Impaired bone defect and fracture healing in dystrophin/utrophin double-knockout mice and the mechanism.

This study investigated the role of muscle damage in bone defect healing using skull and tibial double-defect and tibial fracture models in dystrophin-/-/Utrophin-/- double-knockout (dKO-Hom) mice. The skull and tibia bone defect and fracture healing was monitored using micro-CT, histology, immuohistochemistry and quantitative PCR. We found the skull defect healing is not impaired while the tibial defect healing was delayed at day 7 in the dKO-Hom group compared to wild-type (WT) group as revealed by micro-CT. Mechanistically, the number of osteoclasts and osteoblasts significantly decreased in the defect area in dKO-Hom group compared to WT group on day 21. DKO-Hom mice showed higher mortality after fracture (6/12) and significantly impaired fracture healing compared to the other groups as revealed by the micro-CT parameters of the calluses. Histology showed higher osteoclast number in the calluses of dKO-Hom mice than other groups. Furthermore, dKO-Hom mice showed down-regulation of 15-Pgdh, Il-4, Bmp7, and Bmp9 at 10 days after tibia fracture and BMP6 and 7 in the muscle. In conclusion, the long bone defect and fracture healing are impaired in dKO-Hom mice which demonstrated significantly muscle sarcopenia and related with disturbance of osteoclastogenesis and osteoblastogenesis. The impaired tibial fracture healing was associated with down-regulation of several genes in the muscle.

Gender differences in tibial fractures healing in normal and muscular dystrophic mice.

Duchenne muscular dystrophy (DMD) patients have a high fracture risk and poor fracture healing. The dystrophin-/- (mdx) mouse is a murine model of DMD and exhibits delayed bone fracture healing. Since our research team has shown that adult stem cells, such as muscle-derived stem cells, display a gender difference in their osteogenic potential with the male cells being more osteogenic, we hypothesize that a potential gender differences may exist during bone healing in normal and mdx mice. To test this hypothesis, wild-type (WT) and mdx mice underwent tibial fracture surgery and microCT live scanning biweekly. The mice were sacrificed at 6 weeks post-surgery and the calluses were collected for histological analysis. To further investigate the mechanism, another two sets of mice were sacrificed at 10 days after fracture for RNA extraction and gene expression analysis and histology. MicroCT results showed, at 6 weeks post- surgery, the calluses were larger but showed less remodeling in both normal and mdx male mice when compared to females, at the same time point. However, females had higher callus bone volume density and an increase in osteoclast (OCs) number. At 10 days after fracture surgery, male mice had formed larger calluses, whereas females formed well-remodeled calluses with more osteoblasts and a greater bone area for both WT and mdx mice. Higher IGF-1 expression was observed in male mdx mice when compared to their female counterparts, whereas female WT mice had higher BMP-9 expression when compared to WT males. In conclusion, male mice formed larger bone calluses than females during tibial fracture healing for both WT and mdx mice. This may be attributed to higher IGF-1 expression, activation of Wnt/ß-catennin signaling pathway and greater OB numbers during callus formation. Female mice achieved better bone remodeling in the regenerated bone with higher bone quality due to increased OC numbers that promote faster remodeling of the fracture calluses, and higher BMP-9 expression levels. Therefore, gender is one of many factors that need to be considered for both animal and human bone research.

High bone microarchitecture, strength, and resistance to bone loss in MRL/MpJ mice correlates with activation of different signaling pathways and systemic factors.

The MRL/MpJ mice have demonstrated an enhanced tissue regeneration capacity for various tissues. In the present study, we systematically characterized bone microarchitecture and found that MRL/MpJ mice exhibit higher bone microarchitecture and strength compared to both C57BL/10J and C57BL/6J WT mice at 2, 4, and 10 months of age. The higher bone mass in MRL/MpJ mice was correlated to increased osteoblasts, decreased osteoclasts, higher cell proliferation, and bone formation, and enhanced pSMAD5 signaling earlier during postnatal development (2-month old) in the spine trabecular bone, and lower bone resorption rate at later age. Furthermore, these mice exhibit accelerated fracture healing via enhanced pSMAD5, pAKT and p-P38MAPK pathways compared to control groups. Moreover, MRL/MpJ mice demonstrated resistance to ovariectomy-induced bone loss as evidenced by maintaining higher bone volume/tissue volume (BV/TV) and lower percentage of bone loss later after ovariectomy. The consistently higher serum IGF1 level and lower RANKL level in MRL/MpJ mice may contribute to the maintenance of high bone mass in uninjured and injured bone. In conclusion, our results indicate that enhanced pSMAD5, pAKT, and p-P38MAPK signaling, higher serum IGF-1, and lower RANKL level contribute to the higher bone microarchitecture and strength, accelerated healing, and resistance to osteoporosis in MRL/MpJ mice.

