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.

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.

Depletion of b-series ganglioside prevents limb length discrepancy after growth plate injury.

INTRODUCTION: Growth plate damage in long bones often results in progressive skeletal growth imbalance and deformity, leading to significant physical problems. Gangliosides, key glycosphingolipids in cartilage, are notably abundant in articular cartilage and regulate chondrocyte homeostasis. This suggests their significant roles in regulating growth plate cartilage repair. METHODS: Chondrocytes from 3 to 5 day-old C57BL/6 mice underwent glycoblotting and mass spectrometry. Based on the results of the glycoblotting analysis, we employed GD3 synthase knockout mice (GD3-/-), which lack b-series gangliosides. In 3-week-old mice, physeal injuries were induced in the left tibiae, with right tibiae sham operated. Tibiae were analyzed at 5 weeks postoperatively for length and micro-CT for growth plate height and bone volume at injury sites. Tibial shortening ratio and bone mineral density were measured by micro-CT. RESULTS: Glycoblotting analysis indicated that b-series gangliosides were the most prevalent in physeal chondrocytes among ganglioside series. At 3 weeks, GD3-/- exhibited reduced tibial shortening (14.7 ± 0.2 mm) compared to WT (15.0 ± 0.1 mm, P = 0.03). By 5 weeks, the tibial lengths in GD3-/- (16.0 ± 0.4 mm) closely aligned with sham-operated lengths (P = 0.70). Micro-CT showed delayed physeal bridge formation in GD3-/-, with bone volume measuring 168.9 ± 5.8 HU at 3 weeks (WT: 180.2 ± 3.2 HU, P = 0.09), but normalizing by 5 weeks. CONCLUSION: This study highlights that GD3 synthase knockout mice inhibit physeal bridge formation after growth plate injury, proposing a new non-invasive approach for treating skeletal growth disorders.

Proteomic analysis of exosomal proteins associated with bone healing speed in a rat tibial fracture model.

This study investigates the impact of exosomes on bone fracture healing in a rat tibial model, distinguishing between fast and slow healing processes. Bone healing and protein expression were assessed through X-ray examinations, hematoxylin and eosin staining, and immunohistochemical staining. Exosomes were isolated, characterized and subjected to liquid chromatography-mass spectrometry for protein analysis. Molecular differences were explored using differentially expressed protein analysis, Kyoto Encyclopedia of Genes and Genomes pathway enrichment and protein-protein interaction networks. Differential bone healing patterns and protein expressions were observed between the control and model groups. Exosomes were successfully isolated and characterized, revealing 2004 identified proteins, including distinct expression profiles. Notably, ribosomal proteins, ferritin and beta-actin emerged as pivotal players in bone fracture healing. This study unveils dynamic changes in bone healing and underscores the role of exosomes in the process. Identified proteins and pathways offer valuable insights for developing innovative therapeutic strategies for bone healing.

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.

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.

Podoplanin is dispensable for mineralized tissue formation and maintenance in the Swiss outbred mouse background.

Podoplanin, PDPN, is a mucin-type transmembrane glycoprotein widely expressed in many tissues, including lung, kidney, lymph nodes, and mineralized tissues. Its function is critical for lymphatic formation, differentiation of type I alveolar epithelial lung cells, and for bone response to biomechanical loading. It has previously been shown that Pdpn null mice die at birth due to respiratory failure emphasizing the importance of Pdpn in alveolar lung development. During the course of generation of Pdpn mutant mice, we found that most Pdpn null mice in the 129S6 and C57BL6/J mixed genetic background die at the perinatal stage, similar to previously published studies with Pdpn null mice, while all Pdpn null mice bred with Swiss outbred mice survived. Surviving mutant mice in the 129S6 and C57BL6/J mixed genetic background showed alterations in the osteocyte lacunocanalicular network, especially reduced osteocyte canaliculi in the tibial cortex with increased tibial trabecular bone. However, adult Pdpn null mice in the Swiss outbred background showed no overt differences in their osteocyte lacunocnalicular network, bone density, and no overt differences when challenged with exercise. Together, these data suggest that genetic variations present in the Swiss outbred mice compensate for the loss of function of PDPN in lung, kidney, and bone.

Multipotential stromal cells in the talus and distal tibia in ankle osteoarthritis - Presence, potency and relationships to subchondral bone changes.

