Preclinical models for studying corticosteroid-induced osteonecrosis of the femoral head.

Nontraumatic osteonecrosis of the femoral head (ONFH) is a refractory condition that commonly results in femoral head collapse and degenerative arthritis of the hip. In the early stages, surgical procedures for hip preservation, including core decompression (CD), have been developed to prevent progressive collapse of the femoral head. Optimization of bone regeneration and biological augmentation may further enhance the therapeutic efficacy of CD for ONFH. Thus, combining CD with cell-based therapy has recently been proposed. In fact, patients treated with cell-based therapy using autologous bone marrow concentrate demonstrate improved survivorship of the femoral head, compared with conventional CD alone. Preclinical research studies to investigate adjunctive therapies for CD often utilize the rabbit model of corticosteroid-induced ONFH. Mesenchymal stem cells (MSCs) are known to promote osteogenesis and angiogenesis, and decrease inflammation in bone. Local drug delivery systems have the potential to achieve targeted therapeutic effects by precisely controlling the drug release rate. Scaffolds can provide an osteoconductive structural framework to facilitate the repair of osteonecrotic bone tissue. We focused on the combination of both cell-based and scaffold-based therapies for bone tissue regeneration in ONFH. We hypothesized that combining CD and osteoconductive scaffolds would provide mechanical strength and structural cell guidance; and that combining CD and genetically modified (GM) MSCs to express relevant cytokines, chemokines, and growth factors would promote bone tissue repair. We developed GM MSCs that overexpress the anti-inflammatory, pro-reconstructive cytokines platelet-derived growth factor-BB to provide MSCs with additional benefits and investigated the efficacy of combinations of these GM MSCs and scaffolds for treatment of ONFH in skeletally mature male New Zealand white rabbits. In the future, the long-term safety, efficacy, durability, and cost-effectiveness of these and other biological and mechanical treatments must be demonstrated for the patients affected by ONFH.

Autophagy modulates the stability of Wee1 and cell cycle G2/M transition.

The mammalian cell cycle is divided into four sequential phases, namely G1 (Gap 1), S (synthesis), G2 (Gap 2), and M (mitosis). Wee1, whose turnover is tightly and finely regulated, is a well-known kinase serving as a gatekeeper for the G2/M transition. However, the mechanism underlying the turnover of Wee1 is not fully understood. Autophagy, a highly conserved cellular process, maintains cellular homeostasis by eliminating intracellular aggregations, damaged organelles, and individual proteins. In the present study, we found autophagy deficiency in mouse liver caused G2/M arrest in two mouse models, namely Fip200 and Atg7 liver-specific knockout mice. To uncover the link between autophagy deficiency and G2/M transition, we combined transcriptomic and proteomic analysis for liver samples from control and Atg7 liver-specific knockout mice. The data suggest that the inhibition of autophagy increases the protein level of Wee1 without any alteration of its mRNA abundance. Serum starvation, an autophagy stimulus, downregulates the protein level of Wee1 in vitro. In addition, the half-life of Wee1 is extended by the addition of chloroquine, an autophagy inhibitor. LC3, a central autophagic protein functioning in autophagy substrate selection and autophagosome biogenesis, interacts with Wee1 as assessed by co-immunoprecipitation assay. Furthermore, overexpression of Wee1 leads to G2/M arrest both in vitro and in vivo. Collectively, our data indicate that autophagy could degrade Wee1-a gatekeeper of the G2/M transition, whereas the inhibition of autophagy leads to the accumulation of Wee1 and causes G2/M arrest in mouse liver.

An osteoinductive and biodegradable intramedullary implant accelerates bone healing and mitigates complications of bone transport in male rats.