A comparison of BMP2 delivery by coacervate and gene therapy for promoting human muscle-derived stem cell-mediated articular cartilage repair.

BACKGROUND: Osteoarthritis and cartilage injury treatment is an unmet clinical need. Therefore, development of new approaches to treat these diseases is critically needed. Previous work in our laboratory has shown that murine muscle-derived stem cells (MDSCs) can efficiently repair articular cartilage in an osteochondral and osteoarthritis model. However, the cartilage repair capacity of human muscle-derived stem cells has not been studied which prompt this study. METHOD: In this study, we tested the in vitro chondrogenesis ability of six populations of human muscle-derived stem cells (hMDSCs), before and after lenti-BMP2/GFP transduction using pellet culture and evaluated chondrogenic differentiation of via histology and Raman spectroscopy. We further compared the in vivo articular cartilage repair of hMDSCs stimulated with BMP2 delivered through coacervate sustain release technology and lenti-viral gene therapy-mediated gene delivery in a monoiodoacetate (MIA)-induced osteoarthritis (OA) model. We used microCT and histology to evaluate the cartilage repair. RESULTS: We observed that all hMDSCs were able to undergo chondrogenic differentiation in vitro. As expected, lenti-BMP2/GFP transduction further enhanced the chondrogenic differentiation capacities of hMDSCs, as confirmed by Alcian blue and Col2A1staining as well as Raman spectroscopy analysis. We observed through micro-CT scanning, Col2A1 staining, and histological analyses that delivery of BMP2 with coacervate could achieve a similar articular cartilage repair to that mediated by hMDSC-LBMP2/GFP. We also found that the addition of soluble fms-like tyrosine kinase-1 (sFLT-1) protein further improved the regenerative potential of hMDSCs/BMP2 delivered through the coacervate sustain release technology. Donor cells did not primarily contribute to the repaired articular cartilage since most of the repair cells are host derived as indicated by GFP staining. CONCLUSIONS: We conclude that the delivery of hMDSCs and BMP2 with the coacervate technology can achieve a similar cartilage repair relative to lenti-BMP2/GFP-mediated gene therapy. The use of coacervate technology to deliver BMP2/sFLT1 with hMDSCs for cartilage repair holds promise for possible clinical translation into an effective treatment modality for osteoarthritis and traumatic cartilage injury.

Characterization of articular cartilage homeostasis and the mechanism of superior cartilage regeneration of MRL/MpJ mice.

This study investigated articular cartilage (AC) homeostasis and different signaling pathways involved in the superior cartilage regeneration of Murphy Roths large (MRL/MpJ) mice previously reported. We collected uninjured and destabilized medial meniscus (DMM)-injured knees from 8-wk-old C57BL/6J and MRL/MpJ mice. We used micro-computed tomography (microCT), histology, and immunohistochemistry to evaluate AC homeostasis and repair. We used the ear punch model to investigate the role of angiogenesis and inflammation in the superior healing of MRL/MpJ mice. We found fewer ß-catenin and more pSMAD5 positive cells in the uninjured AC of MRL/MpJ mice than that from C57BL/6J mice. MRL/MpJ mice exhibited better AC repair in DMM-induced OA, as indicated by microCT results, Alcian blue, and Safranin O staining. Mechanistically, fewer ß-catenin, pSMAD2-, pSMAD3-, a disintegrin and metalloproteinase with thrombospondin motifs 4-, matrix metalloproteinase (MMP) 9-, and MMP13-positive cells and more proliferating cell nuclear antigen- and pSMAD5-positive cells were found in the DMM-injured AC in MRL/MpJ mice than in normal mice. The accelerated ear wound healing of MRL/MpJ mice correlated with enhanced angiogenesis and macrophage polarization toward the M2a phenotype through elevated IL-10 and IL-4 expressing cells. Collectively, our study revealed that down-regulation of pSMAD2/3, ß-catenin, and MMPs and up-regulation of pSMAD5 and M2a macrophage polarization contribute to the enhanced cartilage repair observed in MRL/MpJ mice.-Deng, Z., Gao, X., Sun, X., Amra, S., Lu, A., Cui, Y., Eltzschig, H. K., Lei, G., Huard, J. Characterization of articular cartilage homeostasis and the mechanism of superior cartilage regeneration of MRL/MpJ mice.

Oncogenic and osteolytic functions of histone demethylase NO66 in castration-resistant prostate cancer.