A large proportion of ankle osteoarthritis (OA) has an early onset and is post-traumatic. Surgical interventions have low patient satisfaction and relatively poor clinical outcome, whereas joint-preserving treatments, which rely on endogenous multipotential stromal cells (MSCs), result in suboptimal repair. This study investigates MSC presence and potency in OA-affected talocrural osteochondral tissue. Bone volume fraction (BV/TV) changes for the loading region trabecular volume and subchondral bone plate (SBP) thickness in OA compared with healthy tissue were investigated using microcomputed tomography. CD271-positive MSC topography was related to bone and cartilage damage in OA tissue, and in vitro MSC potency was compared with control healthy iliac crest (IC) MSCs. A 1.3- to 2.5-fold SBP thickening was found in both OA talus and tibia, whereas BV/TV changes were depth-dependent. MSCs were abundant in OA talus and tibia, with similar colony characteristics. Tibial and talar MSCs were tripotential, but talar MSCs had 10-fold lower adipogenesis and twofold higher chondrogenesis than IC MSCs (P = .01 for both). Cartilage damage in both OA tibia and talus correlated with SBP thickening and CD271+ MSCs was 1.4- to twofold more concentrated near the SBP. This work shows multipotential MSCs are present in OA talocrural subchondral bone, with their topography suggesting ongoing involvement in SBP thickening. Potentially, biomechanical stimulation could augment the chondrogenic differentiation of MSCs for joint-preserving treatments.

Arginine site 264 in murine estrogen receptor-α is dispensable for the regulation of the skeleton.

Mutation of arginine 264 in ERα has been shown to abrogate rapid membrane ERα-mediated endothelial effects. Our novel finding that mutation of R264 is dispensable for ERα-mediated skeletal effects supports the concept that R264 determines tissue specificity of ERα. Estrogen protects against bone loss but is not a suitable treatment due to adverse effects in other tissues. Therefore, increased knowledge regarding estrogen signaling in estrogen-responsive tissues is warranted to aid the development of bone-specific estrogen treatments. Estrogen receptor-α (ERα), the main mediator of estrogenic effects in bone, is widely subjected to posttranslational modifications (PTMs). In vitro studies have shown that methylation at site R260 in the human ERα affects receptor localization and intracellular signaling. The corresponding amino acid R264 in murine ERα has been shown to have a functional role in endothelium in vivo, although the methylation of R264 in the murine gene is yet to be empirically demonstrated. The aim of this study was to investigate whether R264 in ERα is involved in the regulation of the skeleton in vivo. Dual-energy X-ray absorptiometry (DEXA) analysis at 3, 6, 9, and 12 mo of age showed no differences in total body areal bone mineral density (BMD) between R264A and wild type (WT) in either female or male mice. Furthermore, analyses using computed tomography (CT) demonstrated that trabecular bone mass in tibia and vertebra and cortical thickness in tibia were similar between R264A and WT mice. In addition, R264A females displayed a normal estrogen treatment response in trabecular bone mass as well as in cortical thickness. Furthermore, uterus, thymus, and adipose tissue responded similarly in R264A and WT female mice after estrogen treatment. In conclusion, our novel finding that mutation of R264 in ERα does not affect the regulation of the skeleton, together with the known role of R264 for ERα-mediated endothelial effects, supports the concept that R264 determines tissue specificity of ERα.NEW & NOTEWORTHY Mutation of arginine 264 in ERα has been shown to abrogate rapid membrane ERα-mediated endothelial effects. Our novel finding that mutation of R264 is dispensable for ERα-mediated skeletal effects supports the concept that R264 determines tissue specificity of ERα.

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.

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.

Expression and enhancement of FABP4 in septoclasts of the growth plate in FABP5-deficient mouse tibiae.

In our previous study, fatty acid-binding protein 5 (FABP5) was expressed in septoclasts with long processes which are considered to resorb uncalcified matrix of the growth plate (GP) cartilage, and no apparent abnormalities were detected in the histo-architecture of the GP of FABP5-deficient (FABP5-/-) mice. Those finding lead us to hypothesize that another FABP can compensate the deletion of FABP5 in septoclasts of its gene-mutant mice. Based on the hypothesis, the present study examined the expression levels of several other FABPs in septoclasts and their morphology in FABP5-/- mouse tibiae. Processes of FABP5-/- septoclasts tend to be shorter than wild septoclasts. FABP4-positive septoclasts in FABP5-/- mice were more numerous than those cells in wild mice.Peroxisome proliferator-activated receptor (PPAR) γ was expressed in FABP4-positive septoclasts of FABP5-/- mice as well as mice administered with GW1929, a PPARγ agonist, suggesting that the occurrence of PPARγ induces an increase of FABP4-positive septoclasts. The present finding suggests that the functional exertion of FABP5 in septoclasts is supplemented by FABP4 in normal and FABP5-/- mice, and that the expression of FABP4 is up-regulated in accompany with PPARγ in FABP5-/- for maintenance of resorptive activity in the GP.

