Fast track surgery for children with congenital pseudarthrosis of the tibia: A prospective interventional study.

OBJECTIVE: The purpose of this study was to explore the effect of fast-track surgery combined with a clinical nursing pathway on the recovery and complications of congenital pseudarthrosis of tibia. METHODS: 82 children with congenital pseudarthrosis of tibia admitted from January 2019 to December 2020 were selected as the study subjects. The control group received routine clinical nursing pathway while the intervention group received a fast-track surgery combined with a clinical nursing pathway. The fasting and water deprivation time were arranged according to the pre operation time, and the accelerated rehabilitation nursing models such as progressive diet management, multi-functional analgesia, and early sequential functional exercise were given after the operation. After collecting data on perioperative diet, postoperative recovery, postoperative complications, and family satisfaction from both groups of patients in a large hospital in China, a comparative analysis was conducted. RESULTS: The retention time of negative pressure drainage tube, urinary catheter and hospital stay in the intervention group were shorter than those in the control group (P < 0.05); The incidence of complications in the intervention group (5%) was significantly lower than that in the control group (21.42%) (P = 0.029). The family satisfaction of the intervention group (95.00%) was higher than that of the control group (80.95%). CONCLUSION: Strengthening the concept of fast-track surgery nursing in the combined operation of congenital pseudarthrosis of tibia can shorten the hospitalization time of children, reduce the occurrence of postoperative complications and improve their family satisfaction.

Research on anterior minimally invasive approach in the treatment of children with developmental dysplasia of the hip.

OBJECTIVES: To investigate the clinical efficacy and safety of open reduction through anterior minimally invasive approach in the treatment of children with developmental dysplasia of the hip. METHOD: A total of 23 patients (25 hips) less than 2 years with developmental dysplasia of the hip treated by open reduction through anterior minimally invasive approach were treated in our hospital from August 2016 to March 2019. Through the anterior minimally invasive approach, we enter from the gap between sartorius muscle and tensor fasciae lata without cutting off rectus femoris muscle, which can effectively expose the joint capsule and reduce the damage to medial blood vessels and nerves. The operation time, incision length, intraoperative bleeding, hospital stay and surgical complications were observed. The progression of developmental dysplasia of the hip and avascular necrosis of the femoral head were evaluated by imaging examination. RESULT: All patients were performed with follow-up visit for an average of 22 months. The average incision length was 2.5 cm, the average operation time was 26 min, the average intraoperative bleeding was 12ml, and the average hospital stay was 4.9 days. All patients received concentric reduction immediately after operation, and no re-dislocation occurred. At the last follow-up visit, the acetabular index was (25.8 ± 6.4°). During the follow-up visit, X-ray showed avascular necrosis of the femoral head in 4 hips (16%). CONCLUSION: open reduction through anterior minimally invasive approach can achieve good clinical effect in the treatment of infantile developmental dysplasia of the hip.

Design of a New "U"-shaped Staple and Its Clinical Application in Postoperative Ankle Valgus of Congenital Pseudarthrosis of the Tibia in Children.

OBJECTIVE: There has been a lack of suitable epiphysis blocking materials due to the characteristics of less tissue coverage and narrow epiphysis in children's distal tibial medial malleolus. Therefore, this study is to investigate the clinical efficacy and safety of a new "U"-shaped staple in the treatment of postoperative ankle valgus of congenital pseudarthrosis of the tibia (CPT). METHOD: According to the inclusion and exclusion criteria, 33 patients with postoperative ankle valgus of CPT were treated with new "U"-shaped staples from May 2013 to September 2019. The deformity of ankle valgus was gradually corrected by implanting a new "U"-shaped staple on the medial side of the distal tibia. Clinical indexes such as the operation time, intraoperative bleeding and hospital stay were observed. Tibiotalar angle was selected as the evaluation index of ankle valgus. American Orthopedic Foot & Ankle Society (AOFAS) scale was used for clinical evaluation of ankle function. The tibiotalar angle, deformity correction rate and complications were evaluated by preoperative, postoperative and last follow-up imaging data. Student's t-test was used for statistical analysis. RESULTS: Thirty-three patients, including 12 males and 21 females were included. All the patients were followed up for at least 14 months, with an average of 35 months. The average operation time was 23 (15-40) min, the average amount of intraoperative bleeding was 7.5 (4-10) mL, and the average hospital stay was 4.2 (3-6) days. The intraoperative tibiotalar angles of all patients were 74.2° ± 4.6°, the tibiotalar angle were 86.8° ± 4.9° when internal fixation was removed, and the tibiotalar angles at the last follow-up were 84.3° ± 5.9°. The average orthopedic rate was 0.68° per month. No patients suffered from serious complications such as screw prolapse, osteomyelitis, wound infection, etc. Postoperative wound pain complications occurred in two patients, which were relieved after conservative treatment. The AOFAS score improved from 46.2 ± 9.4 before the operation to 74.6 ± 5.7 at the last follow-up (P < 0.01). The ankle movement was good without joint stiffness. There was no epiphyseal plate injury after the removal of internal fixation. CONCLUSION: The new "U"-shaped staple is characterized by simple implantation, low notch, lower risk of fixation failure and close fitting with cortical bone. It is a safe and effective internal fixation system for the treatment of ankle valgus in children.