Epigenetic changes that cause dysregulated gene expression during progression of androgen-independent prostate cancer (PCa) and metastatic skeletal lesions remain elusive. Here, we explored the role of histone demethylase NO66 in the pathogenesis of PCa and bone metastasis-related skeletal lesions. Tissue and cDNA microarrays of PCa were analyzed for NO66 mRNA and protein levels. We examined the effects of gain and loss of NO66 function on cell viability, colony formation, migration, invasion, and tumor-induced skeletal lesions in femoral bone. RNAseq and ChIPseq were performed to elucidate NO66-target genes in PCa. We report that NO66 levels were upregulated in advanced primary prostate tumors compared to normal tissue or tumors with low Gleason scores. Forced expression of NO66 promoted cell survival and invasion of PCa cells; whereas, knockdown of NO66 resulted in decreased cell survival and increased sensitivity to docetaxel. NO66-overexpressing PC3 cells implanted into the femoral bone of male SCID mice caused massive bone loss and stimulation of mouse osteoclast-promoting genes, including Dickkopf1, Cathepsin K, Nf-kß,; and Calcr, suggesting a role for NO66 in tumor growth in bone and osteoclast activity. Combined RNAseq and ChIP-seq revealed that NO66 activates the survival gene MCL1, the invasion-associated genes IGFBP5 and MMP3, the pro-oncogenic genes CTNNB1 and CCND1, and the epigenetic modifier gene KMT2A in androgen-independent PCa. Our findings uncover the role of NO66 as a key oncogenic driver in PCa, causing osteolytic lesions through upstream epigenetic regulation of key genes for survival, invasion and metastasis, and pro-osteoclastic factors.

Systemic investigation of bone and muscle abnormalities in dystrophin/utrophin double knockout mice during postnatal development and the mechanisms.

The dystrophin-/-/utrophin-/-/ double knockout (dKO-Hom) mouse is a murine model of human Duchenne muscular dystrophy. This study investigated the bone and muscle abnormalities of dKO-Hom mouse and mechanisms. We collected bone and skeletal muscle samples from control mice and three muscular dystrophic mouse models at different ages and performed micro-computer tomography and histological analyses of both bone and skeletal muscle tissues. Serum receptor activator of nuclear factor kappa-Β ligand (RANKL) and sclerostin (SOST) levels, osteoclastogenesis and serum proteomics were also analyzed. Our results indicated that dKO-Hom mice developed skeletal muscle histopathologies by 5 days of age, whereas bone abnormalities developed at 4 weeks of age. Furthermore, our results indicated that the numbers of osteoblasts and osteoclasts were decreased in the proximal tibia and spine trabecular bone of dKO-Hom mice compared to wild-type (WT) mice, which correlated with a significant reduction in serum RANKL levels. The number of tibia cortical osteocytes also decreased, whereas serum SOST levels increased significantly in dKO-Hom mice than WT mice. Osteoblastic number was significantly lower, but osteoclast number increased, in the spine L6 of dKO-Hom mice than WT mice at 6 weeks of age, resulting in a decrease in bone formation and an increase in bone resorption. Serum proteomics results revealed abnormal proteome profiles in dKO-Hom mice compared to control mice. In conclusion, our study elucidated the timing of development of bone and muscle abnormalities. The bone abnormalities in dKO-Hom mice are correlated with lower serum RANKL and higher SOST levels that resulted in dysregulation of osteogenesis and osteoclastogenesis and bone loss.

TIPE2 gene transfer with adeno-associated virus 9 ameliorates dystrophic pathology in mdx mice.

Duchenne muscle dystrophy (DMD), characterized by progressive loss of muscle architecture and function, is caused by lack of dystrophin expression in the sarcolemma of myofibers. Recurrent muscle damages in DMD patients and DMD mouse model, mdx, lead to chronic inflammation, which further exacerbate the muscle histopathology. It is critical to find a successful therapy that will improve the histopathology of muscles of DMD patients and restore skeletal muscle function. TIPE2 (tumor necrosis factor α-induced-protein 8-like 2), identified as a negative regulator of immune response, has been found to be expressed in various types of immune cells including macrophages. However, whether and how TIPE2 plays a role in the DMD-related inflammation remains unknown. In this study, we found the basal expression levels of TIPE2 in skeletal muscle from mdx mice are significantly lower than wild-type (WT) mice. To investigate the potential beneficial effect of TIPE2 in muscular dystrophy, we performed intramuscular injection of adeno-associated virus 9 carrying the TIPE2 gene in mdx mice. Our results indicate that the restoration of TIPE2 ameliorates muscular dystrophy phenotype through a reduction in inflammation and fibrosis. In addition, TIPE2 overexpression dramatically decreased the proliferation and migration rate of macrophages, as well as repressed the secretion of pro-inflammatory factors induced by tumor necrotic factor alpha. Taken together, our results indicate that a reduction of TIPE2 expression is observed in dystrophic skeletal muscle, when compared to WT and more importantly that TIPE2 gene delivery may provide as a novel anti-inflammatory therapy to alleviate the muscle weakness in DMD patients.

