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.

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.

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.

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.

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.

Intravital Imaging of Bone Marrow Microenvironment in the Mouse Calvaria and Tibia.

The complex bone marrow microenvironment or niche is an important anatomical structure responsible for hematopoiesis and providing support to the immune cells function. Being the source of immune and blood cells, the interaction of these hematopoietic stem and progenitor cells with the cellular niches regulates their ability for self-renewal, proliferation, and differentiation. Dynamic imaging not only provides spatiotemporal information of cell motility but also the morphological changes due to cell-cell interactions in the bone marrow, providing insights into the ongoing physiological activities within the tissue. Here, we describe customized stages with compatible equipment best suited for the upright two-photon microscope, accompanied by detailed methods for both calvarial and tibial intravital imaging. We demonstrate a general protocol for calvarial imaging using a minimally invasive surgical approach, and introduce a bone shaving-based tibial imaging as a complementary method. To demonstrate the applicability of our method we used Lyz2-EGFP transgenic mice to track bone marrow neutrophil activities as an example.

Total flavonoids of rhizoma drynariae ameliorates bone formation and mineralization in BMP-Smad signaling pathway induced large tibial defect rats.

Osteogenesis and angiogenesis acts as an essential role in repairing large tibial defects (LTDs). Total flavonoids of rhizoma drynariae (TFRD), a traditional Chinese medicinal herb, is reported to show anabolic effects on fracture healing. However, whether TFRD could improve the bone formation and angiogenesis in LTDs remains unknown. The purpose of this study was to evaluate the effect of TFRD on bone formation and angiogenesis in LTDs in distraction osteogenesis (DO). Using a previously established fracture model, LTD rats was established with circular external fixator (CEF). All rats then randomly divided into TFRD low dosage group (with DO), TFRD medium dosage group (with DO), TFRD high dosage group (with DO), model group (with DO) and blank group (without DO). Twelve weeks after treatment, according to X-ray and Micro-CT, TFRD groups (especially in medium dosage group) can significantly promote the formation of a large number of epiphyses and improve new bone mineralization compared with model group, and the results of HE and Masson staining and in vitro ALP level of BMSC also demonstrated the formation of bone matrix and mineralization in the TFRD groups. Also, angiographic imaging suggested that total flavonoids of TFRD was able to promote angiogenesis in the defect area. Consistently, TFRD significantly increased the levels of BMP-2, SMAD1, SMAD4, RUNX-2, OSX and VEGF in LTD rats based on ELISA and Real-Time PCR. In addition, we found that ALP activity of TFRD medium dosage group reached a peak after 10 days of induction through BMSC cell culture in vitro experiment. TFRD promoted bone formation in LTD through activation of BMP-Smad signaling pathway, which provides a promising new strategy for repairing bone defects in DO surgeries.

Janus kinase/signal transducer and activator of transcription signaling pathway-related genes STAT3, SOCS3 and their role in thiram induced tibial dyschondroplasia chickens.

Pathogenicity of tibial dyschondroplasia (TD) in broiler chickens is not detected yet. Janus Kinase/Signal Transducer and Activator of Transcription (JAK-STAT) signaling pathway-related genes were investigated in thiram induced TD chickens. Real-time qPCR and immunohistochemical (IHC) technique were used to observe the expression changes of STAT3 and SOSC3 gene on days 1, 2, 4, 6 after feeding 100 mg·kg-1 thiram. Morphological, pathological, and histological results of this study suggested that chondrocyte cells were observed more damaged on day 6 than day 1, 2, and 4. Therefore, Lameness and damaged chondrocytes gradually increased from day 1 to 6. The mRNA expression level of STAT3 was observed insignificant (P > 0.05) in thiram induced TD chickens' group of day 1. However, on days 2, 4, and 6, the expression was significant (P < 0.05). SOCS3 increased in thiram group on days 1, 2 and 6, decreased on day 4 (P < 0.05). The p-STAT3 and SOCS3 protein's protein localization was evaluated in the control and thiram-induced TD broiler chickens through IHC, suggesting that SOSC3 protein was observed significantly higher on days 1, 2, and 6 and down-regulated on day 4. p-STAT3 protein on thiram induced group was observed significantly upregulated on days 4 and 6. In conclusion, the differential expression of STAT3 and SOCS3 showed that the JAK-STAT signaling pathway might play an important role in regulating an abnormal proliferation, differentiation, or apoptosis of chondrocytes in TD at an early stage.

Intramedullary injury combined with osteoporosis therapeutics regulates targeted local osteogenesis.

Bone marrow ablation prompts transient bone formation in nearly the entire medullary cavity before marrow regeneration occurs. Here, we establish a procedure to direct bone formation in a desired particular site within the medullary cavity for support of biomedical devices. Local intramedullary injury was performed in the tibiae of rats and parathyroid hormone (PTH), alendronate, or saline was administered. Newly generated bone in the medulla was assessed by micro-CT and histology. To evaluate the function of newly generated bone, animals received intramedullary injury in tibiae followed by daily PTH. At day-14, implants were placed in the endocortical bone and the bone response to the implants was assessed. The fate of newly generated bone was compared with and without implants. We found that neither intramedullary injury nor medication alone resulted in bone formation. However, when combined, substantial bone was generated locally inside the diaphyseal medulla. Newly formed bone disappeared without implant placement but was retained with implants. Bone was especially retained around and between the implants. This study found that local bone marrow disruption followed by PTH or alendronate generated substantial cancellous bone locally in the diaphyseal medulla. This approach offers promise as a tissue engineering tool in medicine and dentistry.