Aged bone matrix-derived extracellular vesicles as a messenger for calcification paradox.

Adipocyte differentiation of bone marrow mesenchymal stem/stromal cells (BMSCs) instead of osteoblast formation contributes to age- and menopause-related marrow adiposity and osteoporosis. Vascular calcification often occurs with osteoporosis, a contradictory association called "calcification paradox". Here we show that extracellular vesicles derived from aged bone matrix (AB-EVs) during bone resorption favor BMSC adipogenesis rather than osteogenesis and augment calcification of vascular smooth muscle cells. Intravenous or intramedullary injection of AB-EVs promotes bone-fat imbalance and exacerbates Vitamin D3 (VD3)-induced vascular calcification in young or old mice. Alendronate (ALE), a bone resorption inhibitor, down-regulates AB-EVs release and attenuates aging- and ovariectomy-induced bone-fat imbalance. In the VD3-treated aged mice, ALE suppresses the ovariectomy-induced aggravation of vascular calcification. MiR-483-5p and miR-2861 are enriched in AB-EVs and essential for the AB-EVs-induced bone-fat imbalance and exacerbation of vascular calcification. Our study uncovers the role of AB-EVs as a messenger for calcification paradox by transferring miR-483-5p and miR-2861.

Harmine targets inhibitor of DNA binding-2 and activator protein-1 to promote preosteoclast PDGF-BB production.

Osteoporosis is one of the most common metabolic bone diseases affecting millions of people. We previously found that harmine prevents bone loss in ovariectomized mice via increasing preosteoclast platelet-derived growth factor-BB (PDGF-BB) production and type H vessel formation. However, the molecular mechanisms by which harmine promotes preosteoclast PDGF-BB generation are still unclear. In this study, we revealed that inhibitor of DNA binding-2 (Id2) and activator protein-1 (AP-1) were important factors implicated in harmine-enhanced preosteoclast PDGF-BB production. Exposure of RANKL-induced Primary bone marrow macrophages (BMMs), isolated from tibiae and femora of mice, to harmine increased the protein levels of Id2 and AP-1. Knockdown of Id2 by Id2-siRNA reduced the number of preosteoclasts as well as secretion of PDGF-BB in RANKL-stimulated BMMs administrated with harmine. Inhibition of c-Fos or c-Jun (components of AP-1) both reversed the stimulatory effect of harmine on preosteoclast PDGF-BB production. Dual-luciferase reporter assay analyses determined that PDGF-BB was the direct target of AP-1 which was up-regulated by harmine treatment. In conclusion, our data demonstrated a novel mechanism involving in the production of PDGF-BB increased by harmine, which may provide potential therapeutic targets for bone loss diseases.

Extracellular Vesicles from Akkermansia muciniphila Elicit Antitumor Immunity Against Prostate Cancer via Modulation of CD8+ T Cells and Macrophages.