Influences of donor and host age on human muscle-derived stem cell-mediated bone regeneration.

BACKGROUND: Human muscle-derived stem cells (hMDSCs) have been shown to regenerate bone efficiently when they were transduced with Lenti-viral bone morphogenetic protein 2 (LBMP2). However, whether the age of hMDSCs and the animal host affect the bone regeneration capacity of hMDSCs and mechanism are unknown which prompted the current study. METHODS: We isolated three gender-matched young and old populations of skeletal muscle stem cells, and tested the influence of cells' age on in vitro osteogenic differentiation using pellet culture before and after Lenti-BMP2/green fluorescent protein (GFP) transduction. We further investigated effects of the age of hMDSCs and animal host on hMDSC-mediated bone regeneration in a critical-size calvarial bone defect model in vivo. Micro-computer tomography (CT), histology, and immunohistochemistry were used to evaluate osteogenic differentiation and mineralization in vitro and bone regeneration in vivo. Western blot, quantitative polymerase chain reaction (PCR), and oxidative stress assay were performed to detect the effects of age of hMDSCs on cell survival and osteogenic-related genes. Serum insulin-like growth factor 1 (IGF1) and receptor activator of nuclear factor-kappa B ligand (RANKL) were measured with an enzyme-linked immunosorbent assay (ELISA). RESULTS: We found LBMP2/GFP transduction significantly enhanced osteogenic differentiation of hMDSCs in vitro, regardless of donor age. We also found old were as efficient as young LBMP2/GFP-transduced hMDSCs for regenerating functional bone in young and old mice. These findings correlated with lower phosphorylated p38MAPK expression and similar expression levels of cell survival genes and osteogenic-related genes in old hMDSCs relative to young hMDSCs. Old cells exhibited equivalent resistance to oxidative stress. However, both young and old donor cells regenerated less bone in old than young hosts. Impaired bone regeneration in older hosts was associated with high bone remodeling due to higher serum levels of RANKL and lower level of IGF-1. CONCLUSION: hMDSC-mediated bone regeneration was not impaired by donor age when hMDSCs were transduced with LBMP2/GFP, but the age of the host adversely affected hMDSC-mediated bone regeneration. Regardless of donor and host age, hMDSCs formed functional bone, suggesting a promising cell resource for bone regeneration.

Size of the nanovectors determines the transplacental passage in pregnancy: study in rats.

OBJECTIVE: The objective of the study was to examine whether the size of silicon nanovectors (SNVs) inhibits their entrance into the fetal circulation. STUDY DESIGN: Pregnant rats were intravenously administered with SNVs or saline. The SNVs were spherical particles with 3 escalating diameters: 519 nm, 834 nm, and 1000 nm. The maternal and fetal distribution of SNVs was assessed. RESULTS: In animals that received 1000 or 834 nm SNV, silicon (Si) levels were significantly higher in the maternal organs vs the saline group, whereas the silicon levels in fetal tissues were similar to controls. However, in animals receiving 519 nm SNVs, fetal silicon levels were significantly higher in the SNV group compared with the saline group (5.93 ± 0.67 µg Si per organ vs 4.80 ± 0.33, P = .01). CONCLUSION: Larger SNVs do not cross the placenta to the fetus and, remaining within the maternal circulation, can serve as carriers for harmful medications in order to prevent fetal exposure.

Proteomic analysis of serum opsonins impacting biodistribution and cellular association of porous silicon microparticles.

Mass transport of drug delivery vehicles is guided by particle properties, such as size, shape, composition, and surface chemistry, as well as biomolecules and serum proteins that adsorb to the particle surface. In an attempt to identify serum proteins influencing cellular associations and biodistribution of intravascularly injected particles, we used two-dimensional gel electrophoresis and mass spectrometry to identify proteins eluted from the surface of cationic and anionic silicon microparticles. Cationic microparticles displayed a 25-fold greater abundance of Ig light variable chain, fibrinogen, and complement component 1 compared to their anionic counterparts. Anionic microparticles were found to accumulate in equal abundance in murine liver and spleen, whereas cationic microparticles showed preferential accumulation in the spleen. Immunohistochemistry supported macrophage uptake of both anionic and cationic microparticles in the liver, as well as evidence of association of cationic microparticles with hepatic endothelial cells. Furthermore, scanning electron micrographs supported cellular competition for cationic microparticles by endothelial cells and macrophages. Despite high macrophage content in the lungs and tumor, microparticle uptake by these cells was minimal, supporting differences in the repertoire of surface receptors expressed by tissue-specific macrophages. In summary, particle surface chemistry drives selective binding of serum components impacting cellular interactions and biodistribution.