Bone transport is a surgery-driven procedure for the treatment of large bone defects. However, challenging complications include prolonged consolidation, docking site nonunion and pin tract infection. Here, we develop an osteoinductive and biodegradable intramedullary implant by a hybrid tissue engineering construct technique to enable sustained delivery of bone morphogenetic protein-2 as an adjunctive therapy. In a male rat bone transport model, the eluting bone morphogenetic protein-2 from the implants accelerates bone formation and remodeling, leading to early bony fusion as shown by imaging, mechanical testing, histological analysis, and microarray assays. Moreover, no pin tract infection but tight osseointegration are observed. In contrast, conventional treatments show higher proportion of docking site nonunion and pin tract infection. The findings of this study demonstrate that the novel intramedullary implant holds great promise for advancing bone transport techniques by promoting bone regeneration and reducing complications in the treatment of bone defects.

Metabolic profile of mesenchymal stromal cells and macrophages in the presence of polyethylene particles in a 3D model.

BACKGROUND: Continuous cross talk between MSCs and macrophages is integral to acute and chronic inflammation resulting from contaminated polyethylene particles (cPE); however, the effect of this inflammatory microenvironment on mitochondrial metabolism has not been fully elucidated. We hypothesized that (a) exposure to cPE leads to impaired mitochondrial metabolism and glycolytic reprogramming and (b) macrophages play a key role in this pathway. METHODS: We cultured MSCs with/without uncommitted M0 macrophages, with/without cPE in 3-dimensional gelatin methacrylate (3D GelMA) constructs/scaffolds. We evaluated mitochondrial function (membrane potential and reactive oxygen species-ROS production), metabolic pathways for adenosine triphosphate (ATP) production (glycolysis or oxidative phosphorylation) and response to stress mechanisms. We also studied macrophage polarization toward the pro-inflammatory M1 or the anti-inflammatory M2 phenotype and the osteogenic differentiation of MSCs. RESULTS: Exposure to cPE impaired mitochondrial metabolism of MSCs; addition of M0 macrophages restored healthy mitochondrial function. Macrophages exposed to cPE-induced glycolytic reprogramming, but also initiated a response to this stress to restore mitochondrial biogenesis and homeostatic oxidative phosphorylation. Uncommitted M0 macrophages in coculture with MSC polarized to both M1 and M2 phenotypes. Osteogenesis was comparable among groups after 21 days. CONCLUSION: This work confirmed that cPE exposure triggers impaired mitochondrial metabolism and glycolytic reprogramming in a 3D coculture model of MSCs and macrophages and demonstrated that macrophages cocultured with MSCs undergo metabolic changes to maintain energy production and restore homeostatic metabolism.

EPHA3 Could Be a Novel Prognosis Biomarker and Correlates with Immune Infiltrates in Bladder Cancer.

PURPOSE: To assess the mechanism of EPH receptor A3 (EPHA3) and its potential value for immunotherapy in BLCA. MATERIALS AND METHODS: The Cancer Genome Atlas (TCGA) bladder cancer (BLCA) database and the Gene Expression Omnibus (GEO) database were used for assessing whether EHPA3 could be used to predict BLCA prognosis. This work carried out in vitro and in vivo assays for exploring how EPHA3 affected the biological behaviors. The downstream pathway was explored using a Western blotting technique. The CIBERSORT, ESTIMATE, TIMER, and TIDE tools were used to predict the immunotherapy value of EPHA3 in BLCA. RESULTS: EPHA3 was poorly expressed in BLCA (p < 0.05), its high expression is related to a good survival prognosis (p = 0.027 and p = 0.0275), and it has a good predictive ability for the histologic grade and status of BLCA (area under curve = 0.787 and 0.904). Overexpressed EPHA3 could inhibit BLCA cell biological behaviors, and it be associated with the downregulation of the Ras/pERK1/2 pathway. EPHA3 was correlated with several immune-infiltrating cells and the corresponding marker genes. CONCLUSIONS: EPHA3 could be regarded as an acceptable anti-cancer biomarker in BLCA. EPHA3 plays an inhibiting role in BLCA, and it could be the candidate immunotherapeutic target for BLCA.