PURPOSE: Prostate cancer (PCa) is one of the most common malignancies in males. Despite the success of immunotherapy in many malignant cancers, strategies are still needed to improve therapeutic efficacy in PCa. This study aimed to investigate the effects of Akkermansia muciniphila-derived extracellular vesicles (Akk-EVs) on PCa and elucidate the underlying immune-related mechanism. METHODS: Akk-EVs were isolated by ultracentrifugation and intravenously injected to treat syngeneic PCa-bearing immune-competent mice. Immunophenotypic changes in immune cells, such as cytotoxic T lymphocytes and macrophages, were measured via flow cytometry analysis. Histological examination was used to detect morphological changes in major organs after Akk-EVs treatments. In vitro, flow cytometry was performed to confirm the effects of Akk-EVs on the activation of CD8+ T cells. Quantitative PCR and immunofluorescence staining were carried out to test the impact of Akk-EVs on macrophage polarization. Cell counting kit-8 (CCK-8) analysis, colony formation assays, and scratch wound healing assays were conducted to assess the effects of Akk-EVs-treated macrophages on the proliferation and invasion of PCa cells. CCK-8 assays also confirmed the impact of Akk-EVs on the viability of normal cells. RESULTS: Intravenous injection of Akk-EVs in immune-competent mice reduced the tumor burden of PCa without inducing obvious toxicity in normal tissues. This treatment elevated the proportion of granzyme B-positive (GZMB+) and interferon γ-positive (IFN-γ+) lymphocytes in CD8+ T cells and caused macrophage recruitment, with increased tumor-killing M1 macrophages and decreased immunosuppressive M2 macrophages. In vitro, Akk-EVs increased the number of GZMB+CD8+ and IFN-γ+CD8+ T cells and M1-like macrophages. In addition, conditioned medium from Akk-EVs-treated macrophages suppressed the proliferation and invasion of prostate cells. Furthermore, the effective dose of Akk-EVs was well-tolerated in normal cells. CONCLUSION: Our study revealed the promising prospects of Akk-EVs as an efficient and biocompatible immunotherapeutic agent for PCa treatment.

Inhibition of miR-331-3p and miR-9-5p ameliorates Alzheimer's disease by enhancing autophagy.

Alzheimer's disease (AD) is currently ranked as the third leading cause of death for eldly people, just behind heart disease and cancer. Autophagy is declined with aging. Our study determined the biphasic changes of miR-331-3p and miR-9-5p associated with AD progression in APPswe/PS1dE9 mouse model and demonstrated inhibiting miR-331-3p and miR-9-5p treatment prevented AD progression by promoting the autophagic clearance of amyloid beta (Aß). Methods: The biphasic changes of microRNAs were obtained from RNA-seq data and verified by qRT-PCR in early-stage (6 months) and late-stage (12 months) APPswe/PS1dE9 mice (hereinafter referred to as AD mice). The AD progression was determined by analyzing Aß levels, neuron numbers (MAP2+) and activated microglia (CD68+IBA1+) in brain tissues using immunohistological and immunofluorescent staining. MRNA and protein levels of autophagic-associated genes (Becn1, Sqstm1, LC3b) were tested to determine the autophagic activity. Morris water maze and object location test were employed to evaluate the memory and learning after antagomirs treatments in AD mice and the Aß in the brain tissues were determined. Results: MiR-331-3p and miR-9-5p are down-regulated in early-stage of AD mice, whereas up-regulated in late-stage of AD mice. We demonstrated that miR-331-3p and miR-9-5p target autophagy receptors Sequestosome 1 (Sqstm1) and Optineurin (Optn), respectively. Overexpression of miR-331-3p and miR-9-5p in SH-SY5Y cell line impaired autophagic activity and promoted amyloid plaques formation. Moreover, AD mice had enhanced Aß clearance, improved cognition and mobility when treated with miR-331-3p and miR-9-5p antagomirs at late-stage. Conclusion: Our study suggests that using miR-331-3p and miR-9-5p, along with autophagic activity and amyloid plaques may distinguish early versus late stage of AD for more accurate and timely diagnosis. Additionally, we further provide a possible new therapeutic strategy for AD patients by inhibiting miR-331-3p and miR-9-5p and enhancing autophagy.

Autophagy receptor OPTN (optineurin) regulates mesenchymal stem cell fate and bone-fat balance during aging by clearing FABP3.