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.

A Novel Orthotopic Mouse Model of Lung Metastasis Using Fluorescent Patient-derived Osteosarcoma Cells.

BACKGROUND: A mouse model of metastatic osteosarcoma is imperative to identify effective agents for metastatic osteosarcoma, which is a recalcitrant disease. In the present study, we established osteosarcoma patient-derived cells (OS-PDCs) and transfected them with green fluorescent protein (GFP). MATERIALS AND METHODS: The OS-PDCs were transfected with GFP-lentivirus. GFP-expressing OS-PDCs (2.0×105) were then injected into the tibia of nude mice to establish the patient-derived orthotopic cell (PDOC) model (n=3). Six weeks after injection, the primary tumor and each organ were resected and imaged. RESULTS: Primary orthotopic tumors were established in two out of three mice. The GFP-expressing OS-PDCs in the PDOC model were visualized. Multiple GFP-expressing lung metastases were detected in one of the two mice with primary tumor. CONCLUSION: The present study proves the concept that a GFP-expressing PDOC model can mimic clinical lung-metastatic osteosarcoma. This model can serve as a paradigm to screen for effective drugs for osteosarcoma lung metastasis.

Knee Joint Distraction Compared with High Tibial Osteotomy and Total Knee Arthroplasty: Two-Year Clinical, Radiographic, and Biochemical Marker Outcomes of Two Randomized Controlled Trials.

OBJECTIVE: Both, knee joint distraction (KJD) and high tibial osteotomy (HTO) are joint-preserving surgeries that postpone total knee arthroplasty (TKA) in younger osteoarthritis (OA) patients. Here we evaluate the 2-year follow-up of KJD versus TKA and KJD versus HTO in 2 noninferiority studies. DESIGN: Knee OA patients indicated for TKA were randomized to KJD (n = 20; KJDTKA) or TKA (n = 40). Medial compartmental knee OA patients considered for HTO were randomized to KJD (n = 23; KJDHTO) or HTO (n = 46). Patient-reported outcome measures were assessed over 2 years of follow-up. The radiographic joint space width (JSW) was measured yearly. In the KJD groups, serum-PIIANP and urinary-CTXII levels were measured as collagen type-II synthesis and breakdown markers. It was hypothesized that there was no clinically important difference in the primary outcome, the total WOMAC, when comparing KJD with HTO and with TKA. RESULTS: Both trials were completed, with 114 patients (19 KJDTKA; 34 TKA; 20 KJDHTO; 41 HTO) available for 2-year analyses. At 2 years, the total WOMAC score (KJDTKA: +30.4 [95% CI 23.0-37.9] points; TKA: +42.4 [95% CI 38.1-46.8]; KJDHTO: +21.6 [95% CI 13.8-29.4]; HTO: +29.2 [95% CI 23.6-34.8]; all: P < 0.05) and radiographic minimum JSW (KJDTKA: +0.9 [95% CI 0.2-1.6] mm; KJDHTO: +0.9 [95% CI 0.5-1.4]; HTO: +0.6 [95% CI 0.3-0.9]; all: P < 0.05) were still increased for all groups. The net collagen type-II synthesis 2 years after KJD was increased (P < 0.05). Half of KJD patients experienced pin tract infections, successfully treated with oral antibiotics. CONCLUSIONS: Sustained improvement of clinical benefit and (hyaline) cartilage thickness increase after KJD is demonstrated. KJD was clinically noninferior to HTO and TKA in the primary outcome.

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.

Inhibition of the activin receptor signaling pathway: A novel intervention against osteosarcoma.

Osteosarcoma is a cancer of pathological bone remodeling with high mortality and severe comorbidity. New therapies are urgently needed. Activin A, a member of the transforming growth factor ß (TGFß) superfamily, has been suggested to stimulate proliferation and invasion of osteosarcoma cells in vitro, thus representing a potential therapeutic target. In this study, inhibition of the activin receptor signaling pathway was explored as a therapy for osteosarcoma. In a murine intratibial osteosarcoma xenograft model, two types of inhibitors were tested: (a) a soluble activin type IIA decoy receptor (ActRIIA-mFc), or (b) a modified variant of follistatin (FSTΔHBS -hFc), either alone or in combination with a bisphosphonate. Both inhibitors reduced primary tumor development by nearly 50% compared to vehicle treatment. When ActRIIA-mFc was combined with bisphosphonate, the effect on tumor size became even more pronounced (78% reduction vs. vehicle). Moreover, FSTΔHBS -hFc increased body weight in the face of tumor progression (14% increase vs. vehicle), and ActRIIA-mFc reduced the number of lung metastases when combined with bisphosphonate. The present study demonstrates a novel approach to treating osteosarcoma and encourages further investigation of inhibition of the activin receptor signaling pathway as an intervention against the disease.