The efficiency of genetically modified mesenchymal stromal cells combined with a functionally graded scaffold for bone regeneration in corticosteroid-induced osteonecrosis of the femoral head in rabbits.

Core decompression (CD) with mesenchymal stromal cells (MSCs) is an effective therapy for early-stage osteonecrosis of the femoral head (ONFH). Preconditioning of MSCs, using inflammatory mediators, is widely used in immunology and various cell therapies. We developed a three-dimensional printed functionally graded scaffold (FGS), made of ß-TCP and PCL, for cell delivery at a specific location. The present study examined the efficacy of CD treatments with genetically modified (GM) MSCs over-expressing PDGF-BB (PDGF-MSCs) or GM MSCs co-over-expressing IL-4 and PDGF-BB and preconditioned for three days of exposure to lipopolysaccharide and tumor necrosis factor-alpha (IL-4-PDGF-pMSCs) using the FGS for treating steroid-induced ONFH in rabbits. We compared CD without cell-therapy, with IL-4-PDGF-pMSCs alone, and with FGS loaded with PDGF-MSCs or IL-4-PDGF-pMSCs. For the area inside the CD, the bone volume in the CD alone was higher than in both FGS groups. The IL-4-PDGF-pMSCs alone and FGS + PDGF-MSCs reduced the occurrence of empty lacunae and improved osteoclastogenesis. There was no significant difference in angiogenesis among the four groups. The combined effect of GM MSCs or pMSCs and the FGS was not superior to the effect of each alone. To establish an important adjunctive therapy for CD for early ONFH in the future, it is necessary and essential to develop an FGS that delivers biologics appropriately and provides structural and mechanical support.

Growth and differentiation factor-7 immobilized, mechanically strong quadrol-hexamethylene diisocyanate-methacrylic anhydride polyurethane polymer for tendon repair and regeneration.

Biological and mechanical cues are both vital for biomaterial aided tendon repair and regeneration. Here, we fabricated mechanically tendon-like (0 s UV) QHM polyurethane scaffolds (Q: Quadrol, H: Hexamethylene diisocyanate; M: Methacrylic anhydride) and immobilized them with Growth and differentiation factor-7 (GDF-7) to produce mechanically strong and tenogenic scaffolds. In this study, we assessed QHM polymer cytocompatibility, amenability to fibrin-coating, immobilization and persistence of GDF-7, and capability to support GDF-7-mediated tendon differentiation in vitro as well as in vivo in mouse subcutaneous and acute rat rotator cuff tendon resection models. Cytocompatibility studies showed that QHM facilitated cell attachment, proliferation, and viability. Fibrin-coating and GDF-7 retention studies showed that mechanically tendon-like 0 s UV QHM polymer could be immobilized with GDF-7 and retained the growth factor (GF) for at least 1-week ex vivo. In vitro differentiation studies showed that GDF-7 mediated bone marrow-derived human mesenchymal stem cell (hMSC) tendon-like differentiation on 0 s UV QHM. Subcutaneous implantation of GDF-7-immobilized, fibrin-coated, QHM polymer in mice for 2 weeks demonstrated de novo formation of tendon-like tissue while implantation of GDF-7-immobilized, fibrin-coated, QHM polymer in a rat acute rotator cuff resection injury model indicated tendon-like tissue formation in situ and the absence of heterotopic ossification. Together, our work demonstrates a promising synthetic scaffold with human tendon-like biomechanical attributes as well as immobilized tenogenic GDF-7 for tendon repair and regeneration. STATEMENT OF SIGNIFICANCE: Biological activity and mechanical robustness are key features required for tendon-promoting biomaterials. While synthetic biomaterials can be mechanically robust, they often lack bioactivity. To biologically augment synthetic biomaterials, numerous drug and GF delivery strategies exist but the large tissue space within the shoulder is constantly flushed with saline during arthroscopic surgery, hindering efficacious controlled release of therapeutic molecules. Here, we coated QHM polymer (which exhibits human tendon-to-bone-like biomechanical attributes) with fibrin for GF binding. Unlike conventional drug delivery strategies, our approach utilizes immobilized GFs as opposed to released GFs for sustained, localized tissue regeneration. Our data demonstrated that GF immobilization can be broadly applied to synthetic biomaterials for enhancing bioactivity, and GDF-7-immobilized QHM exhibit high clinical translational potential for tendon repair.