Senile osteoporosis (OP) is often concomitant with decreased autophagic activity. OPTN (optineurin), a macroautophagy/autophagy (hereinafter referred to as autophagy) receptor, is found to play a pivotal role in selective autophagy, coupling autophagy with bone metabolism. However, its role in osteogenesis is still mysterious. Herein, we identified Optn as a critical molecule of cell fate decision for bone marrow mesenchymal stem cells (MSCs), whose expression decreased in aged mice. Aged mice revealed osteoporotic bone loss, elevated senescence of MSCs, decreased osteogenesis, and enhanced adipogenesis, as well as optn-/ - mice. Importantly, restoring Optn by transplanting wild-type MSCs to optn-/ - mice or infecting optn-/ - mice with Optn-containing lentivirus rescued bone loss. The introduction of a loss-of-function mutant of OptnK193R failed to reestablish a bone-fat balance. We further identified FABP3 (fatty acid binding protein 3, muscle and heart) as a novel selective autophagy substrate of OPTN. FABP3 promoted adipogenesis and inhibited osteogenesis of MSCs. Knockdown of FABP3 alleviated bone loss in optn-/ - mice and aged mice. Our study revealed that reduced OPTN expression during aging might lead to OP due to a lack of FABP3 degradation via selective autophagy. FABP3 accumulation impaired osteogenesis of MSCs, leading to the occurrence of OP. Thus, reactivating OPTN or inhibiting FABP3 would open a new avenue to treat senile OP.Abbreviations: ADIPOQ: adiponectin, C1Q and collagen domain containing; ALPL: alkaline phosphatase, liver/bone/kidney; BGLAP/OC/osteocalcin: bone gamma carboxyglutamate protein; BFR/BS: bone formation rate/bone surface; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CDKN1A/p21: cyclin-dependent kinase inhibitor 1A; CDKN2A/p16: cyclin dependent kinase inhibitor 2A; CDKN2B/p15: cyclin dependent kinase inhibitor 2B; CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha; COL1A1: collagen, type I, alpha 1; Ct. BV/TV: cortical bone volume fraction; Ct. Th: cortical thickness; Es. Pm: endocortical perimeter; FABP4/Ap2: fatty acid binding protein 4, adipocyte; H2AX: H2A.X variant histone; HE: hematoxylin and eosin; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MAR: mineral apposition rate; MSCs: bone marrow mesenchymal stem cells; NBR1: NBR1, autophagy cargo receptor; OP: osteoporosis; OPTN: optineurin; PDB: Paget disease of bone; PPARG: peroxisome proliferator activated receptor gamma; Ps. Pm: periosteal perimeter; qRT-PCR: quantitative real-time PCR; γH2AX: Phosphorylation of the Serine residue of H2AX; ROS: reactive oxygen species; RUNX2: runt related transcription factor 2; SA-GLB1: senescence-associated (SA)-GLB1 (galactosidase, beta 1); SP7/Osx/Osterix: Sp7 transcription factor 7; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 (human T cell leukemia virus type I) binding protein 1; Tb. BV/TV: trabecular bone volume fraction; Tb. N: trabecular number; Tb. Sp: trabecular separation; Tb. Th: trabecular thickness; µCT: micro computed tomography.

Ångstrom-scale silver particle-embedded carbomer gel promotes wound healing by inhibiting bacterial colonization and inflammation.

Poor wound healing after diabetes or extensive burn remains a challenging problem. Recently, we presented a physical approach to fabricate ultrasmall silver particles from Ångstrom scale to nanoscale and determined the antitumor efficacy of Ångstrom-scale silver particles (AgÅPs) in the smallest size range. Here we used the medium-sized AgÅPs (65.9 ± 31.6 Å) to prepare carbomer gel incorporated with these larger AgÅPs (L-AgÅPs-gel) and demonstrated the potent broad-spectrum antibacterial activity of L-AgÅPs-gel without obvious toxicity on wound healing-related cells. Induction of reactive oxygen species contributed to L-AgÅPs-gel-induced bacterial death. Topical application of L-AgÅPs-gel to mouse skin triggered much stronger effects than the commercial silver nanoparticles (AgNPs)-gel to prevent bacterial colonization, reduce inflammation, and accelerate diabetic and burn wound healing. L-AgÅPs were distributed locally in skin without inducing systemic toxicities. This study suggests that L-AgÅPs-gel represents an effective and safe antibacterial and anti-inflammatory material for wound therapy.

Fructose-coated Angstrom silver inhibits osteosarcoma growth and metastasis via promoting ROS-dependent apoptosis through the alteration of glucose metabolism by inhibiting PDK.