Assessment of quantitative [18F]Sodium fluoride PET measures of knee subchondral bone perfusion and mineralization in osteoarthritic and healthy subjects.

OBJECTIVE: Molecular information derived from dynamic [18F]sodium fluoride ([18F]NaF) PET imaging holds promise as a quantitative marker of bone metabolism. The objective of this work was to evaluate physiological mechanisms of [18F]NaF uptake in subchondral bone of individuals with and without knee osteoarthritis (OA). METHODS: Eleven healthy volunteers and twenty OA subjects were included. Both knees of all subjects were scanned simultaneously using a 3T hybrid PET/MRI system. MRI MOAKS assessment was performed to score the presence and size of osteophytes, bone marrow lesions, and cartilage lesions. Subchondral bone kinetic parameters of bone perfusion (K1), tracer extraction fraction, and total tracer uptake into bone (Ki) were evaluated using the Hawkins 3-compartment model. Measures were compared between structurally normal-appearing bone regions and those with structural findings. RESULTS: Mean and maximum SUV and kinetic parameters Ki, K1, and extraction fraction were significantly different between Healthy subjects and subjects with OA. Between-group differences in metabolic parameters were observed both in regions where the OA group had degenerative changes as well as in regions that appeared structurally normal. CONCLUSIONS: Results suggest that bone metabolism is altered in OA subjects, including bone regions with and without structural findings, compared to healthy subjects. Kinetic parameters of [18F]NaF uptake in subchondral bone show potential to quantitatively evaluate the role of bone physiology in OA initiation and progression. Objective measures of bone metabolism from [18F]NaF PET imaging can complement assessments of structural abnormalities observed on MRI.

FK506 ameliorates osteoporosis caused by osteoblast apoptosis via suppressing the activated CaN/NFAT pathway during oxidative stress.

OBJECTIVE: Osteoporosis is affecting the health of postmenopausal women in the world. In case of that, we explored whether FK-506 could ameliorate osteoporosis by inhibiting the activated CaN/NFAT pathway during oxidative stress. METHODS: First, the castrated rat model is constructed through the bilateral ovariectomy. Hologic Discovery (S/N 80347) dual-energy X-ray absorptiometry assessed bone mineral density (BMD) implemented at left femur of rats. Next, hematoxylin-eosin (H&E) staining observed and calculated the changes of bone trabecular, mean trabecular plate separation (Tb.Sp), mean trabecular plate thickness (Tb.Th), and bone volume fraction (BV/TV). Then, CCK-8 assay, TUNEL assay, ALP kit and alizarin red staining detected the viability, apoptosis, alkaline phosphatase (ALP) activity, and capacity of mineralization respectively. At last, commercially available kits detected the levels of ROS and SOD in transfected MC3T3-E1 cells and bone tissues, and Western blot analysis detected proteins related to apoptosis and CaN/NFAT pathway. RESULTS: FK-506 increased the BMD and changes of bone trabecular in female castrated rats. FK-506 inhibited the oxidative stress and apoptosis by suppressing the activated CaN/NFAT pathway. Low dose of FK-506 improved the viability, ALP activity, and mineralization capacity. What's more, it suppressed the apoptosis of H2O2-induced MC3T3-E1 cells, which was deteriorated by the high dose of FK-506. Briefly, low dose of FK-506 inhibited the oxidative stress by suppressing the activated CaN/NFAT pathway, while high dose of that further inhibited the oxidative stress by suppressing the CaN/NFAT pathway. CONCLUSION: FK-506 ameliorates osteoporosis resulted from osteoblastic apoptosis which caused by suppressing the activated CaN/NFAT pathway during oxidative stress.

Comparison of bone single-photon emission computed tomography (SPECT)/CT and bone scintigraphy in assessing knee joints.