Ubiquitination and degradation of MGMT by TRIM72 increases the sensitivity of uveal melanoma cells to Dacarbazine treatment.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults with high metastasis rates. The O6-methylguanine DNA methyl transferase (MGMT) is involved in chemoresistance of Dacarbazine (DTIC) treatment. Our previous study found that the combination of oncolytic adenovirus H101 and DTIC in the treatment of UM cells shows a synergistic antitumor effect mainly though down-regulation of MGMT. MGMT knockdown by shRNAs increases the sensitivity of uveal melanoma cells to DTIC treatment. The protein hemostasis of MGMT is important for the antitumor effect of DTIC. Tripartite motif-containing protein 72 (TRIM72) belongs to the tripartite motif (TRIM) proteins family and was identified as a novel E3 ligase for MGMT, which interacts with and mediates the ubiquitination of MGMT. TRIM72 knockdown increases the protein levels of MGMT, while reduces the ubiquitination of MGMT. Further study indicated that MGMT is highly expressed in UM cells, and the protein levels of MGMT and TRIM72 shows a negative correlation. UM cells that ectopically expressing TRIM72 shows increased sensitivity to DTIC treatment, which is consistent with the antitumor affect exhibited by H101. These results suggest that TRIM72 is a promising therapeutic target for UM treatment.

Probing the role of methyl methacrylate release from spacer materials in induced membrane bone healing.

In the induced membrane (IM) technique for bone reconstruction, a poly(methyl methacrylate) (PMMA) spacer is implanted to induce formation of a foreign body membrane around the defect site. Membrane development is essential for later bone grafting success, yet the mechanism by which the IM promotes bone regeneration remains unknown, as are the ways that spacer composition plays a role in the membrane's healing potential. This study investigated the impact of leached methyl methacrylate (MMA)-the major monomeric component of PMMA-on IM development. In vitro cell culture found that MMA elution did not impact endothelial cell or mesenchymal stem cell proliferation. For in vivo analysis, we advanced a streamlined rat femoral model to efficiently study the influence of spacer properties on IM characteristics. Comparison of membrane formation around polycaprolactone (PCL), MMA-eluting PCL (high-dose PCL-MMA and low-dose PCL-MMA), and surgical PMMA revealed robust membranes enveloped all groups after 4 weeks in vivo, with elevated expression of osteogenic bone morphogenetic protein-2 and angiogenic vascular endothelial growth factor compared with the surrounding muscle and bone tissues. Growth factor quantitation in IM tissue found no statistically significant difference between groups. New bone growth, vascularization, and CD163+ macrophage populations surrounding the polymer implants were also quantified; and blood vessel formation around high-dose PCL-MMA was found to be significantly decreased compared with PCL alone. To the best of our knowledge, these findings represent the first time that results have been obtained about the characteristics of membranes formed around PCL in the IM setting.

The effect of genetically modified platelet-derived growth factor-BB over-expressing mesenchymal stromal cells during core decompression for steroid-associated osteonecrosis of the femoral head in rabbits.