Osteosarcoma is a common malignant bone cancer easily to metastasize. Much safer and more efficient strategies are still needed to suppress osteosarcoma growth and lung metastasis. We recently presented a pure physical method to fabricate Ångstrom-scale silver particles (AgÅPs) and determined the anti-tumor efficacy of fructose-coated AgÅPs (F-AgÅPs) against lung and pancreatic cancer. Our study utilized an optimized method to obtain smaller F-AgÅPs and aimed to assess whether F-AgÅPs can be used as an efficient and safe agent for osteosarcoma therapy. We also investigated whether the induction of apoptosis by altering glucose metabolic phenotype contributes to the F-AgÅPs-induced anti-osteosarcoma effects. Methods: A modified method was developed to prepare smaller F-AgÅPs. The anti-tumor, anti-metastatic and pro-survival efficacy of F-AgÅPs and their toxicities on healthy tissues were compared with that of cisplatin (a first-line chemotherapeutic drug for osteosarcoma therapy) in subcutaneous or orthotopic osteosarcoma-bearing nude mice. The pharmacokinetics, biodistribution and excretion of F-AgÅPs were evaluated by testing the levels of silver in serum, tissues, urine and feces of mice. A series of assays in vitro were conducted to assess whether the induction of apoptosis mediates the killing effects of F-AgÅPs on osteosarcoma cells and whether the alteration of glucose metabolic phenotype contributes to F-AgÅPs-induced apoptosis. Results: The newly obtained F-AgÅPs (9.38 ± 4.11 nm) had good stability in different biological media or aqueous solutions and were more effective than cisplatin in inhibiting tumor growth, improving survival, attenuating osteolysis and preventing lung metastasis in osteosarcoma-bearing nude mice after intravenous injection, but were well tolerated in normal tissues. One week after injection, about 68% of F-AgÅPs were excreted through feces. F-AgÅPs induced reactive oxygen species (ROS)-dependent apoptosis of osteosarcoma cells but not normal cells, owing to their ability to selectively shift glucose metabolism of osteosarcoma cells from glycolysis to mitochondrial oxidation by inhibiting pyruvate dehydrogenase kinase (PDK). Conclusion: Our study suggests the promising prospect of F-AgÅPs as a powerful selective anticancer agent for osteosarcoma therapy.

Extracellular vesicles from human urine-derived stem cells inhibit glucocorticoid-induced osteonecrosis of the femoral head by transporting and releasing pro-angiogenic DMBT1 and anti-apoptotic TIMP1.

Osteonecrosis of the femoral head (ONFH) frequently occurs after glucocorticoid (GC) treatment. Extracellular vesicles (EVs) are important nano-sized paracrine mediators of intercellular crosstalk. This study aimed to determine whether EVs from human urine-derived stem cells (USC-EVs) could protect against GC-induced ONFH and focused on the impacts of USC-EVs on angiogenesis and apoptosis to explore the mechanism by which USC-EVs attenuated GC-induced ONFH. The results in vivo showed that the intravenous administration of USC-EVs at the early stage of GC exposure could rescue angiogenesis impairment, reduce apoptosis of trabecular bone and marrow cells, prevent trabecular bone destruction and improve bone microarchitecture in the femoral heads of rats. In vitro, USC-EVs reversed the GC-induced suppression of endothelial angiogenesis and activation of apoptosis. Deleted in malignant brain tumors 1 (DMBT1) and tissue inhibitor of metalloproteinases 1 (TIMP1) proteins were enriched in USC-EVs and essential for the USC-EVs-induced pro-angiogenic and anti-apoptotic effects in GC-treated cells, respectively. Knockdown of TIMP1 attenuated the protective effects of USC-EVs against GC-induced ONFH. Our study suggests that USC-EVs are a promising nano-sized agent for the prevention of GC-induced ONFH by delivering pro-angiogenic DMBT1 and anti-apoptotic TIMP1. STATEMENT OF SIGNIFICANCE: This study demonstrates that the intravenous injection of extracellular vesicles from human urine-derived stem cells (USC-EVs) at the early stage of glucocorticoid (GC) exposure efficiently protects the rats from the GC-induced osteonecrosis of the femoral head (ONFH). Moreover, this study identifies that the promotion of angiogenesis and inhibition of apoptosis by transferring pro-angiogenic DMBT1 and anti-apoptotic TIMP1 proteins contribute importantly to the USC-EVs-induced protective effects against GC-induced ONFH. This study suggests the promising prospect of USC-EVs as a new nano-sized agent for protecting against GC-induced ONFH, and the potential of DMBT1 and TIMP1 as the molecular targets for further augmenting the protective function of USC-EVs.