BACKGROUND: This study attempted to compare the radiopharmaceutical uptake findings of planar bone scintigraphy (BS) and single photon emission computed tomography (SPECT)/computed tomography (CT) performed on knee joints. METHODS: We retrospectively included 104 patients who underwent bone SPECT/CT and BS 4 h after the intravenous administration of technetium-99m-hydroxymethylene diphosphonate (99mTc-HDP) for pain in the knee joint. The uptake degree of each of the knee regions (medial femoral, lateral femoral, medial tibial, lateral tibial, and patellar area) in planar images and SPECT/CT were evaluated by visual (grades 0 to 2) and quantitative analyses (uptake counts for planar image and standardized uptake values [SUVs] for SPECT/CT). RESULTS: The uptake grades assessed visually on the planar images differed significantly from the uptake grades on SPECT/CT images in all areas of the knee (all p < 0.001), and SPECT/CT imaging revealed a larger number of uptake lesions than those noted in planar imaging for each patient (3.3 ± 2.0 vs 2.4 ± 2.3, p < 0.0001). In all regions of the knee, all of the quantitative values, including uptake counts obtained from the planar image as well as the maximum SUV (SUVmax) and mean SUV (SUVmean) obtained from SPECT/CT, showed statistically higher values as their visual grades increased (all p < 0.001). However, when analyzed for each area, only the SUVmax showed a significant difference by grade in all knee regions. Quantitative uptake values obtained from planar images were moderately correlated with SUVs of SPECT/CT images (r = 0.58 for SUVmean and r = 0.53 for SUVmax, all p < 0.001) in the total knee regions. Looking at each area, there was a significant but low correlation between the uptake counts of the planar images and the SUVs on SPECT/CT in the right lateral tibial region (r = 0.45 for SUVmean, r = 0.31 for SUVmax, all p < 0.001). CONCLUSIONS: In assessing knee joints, the findings of planar images and SPECT/CT images differ both visually and quantitatively, and more lesions can be found in SPECT/CT than in the planar images. The SUVmax could be a reliable value to evaluate knee joint uptake activity.

Bilateral Looser zones or pseudofractures in the anteromedial tibia as a component of medial tibial stress syndrome in athletes.

PURPOSE: Medial tibial stress syndrome (MTSS) represents a common diagnosis in individuals exposed to repetitive high-stress loads affecting the lower limb, e.g., high-performance athletes. However, the diagnostic approach and therapeutic regimens are not well established. METHODS: Nine patients, diagnosed as MTSS, were analyzed by a comprehensive skeletal analysis including laboratory bone turnover parameters, dual-energy X-Ray absorptiometry (DXA), and high-resolution peripheral quantitative computed tomography (HR-pQCT). RESULTS: In 4/9 patients, bilateral pseudofractures were detected in the mid-shaft tibia. These patients had significantly lower levels of 25-hydroxycholecalciferol compared to patients with MTSS but similar levels of bone turnover parameters. Interestingly, the skeletal assessment revealed significantly higher bone mineral density (BMD) Z-scores at the hip (1.3 ± 0.6 vs. - 0.7 ± 0.5, p = 0.013) in patients with pseudofractures and a trend towards higher bone microarchitecture parameters measured by HR-pQCT at the distal tibia. Vitamin D supplementation restored the calcium-homeostasis in all patients. Combined with weight-bearing as tolerated, pseudofractures healed in all patients and return to competition was achieved. CONCLUSION: In conclusion, deficient vitamin D levels may lead to pseudofractures due to localized deterioration of mineralization, representing a pivotal component of MTSS in athletes with increased repetitive mechanical loading of the lower limbs. Moreover, the manifestation of pseudofractures is not a consequence of an altered BMD nor microarchitecture but appears in patients with exercise-induced BMD increase in combination with reduced 25-OH-D levels. The screening of MTSS patients for pseudofractures is crucial for the initiation of an appropriate treatment such as vitamin D supplementation to prevent a prolonged course of healing or recurrence. LEVEL OF EVIDENCE: III.

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.

Crossfibrillar mineral tessellation in normal and Hyp mouse bone as revealed by 3D FIB-SEM microscopy.