BACKGROUND: Approximately one third of patients undergoing core decompression (CD) for early-stage osteonecrosis of the femoral head (ONFH) experience progression of the disease, and subsequently require total hip arthroplasty (THA). Thus, identifying adjunctive treatments to optimize bone regeneration during CD is an unmet clinical need. Platelet-derived growth factor (PDGF)-BB plays a central role in cell growth and differentiation. The aim of this study was to characterize mesenchymal stromal cells (MSCs) that were genetically modified to overexpress PDGF-BB (PDGF-BB-MSCs) in vitro and evaluate their therapeutic effect when injected into the bone tunnel at the time of CD in an in vivo rabbit model of steroid-associated ONFH. METHODS: In vitro studies: Rabbit MSCs were transduced with a lentivirus vector carrying the human PDGF-BB gene under the control of either the cytomegalovirus (CMV) or phosphoglycerate (PGK) promoter. The proliferative rate, PDGF-BB expression level, and osteogenic differentiation capacity of unmodified MSCs, CMV-PDGF-BB-MSCs, and PGK-PDGF-BB-MSCs were assessed. In vivo studies: Twenty-four male New Zealand white rabbits received an intramuscular (IM) injection of methylprednisolone 20 mg/kg. Four weeks later, the rabbits were divided into four groups: the CD group, the hydrogel [HG, (a collagen-alginate mixture)] group, the MSC group, and the PGK-PDGF-BB-MSC group. Eight weeks later, the rabbits were sacrificed, their femurs were harvested, and microCT, mechanical testing, and histological analyses were performed. RESULTS: In vitro studies: PGK-PDGF-BB-MSCs proliferated more rapidly than unmodified MSCs (P < 0.001) and CMV-PDGF-BB-MSCs (P < 0.05) at days 3 and 7. CMV-PDGF-BB-MSCs demonstrated greater PDGF-BB expression than PGK-PDGF-BB-MSCs (P < 0.01). However, PGK-PDGF-BB-MSCs exhibited greater alkaline phosphatase staining at 14 days (P < 0.01), and osteogenic differentiation at 28 days (P = 0.07) than CMV-PDGF-BB-MSCs. In vivo: The PGK-PDGF-BB-MSC group had a trend towards greater bone mineral density (BMD) than the CD group (P = 0.074). The PGK-PDGF-BB-MSC group demonstrated significantly lower numbers of empty lacunae (P < 0.001), greater osteoclast density (P < 0.01), and greater angiogenesis (P < 0.01) than the other treatment groups. CONCLUSION: The use of PGK-PDGF-BB-MSCs as an adjunctive treatment with CD may reduce progression of osteonecrosis and enhance bone regeneration and angiogenesis in the treatment of early-stage ONFH.

The efficacy of lapine preconditioned or genetically modified IL4 over-expressing bone marrow-derived mesenchymal stromal cells in corticosteroid-associated osteonecrosis of the femoral head in rabbits.

Cell-based therapy for augmentation of core decompression (CD) using mesenchymal stromal cells (MSCs) is a promising treatment for early stage osteonecrosis of the femoral head (ONFH). Recently, the therapeutic potential for immunomodulation of osteogenesis using preconditioned (with pro-inflammatory cytokines) MSCs (pMSCs), or by the timely resolution of inflammation using MSCs that over-express anti-inflammatory cytokines has been described. Here, pMSCs exposed to tumor necrosis factor-alpha and lipopolysaccharide for 3 days accelerated osteogenic differentiation in vitro. Furthermore, injection of pMSCs encapsulated with injectable hydrogels into the bone tunnel facilitated angiogenesis and osteogenesis in the femoral head in vivo, using rabbit bone marrow-derived MSCs and a model of corticosteroid-associated ONFH in rabbits. In contrast, in vitro and in vivo studies demonstrated that genetically-modified MSCs that over-express IL4 (IL4-MSCs), established by using a lentiviral vector carrying the rabbit IL4 gene under the cytomegalovirus promoter, accelerated proliferation of MSCs and decreased the percentage of empty lacunae in the femoral head. Therefore, adjunctive cell-based therapy of CD using pMSCs and IL4-MSCs may hold promise to heal osteonecrotic lesions in the early stage ONFH. These interventions must be applied in a temporally sensitive fashion, without interfering with the mandatory acute inflammatory phase of bone healing.

High-resolution radioluminescence microscopy of FDG uptake in an engineered 3D tumor-stoma model.