Shikonin relieves osteoporosis of ovariectomized mice by inhibiting RANKL-induced NF-κB and NFAT pathways.

Postmenopausal osteoporosis is very common in women. Currently, many kinds of new drugs are being developed for this disease. Postmenopausal osteoporosis is closely related to overactivity of osteoclasts in body. Shikonin is purple red naphthoquinone pigment extracted from lithospermum, which has anti-inflammation, antivirus, anticancer and other bioactivities. At the same time, it has been proved that shikonin can promote the proliferation and differentiation of osteoblasts, but its influence on osteoclasts and molecular mechanism are unknown. Our study showed that shikonin could inhibit the activity and formation of RANKL-mediated osteoclasts depending on dose without affecting the activity of bone marrow macrophages (BMM). In addition, we have also found that shikonin can inhibit the expression of specific marker gene of osteoclasts, including nuclear factor of activated T cells cytoplasmic 1 (NFATc1), cathepsin K (Ctsk), tartrate resistant acid phosphatase (TRAcP) and calcitonin receptor. Shikonin also could promote the proliferation of MC3T3-E1, increasing the expression of mRNA related to osteogenesis, like the expression of bone morphogenetic protein-2 (BMP-2), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN). Luciferase reporter gene assay and Western blot analysis further indicated that shikonin could inhibit the activity of RANKL-induced NF-κB and NFAT receptors. Moreover, shikonin can also slow down bone loss of ovariectomized (OVX) mice by inhibiting the activity of osteoclasts. This work explains the molecular mechanism of shikonin in RANKL-mediated formation of osteoclasts, and reveals the potential of further developing shikonin into a new drug for prevention and treatment of postmenopausal osteoporosis.

Fasting before or after wound injury accelerates wound healing through the activation of pro-angiogenic SMOC1 and SCG2.

Healing of the chronic diabetic ulceration and large burns remains a clinical challenge. Therapeutic fasting has been shown to improve health. Our study tested whether fasting facilitates diabetic and burn wound healing and explored the underlying mechanism. Methods: The effects of fasting on diabetic and burn wound healing were evaluated by analyzing the rates of wound closure, re-epithelialization, scar formation, collagen deposition, skin cell proliferation and neovascularization using histological analyses and immunostaining. In vitro functional assays were conducted to assess fasting and refeeding on the angiogenic activities of endothelial cells. Transcriptome sequencing was employed to identify the differentially expressed genes in endothelial cells after fasting treatment and the role of the candidate genes in the fasting-induced promotion of angiogenesis was demonstrated. Results: Two times of 24-h fasting in a week after but especially before wound injury efficiently induced faster wound closure, better epidermal and dermal regeneration, less scar formation and higher level of angiogenesis in mice with diabetic or burn wounds. In vitro, fasting alone by serum deprivation did not increase, but rather reduced the abilities of endothelial cell to proliferate, migrate and form vessel-like tubes. However, subsequent refeeding did not merely rescue, but further augmented the angiogenic activities of endothelial cells. Transcriptome sequencing revealed that fasting itself, but not the following refeeding, induced a prominent upregulation of a variety of pro-angiogenic genes, including SMOC1 (SPARC related modular calcium binding 1) and SCG2 (secretogranin II). Immunofluorescent staining confirmed the increase of SMOC1 and SCG2 expression in both diabetic and burn wounds after fasting treatment. When the expression of SMOC1 or SCG2 was down-regulated, the fasting/refeeding-induced pro-angiogenic effects were markedly attenuated. Conclusion: This study suggests that fasting combined with refeeding, but not fasting solely, enhance endothelial angiogenesis through the activation of SMOC1 and SCG2, thus facilitating neovascularization and rapid wound healing.

Synechococcus elongatus PCC7942 secretes extracellular vesicles to accelerate cutaneous wound healing by promoting angiogenesis.

