxacin treatment sufficiency.

Enhanced Extracellular Matrix Degradation in Growth Plate Contributes to Manganese Deficiency-Induced Tibial Dyschondroplasia in Broiler Chicks.

Manganese (Mn) is a crucial trace element for poultry nutrition, and its deficiency compromises tibial cartilage development, leading to perosis and a higher incidence of slipped tendon. Tibial dyschondroplasia (TD) is a metabolic cartilage disease characterized by disruption of endochondral bone formation, which is closely related to extracellular matrix (ECM) degradation, in which Mn deficiency plays an important role. Previous studies have confirmed the role of matrix metalloproteinases (MMPs) in the pathogenesis of TD, but whether dysregulated ECM degradation and MMP expression profiles in growth plate are involved in Mn deficiency-induced avian TD has not been fully elucidated yet. Thus, this study was conducted to clarify these issues. Firstly, we successfully established TD model induced by Mn deficiency in broiler chicks. Mn deficiency decreased the number of chondrocytes, contents of proteoglycan, and type II collagen in tibial growth plate, demonstrating the tibial growth plate damage with enhanced ECM degradation. Also, Mn deficiency inhibited the Nrf2 signaling pathway and enhanced the protein levels of NLRP3, active caspase-1, and active IL-1ß in tibial growth plate, indicating the oxidative stress and inflammatory response in Mn deficiency-induced TD. Additionally, upregulated expression levels of MMPs (MMP1, 9, and 13) were observed in tibial growth plate of Mn deficiency group. In summary, these findings suggest that Mn deficiency-enhanced ECM degradation is involved in avian TD, which may be correlated with oxidative stress, inflammatory response, and upregulation of MMPs.

IL4/IL4R signaling promotes the osteolysis in metastatic bone of CRC through regulating the proliferation of osteoclast precursors.

BACKGROUND: Bone metastasis of colorectal cancer (CRC) often indicates a poor prognosis. Osteolysis can be observed in metastatic sites, implying an aberrant activation of osteoclasts. However, how osteoclastogenesis is regulated in metastatic microenvironment caused by colorectal cancer is still unclear. METHODS: In this study, mice bone metastatic model of CRC was established through injection of MC-38 or CT-26 cells. BrdU assays showed primary CD115 ( +) osteoclast precursors (OCPs) proliferated at the first 2 weeks. Transcriptomic profiling was performed to identify differentially expressing genes and pathways in OCPs indirectly co-cultured with CRC cells RESULTS: The expression of IL4Rα was found to be significantly upregulated in OCPs stimulated by tumor conditioned medium (CM). Further investigation indicated that IL-4 signaling regulated proliferation of OPCs through interacting with type I IL4 receptor, and neutrophils were the main source of IL-4 in bone marrow. The proliferation of OCPs can be inhibited in IL4 deficiency mice. In addition, ERK pathway was activated by IL4/IL4R signaling. Ravoxertinib, an ERK antagonists, could significantly prevent bone destruction through inhibiting the proliferation of OCPs. CONCLUSION: Our study indicates the essential role of IL4/IL4R signaling for the proliferation of OCPs in early metastasis of CRC predominantly through activating ERK pathway, which remarkedly impacts the number of osteoclasts in later stage and leads to osteolytic lesions. Moreover, Ravoxertinib could be a new therapeutical target for bone metastasis of CRC.

A Complex Evaluation of the In-Vivo Biocompatibility and Degradation of an Extruded ZnMgSr Absorbable Alloy Implanted into Rabbit Bones for 360 Days.

The increasing incidence of trauma in medicine brings with it new demands on the materials used for the surgical treatment of bone fractures. Titanium, its alloys, and steel are used worldwide in the treatment of skeletal injuries. These metallic materials, although inert, are often removed after the injured bone has healed. The second-stage procedure-the removal of the plates and screws-can overwhelm patients and overload healthcare systems. The development of suitable absorbable metallic materials would help us to overcome these issues. In this experimental study, we analyzed an extruded Zn-0.8Mg-0.2Sr (wt.%) alloy on a rabbit model. From this alloy we developed screws which were implanted into the rabbit tibia. After 120, 240, and 360 days, we tested the toxicity at the site of implantation and also within the vital organs: the liver, kidneys, and brain. The results were compared with a control group, implanted with a Ti-based screw and sacrificed after 360 days. The samples were analyzed using X-ray, micro-CT, and a scanning electron microscope. Chemical analysis revealed only small concentrations of zinc, strontium, and magnesium in the liver, kidneys, and brain. Histologically, the alloy was verified to possess very good biocompatibility after 360 days, without any signs of toxicity at the site of implantation. We did not observe raised levels of Sr, Zn, or Mg in any of the vital organs when compared with the Ti group at 360 days. The material was found to slowly degrade in vivo, forming solid corrosion products on its surface.

Smad2 and Smad3 expressed in skeletal muscle promote immobilization-induced bone atrophy in mice.

Skeletal muscle is known to regulate bone homeostasis through muscle-bone interaction, although factors that control this activity remain unclear. Here, we newly established Smad3-flox mice, and then generated skeletal muscle-specific Smad2/Smad3 double conditional knockout mice (DcKO) by crossing Smad3-flox with skeletal muscle-specific Ckmm Cre and Smad2-flox mice. We show that immobilization-induced gastrocnemius muscle atrophy occurring due to sciatic nerve denervation was partially but significantly inhibited in DcKO mice, suggesting that skeletal muscle cell-intrinsic Smad2/3 is required for immobilization-induced muscle atrophy. Also, tibial bone atrophy seen after sciatic nerve denervation was partially but significantly inhibited in DcKO mice. Bone formation rate in wild-type mouse tibia was significantly inhibited by immobilization, but inhibition was abrogated in DcKO mice. We propose that skeletal muscle regulates immobilization-induced bone atrophy via Smad2/3, and Smad2/3 represent potential therapeutic targets to prevent both immobilization-induced bone and muscle atrophy.