PURPOSE: The increased glucose metabolism of cancer cells is the basis for 18F-fluorodeoxyglucose positron emission tomography (FDG-PET). However, due to its coarse image resolution, PET is unable to resolve the metabolic role of cancer-associated stroma, which often influences the metabolic reprogramming of a tumor. This study investigates the use of radioluminescence microscopy for imaging FDG uptake in engineered 3D tumor models with high resolution. METHOD: Multicellular tumor spheroids (A549 lung adenocarcinoma) were co-cultured with GFP-expressing human umbilical vein endothelial cells (HUVECs) within an artificial extracellular matrix to mimic a tumor and its surrounding stroma. The tumor model was constructed as a 200-µm-thin 3D layer over a transparent CdWO4 scintillator plate to allow high-resolution imaging of the cultured cells. After incubation with FDG, the radioluminescence signal was collected by a highly sensitive widefield microscope. Fluorescence microscopy was performed using the same instrument to localize endothelial and tumor cells. RESULTS: Simultaneous and co-localized brightfield, fluorescence, and radioluminescence imaging provided high-resolution information on the distribution of FDG in the engineered tissue. The microvascular stromal compartment as a whole took up a large fraction of the FDG, comparable to the uptake of the tumor spheroids. In vitro gamma counting confirmed that A549 and HUVEC cells were both highly glycolytic with rapid FDG uptake kinetics. Despite the relative thickness of the tissue constructs, an average spatial resolution of 64 ± 4 µm was achieved for imaging FDG. CONCLUSION: Our study demonstrates the feasibility of imaging the distribution of FDG uptake in engineered in vitro tumor models. With its high spatial resolution, the method can separately resolve tumor and stromal components. The approach could be extended to more advanced engineered cancer models but also to surgical tissue slices and tumor biopsies.

Combining a Vascular Bundle and 3D Printed Scaffold with BMP-2 Improves Bone Repair and Angiogenesis.

Vascularization is currently considered the biggest challenge in bone tissue engineering due to necrosis in the center of large scaffolds. We established a new expendable vascular bundle model to vascularize a three-dimensional printed channeled scaffold with and without bone morphogenetic protein-2 (BMP-2) for improved healing of large segmental bone defects. Bone formation and angiogenesis in an 8 mm critical-sized bone defect in the rat femur were significantly promoted by inserting a bundle consisting of the superficial epigastric artery and vein into the central channel of a large porous polycaprolactone scaffold. Vessels were observed sprouting from the vascular bundle inserted in the central tunnel. Although the regenerated bone volume in the group receiving the scaffold and vascular bundle was similar to that of the healthy femur, the rate of union of the group was not satisfactory (25% at 8 weeks). BMP-2 delivery was found to promote not only bone formation but also angiogenesis in the critical-sized bone defects. Both insertion of the vascular bundle alone and BMP-2 loading alone induced comparable levels of angiogenesis and when used in combination, significantly greater vascular volume was observed. These findings suggest a promising new modality of treatment in large bone defects. Level of Evidence: Therapeutic level I. Impact statement Vascularization is currently the main challenge in bone tissue engineering. The combination of a vascular bundle and an osteoinductive three-dimensional printed graft significantly improved and accelerated bone regeneration and angiogenesis in critical-sized large bone defects, suggesting a promising new modality of treatment in large bone defects.

Regenerative Approaches for the Treatment of Large Bone Defects.

A variety of engineered materials have gained acceptance in orthopedic practice as substitutes for autologous bone grafts, although the regenerative efficacy of these engineered grafts is still limited compared with that of transplanted native tissues. For bone defects greater than 4-5 cm, however, common bone grafting procedures are insufficient and more complicated surgical interventions are required to repair and regenerate the damaged or missing bone. In this review, we describe current grafting materials and surgical techniques for the reconstruction of large bone defects, followed by tissue engineering (TE) efforts to develop improved therapies. Particular emphasis is placed on graft vascularization, because for both autologous bone and engineered alternatives, achieving adequate vascular development within the regenerating bone tissues remains a significant challenge in the context of large bone defects. To this end, TE and surgical strategies to induce development of a vasculature within bone grafts are discussed. Impact statement This review aims to present an accessible and thorough overview of current orthopedic surgical techniques as well as bone tissue engineering and vascularization strategies that might one day offer improvements to clinical therapies for the repair of large bone defects. We consider the lessons that clinical orthopedic reconstructive practices can contribute to the push toward engineered bone.