Poor wound healing affects millions of people worldwide each year and needs better therapeutic strategies. Synechococcus elongatus PCC 7942 is a naturally occurring photoautotrophic cyanobacterium that can be easily obtained and large-scale expanded. Here, we investigated the therapeutic efficacy of this cyanobacterium in a mouse model of acute burn injury and whether the secretion of extracellular vesicles (EVs), important mediators of cell paracrine activity, is a key mechanism of the cyanobacterium-induced regulation of wound healing. Methods: The effects of Synechococcus elongatus PCC 7942 on burn wound healing in mice under light or dark conditions were evaluated by measuring wound closure rates, histological and immunofluorescence analyses. A series of assays in vivo and in vitro were conducted to assess the impact of the cyanobacterium on angiogenesis. GW4869 was used to interfere with the secretion of EVs by the cyanobacterium and the abilities of the GW4869-pretreated and untreated Synechococcus elongatus PCC 7942 to regulate endothelial angiogenesis were compared. The direct effects of the cyanobacterium-derived EVs (S. elongatus-EVs) on angiogenesis, wound healing and expressions of a class of pro-inflammatory factors that have regulatory roles in wound healing were also examined. Results: Synechococcus elongatus PCC 7942 treatment under light and dark conditions both significantly promoted angiogenesis and burn wound repair in mice. In vitro, the cyanobacterium enhanced angiogenic activities of endothelial cells, but the effects were markedly blocked by GW4869 pretreatment. S. elongatus-EVs were capable of augmenting endothelial angiogenesis in vitro, and stimulating new blood vessel formation and burn wound healing in mice. The expression of interleukin 6 (IL-6), which has an essential role in angiogenesis during skin wound repair, was induced in wound tissues and wound healing-related cells by S. elongatus-EVs and Synechococcus elongatus PCC 7942. Conclusion: Synechococcus elongatus PCC 7942 has the potential as a promising strategy for therapeutic angiogenesis and wound healing primarily by the delivery of functional EVs, not by its photosynthetic activity. The promotion of IL-6 expression may be a mechanism of the cyanobacterium and its EVs-induced pro-angiogenic and -wound healing effects.

Harmine enhances type H vessel formation and prevents bone loss in ovariectomized mice.

Recently, researchers identified a distinct vessel subtype called type H vessels that couple angiogenesis and osteogenesis. We previously found that type H vessels are reduced in ovariectomy (OVX)-induced osteoporotic mice, and preosteoclasts are able to secrete platelet-derived growth factor-BB (PDGF-BB) to stimulate type H vessel formation and thereby to promote osteogenesis. This study aimed to explore whether harmine, a ß-carboline alkaloid, is capable of preventing bone loss in OVX mice by promoting preosteoclast PDGF-BB-induced type H vessel formation. METHODS: The impact of harmine on osteoclastogenesis of RANKL-stimulated RAW264.7 cells was verified by gene expression analysis and tartrate-resistant acid phosphatase (TRAP) staining. Enzyme-linked immunosorbent assay (ELISA) was conducted to test PDGF-BB production by preosteoclasts. A series of angiogenesis-related assays in vitro were performed to assess the pro-angiogenic effects of the conditioned media from RANKL-stimulated RAW264.7 cells treated with or without harmine. Meanwhile, the role of PDGF-BB in this process was determined. In vivo, OVX mice were intragastrically administrated with harmine emulsion or an equal volume of vehicle. 2 months later, bone samples were collected for µCT, histological, immunohistochemical and immunofluorescent analyses to evaluate bone mass, osteogenic and osteoclastic activities, as well as the numbers of type H vessels. Bone marrow PDGF-BB concentrations were assessed by ELISA. RESULTS: Exposure of RANKL-stimulated RAW264.7 cells to harmine enhanced the formation of preosteoclasts and the production of PDGF-BB. Harmine augmented the ability of RANKL-stimulated RAW264.7 cells to promote angiogenesis of endothelial cells, whereas the effect was blocked by PDGF-BB inhibition. In vivo, the oral administration of harmine emulsion to OVX mice resulted in enhanced trabecular bone mass and osteogenic responses, increased numbers of preosteoclasts, as well as reduced numbers of osteoclasts and fat cells. Moreover, OVX mice treated with harmine exhibited higher levels of bone marrow PDGF-BB and much more type H vessels in bone. CONCLUSION: Harmine may exert bone-sparing effects by suppression of osteoclast formation and promotion of preosteoclast PDGF-BB-induced angiogenesis.

Exosomal DMBT1 from human urine-derived stem cells facilitates diabetic wound repair by promoting angiogenesis.