In bone, structural components such as mineral extend across length scales to provide essential biomechanical functions. Using X-ray micro-computed tomography (µCT), and focused ion beam scanning electron microscopy (FIB-SEM) in serial-surface-view mode, together with 3D reconstruction, entire mouse skeletons and small bone tissue volumes were examined in normal wildtype (WT) and mutant Hyp mice (an animal model for X-linked hypophosphatemia/XLH, a disease with severe hypomineralization of bone). 3D thickness maps of the skeletons showed pronounced irregular thickening and abnormalities of many skeletal elements in Hyp mice compared to WT mice. At the micro- and nanoscale, near the mineralization front in WT tibial bone volumes, mineralization foci grow as expanding prolate ellipsoids (tesselles) to abut and pack against one another to form a congruent and contiguous mineral tessellation pattern within collagen bundles that contributes to lamellar periodicity. In the osteomalacic Hyp mouse bone, mineralization foci form and begin initial ellipsoid growth within normally organized collagen assembly, but their growth trajectory aborts. Mineralization-inhibiting events in XLH/Hyp (low circulating serum phosphate, and increased matrix osteopontin) combine to result in decreased mineral ellipsoid tessellation - a defective mineral-packing organization that leaves discrete mineral volumes isolated in the extracellular matrix such that ellipsoid packing/tessellation is not achieved. Such a severely altered mineralization pattern invariably leads to abnormal compliance, other aberrant biomechanical properties, and altered remodeling of bone, all of which indubitably lead to macroscopic bone deformities and anomalous mechanical performance in XLH/Hyp. Also, we show the relationship of osteocytes and their cell processes to this mineralization pattern.

A histological study of the medial meniscus posterior root tibial insertion.

Purpose/Aim of the study: Posterior root injury of the medial meniscus often leads to articular cartilage degeneration due to altered biomechanics. To avoid dysfunction, the attachment must be repaired using the transtibial pullout technique. To guide appropriate placement of the tibial tunnel, additional details on the normal anatomy of the meniscus insertion are needed. Therefore, we performed a histological analysis of a tibial bone slice with the medial meniscus posterior insertion obtained during total knee arthroplasty surgery. Materials and methods: Horizontal slices of the proximal tibia were obtained from 7 patients with osteoarthritis who underwent total knee arthroplasty. After decalcification, the region of the posterior horn was cut out and segmented into four pieces (2.0 mm thickness; medial to lateral). Sagittal sections were evaluated by safranin O staining or immunohistochemistry with anti-type collagen antibody. Results: Safranin O staining showed that the insertion of the posterior root consisted primarily of fibrocartilaginous layers in segment 2. Anatomically, segment 2 corresponded to the sagittal plane passing through the peak of the medial intercondylar tubercle. In this section, safranin O staining and immunohistochemistry revealed that the anterior one-third of the posterior root insertion was richer in proteoglycans and type II collagen than the central and posterior one-third. Conclusions: Anatomical insertion of the posterior root of the medial meniscus was located at the sagittal plane passing through the peak of the medial intercondylar tubercle. The structure of the medial meniscus posterior insertion was mainly localized in the anterior one-third.

Mechanosensitive Ca2+ signaling and coordination is diminished in osteocytes of aged mice during ex vivo tibial loading.

Purpose: The osteocyte is considered the major mechanosensor in bone, capable of detecting forces at a cellular level to coordinate bone formation and resorption. The pathology of age-related bone loss, a hallmark of osteoporosis, is attributed in part to impaired osteocyte mechanosensing. However, real-time evidence of the effect of aging on osteocyte responses to mechanical load is lacking. Intracellular calcium (Ca2+) oscillations have been characterized as an early mechanosensitive response in osteocytes in systems of multiple scales and thus can serve as a real-time measure of osteocyte mechanosensitivity. Our objective was to utilize an ex vivo model to investigate potentially altered mechanosensing in the osteocyte network with aging.Methods: Tibiae were explanted from young-adult (5 mo) and aged (22 mo) female mice and incubated with Fluo-8 AM to visualize osteocyte intracellular Ca2+. Whole tibiae were cyclically loaded while in situ osteocyte Ca2+ dynamics were simultaneously imaged with confocal microscopy. Responsive osteocyte percentage and Ca2+ peak characteristics were quantified, as well as signaling synchrony between paired cells in the field of view.Results: Fewer osteocytes responded to mechanical loading in aged mice compared to young-adult and did so in a delayed manner. Osteocytes from aged mice also lacked the well-correlated relationship between Ca2+ signaling synchrony and cell-cell distance exhibited by young-adult osteocytes.Conclusions: We have demonstrated, for the first time, real-time evidence of the diminished mechanosensing and lack of signaling coordination in aged osteocyte networks in tibial explants, which may contribute to pathology of age-induced bone loss.