Sarcopenia as a predictor of mortality in women with breast cancer: a meta-analysis and systematic review.

BACKGROUND: Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in women worldwide. Recently, studies have been published with inconsistent findings regarding whether sarcopenia is a risk factor for mortality in breast cancer patients. Therefore, the aim of this systematic review and meta-analysis was to systematically assess and quantify sarcopenia as a risk factor for mortality in breast cancer patients. METHODS: In a systematic literature review of PubMed, EMBASE, and the Cochrane CENTRAL Library, we searched for observational studies written in English (from database inception until April 30, 2019) that reported an association between sarcopenia and breast cancer in women who were 18 years or older. RESULTS: A total of six studies (5497 participants) were included in this meta-analysis. Breast cancer patients with sarcopenia were associated with a significantly higher risk of mortality, compared to breast cancer patients without sarcopenia (pooled HR-hazard ratio = 1.71, 95% CI: 1.25-2.33, I2 = 59.1%). In addition, the results of age subgroup analysis showed that participants younger than 55 years with sarcopenia had a lower risk of mortality than participants aged 55 years and older with sarcopenia (pooled HR = 1.46, 95% CI: 1.24-1.72 versus pooled HR = 1.99, 95% CI: 1.05-3.78), whereas both have an increased risk of mortality compared to non-sarcopenic patients. Subgroup analyses regarding stage at diagnosis revealed an increased risk of mortality in non-metastatic patients compared to participants without sarcopenia (pooled HR = 1.91, 95% CI: 1.32-2.78), whereas the association was not significant in metastatic breast cancer patients. Other subgroup analyses were performed using different follow-up periods (> 5 years versus ≤5 years) and the results were different (pooled HR = 1.81, 95% CI: 1.23-2.65 versus pooled HR = 1.70, 95% CI: 0.80-3.62). CONCLUSIONS: The present study found that sarcopenia is a risk factor for mortality among female early breast cancer patients. It is imperative that more research into specific interventions aimed at treating sarcopenia be conducted in the near future in order to provide evidence which could lead to decreased mortality rates in breast cancer patients.

Administration of allogeneic mesenchymal stem cells in lengthening phase accelerates early bone consolidation in rat distraction osteogenesis model.

BACKGROUND: Distraction osteogenesis (DO) is a surgical technique to promote bone regeneration which may require long duration for bone consolidation. Bone marrow-derived mesenchymal stem cells (MSCs) have been applied to accelerate bone formation in DO. However, the optimal time point for cell therapy in DO remains unknown. This study sought to determine the optimal time point of cell administration to achieve early bone consolidation in DO. We hypothesized that the ratio of circulating MSCs to peripheral mononuclear cells and the level of cytokines in serum might be indicators for cell administration in DO. METHODS: Unilateral tibial osteotomy with an external fixator was performed in adult Sprague Dawley rats. Three days after osteotomy, the tibia was lengthened at 0.5 mm/12 h for 5 days. At first, 5 rats were used to analyze the blood components at 6 different time points (3 days before lengthening, on the day lengthening began, or 3, 6, 10, or 14 days after lengthening began) by sorting circulating MSCs and measuring serum levels of stromal cell-derived factor 1 (SDF-1) and interleukin 1ß. Then, 40 rats were used for cell therapy study. A single dose of 5 × 105 allogeneic MSCs was locally injected at the lengthening site on day 3, 6, or 10 after lengthening began, or 3 doses of MSCs were injected at the three time points. Sequential X-ray radiographs were taken weekly. Endpoint examinations included micro-computed tomography analysis, mechanical testing, histomorphometry, and histology. RESULTS: The number of circulating MSCs and serum level of SDF-1 were significantly increased during lengthening, and then decreased afterwards. Single injection of MSCs during lengthening phase (on day 3, but not day 6 or 10) significantly increased bone volume fraction, mechanical maximum loading, and bone mineralization of the regenerate. Triple injections of MSCs at three time points also significantly increased bone volume and maximum loading of the regenerates. CONCLUSION: This study demonstrated that bone consolidation could be accelerated by a single injection of MSCs during lengthening when the ratio of peripheral MSCs to mononuclear cells and the serum SDF-1 presented at peak levels concurrently, suggesting that day 3 after lengthening began may be the optimal time point for cell therapy to promote early bone consolidation.

Bursicon homodimers induce the innate immunity via Relish in Procambarus clarkii.

Bursicon (burs) is a neuropeptide hormone consisting of two cystine-knot proteins (burs α and burs ß), and burs α-ß is responsible for cuticle tanning in insects. Further studies show that burs homodimers induce prophylactic immunity. Here, we investigated the hypothesis that burs homodimers act in regulating immunity in the red swamp crayfish Procambarus clarkii. We found that burs α and burs ß are expressed in neural system of crayfish. Treating crayfish with recombinant burs-homodimer proteins led to up-regulation of several anti-microbial peptide (AMP) genes, and RNAi-mediated knockdown of burs led to decreased expression of AMP genes. The burs proteins also facilitated bacterial clearance and decreased crayfish mortality upon bacterial infection. Furthermore, burs proteins activated the transcriptional factor Relish, and knockdown of Relish abolished the influence of recombinant burs homodimers on AMP induction. We infer the burs homodimers induce expression of AMP genes via Relish in crayfish and this study extends this immune signaling pathway from insects to crustaceans.

Identifying deer antler uhrf1 proliferation and s100a10 mineralization genes using comparative RNA-seq.

BACKGROUND: Deer antlers are bony structures that re-grow at very high rates, making them an attractive model for studying rapid bone regeneration. METHODS: To identify the genes that are involved in this fast pace of bone growth, an in vitro RNA-seq model that paralleled the sharp differences in bone growth between deer antlers and humans was established. Subsequently, RNA-seq (> 60 million reads per library) was used to compare transcriptomic profiles. Uniquely expressed deer antler proliferation as well as mineralization genes were identified via a combination of differential gene expression and subtraction analysis. Thereafter, the physiological relevance as well as contributions of these identified genes were determined by immunofluorescence, gene overexpression, and gene knockdown studies. RESULTS: Cell characterization studies showed that in vitro-cultured deer antler-derived reserve mesenchyme (RM) cells exhibited high osteogenic capabilities and cell surface markers similar to in vivo counterparts. Under identical culture conditions, deer antler RM cells proliferated faster (8.6-11.7-fold increase in cell numbers) and exhibited increased osteogenic differentiation (17.4-fold increase in calcium mineralization) compared to human mesenchymal stem cells (hMSCs), paralleling in vivo conditions. Comparative RNA-seq identified 40 and 91 previously unknown and uniquely expressed fallow deer (FD) proliferation and mineralization genes, respectively, including uhrf1 and s100a10. Immunofluorescence studies showed that uhrf1 and s100a10 were expressed in regenerating deer antlers while gene overexpression and gene knockdown studies demonstrated the proliferation contributions of uhrf1 and mineralization capabilities of s100a10. CONCLUSION: Using a simple, in vitro comparative RNA-seq approach, novel genes pertinent to fast bony antler regeneration were identified and their proliferative/osteogenic function was verified via gene overexpression, knockdown, and immunostaining. This combinatorial approach may be applicable to discover unique gene contributions between any two organisms for a given phenomenon-of-interest.