Chronic non-healing wounds represent one of the most common complications of diabetes and need advanced treatment strategies. Exosomes are key mediators of cell paracrine action and can be directly utilized as therapeutic agents for tissue repair and regeneration. Here, we explored the effects of exosomes from human urine-derived stem cells (USC-Exos) on diabetic wound healing and the underlying mechanism. Methods: USCs were characterized by flow cytometry and multipotent differentiation potential analyses. USC-Exos were isolated from the conditioned media of USCs and identified by transmission electron microscopy and flow cytometry. A series of functional assays in vitro were performed to assess the effects of USC-Exos on the activities of wound healing-related cells. Protein profiles in USC-Exos and USCs were examined to screen the candidate molecules that mediate USC-Exos function. The effects of USC-Exos on wound healing in streptozotocin-induced diabetic mice were tested by measuring wound closure rates, histological and immunofluorescence analyses. Meanwhile, the role of the candidate protein in USC-Exos-induced regulation of angiogenic activities of endothelial cells and diabetic wound healing was assessed. Results: USCs were positive for CD29, CD44, CD73 and CD90, but negative for CD34 and CD45. USCs were able to differentiate into osteoblasts, adipocytes and chondrocytes. USC-Exos exhibited a cup- or sphere-shaped morphology with a mean diameter of 51.57 ± 2.93 nm and positive for CD63 and TSG101. USC-Exos could augment the functional properties of wound healing-related cells including the angiogenic activities of endothelial cells. USC-Exos were enriched in the proteins that are involved in regulation of wound healing-related biological processes. Particularly, a pro-angiogenic protein called deleted in malignant brain tumors 1 (DMBT1) was highly expressed in USC-Exos. Further functional assays showed that DMBT1 protein was required for USC-Exos-induced promotion of angiogenic responses of cultured endothelial cells, as well as angiogenesis and wound healing in diabetic mice. Conclusion: Our findings suggest that USC-Exos may represent a promising strategy for diabetic soft tissue wound healing by promoting angiogenesis via transferring DMBT1 protein.

Liraglutide attenuates the osteoblastic differentiation of MC3T3­E1 cells by modulating AMPK/mTOR signaling.

Liraglutide, a synthetic analogue of glucagon-like peptide­1, is utilized in the treatment of type 2 diabetes and obesity. Liraglutide has been previously demonstrated to prevent osteoblastic differentiation of human vascular smooth muscle cells, resulting in the slowing of arterial calcification, however, its effect on bone formation remains unclear. The present study investigated the effect of liraglutide on osteoblastic differentiation using Alizarin Red S staining, and examined the molecular mechanisms underlying the regulatory effect by western blot analysis. The present study demonstrated that protein expression levels of phosphorylated adenosine monophosphate­activated protein kinase (p­AMPK) were downregulated in MC3T3­E1 cells during osteoblastic differentiation in commercial osteogenic differentiation medium, whereas protein expression levels of transforming growth factor­ß (TGF­ß) and phosphorylated mammalian target of rapamycin (p­mTOR) increased. Liraglutide was subsequently demonstrated to dose­dependently attenuate the osteoblastic differentiation of MC3T3­E1 cells, to upregulate p­AMPK, and downregulate p­mTOR and TGF­ß protein expression levels. Treatment with an AMPK­specific inhibitor, Compound C, eradicated the effect of liraglutide on osteoblastic differentiation, and p­mTOR and TGF­ß downregulation. An mTOR activator, MHY1485, also abolished the inhibitory effect of liraglutide on osteoblastic differentiation, and resulted in p­mTOR and TGF­ß downregulation, but did not attenuate the liraglutide­induced increase in p­AMPK protein expression levels. The results of the present study demonstrate that liraglutide attenuates osteoblastic differentiation of MC3T3­E1 cells via modulation of AMPK/mTOR signaling. The present study revealed a novel function of liraglutide, which contributes to the understanding of its pharmacological and physiological effects in clinical settings.

One-stage posterior focus debridement, fusion, and instrumentation in the surgical treatment of lumbar spinal tuberculosis with kyphosis in children.

OBJECTIVE: The purpose of this study is to investigate the clinical efficacy and feasibility of one-stage posterior focus debridement, fusion, and instrumentation in the surgical treatment of lumbar spinal tuberculosis with kyphosis in children. METHODS: From December 2007 to May 2012, 13 patients (six males and seven females) suffering from lumbar spinal tuberculosis with kyphosis were admitted. All patients were treated with one-stage posterior focus debridement, fusion, and instrumentation. Then, the clinical efficacy was estimated by statistical analysis based on the data about Frankel grade, the Cobb angle of kyphosis, and erythrocyte sedimentation rate (ESR), which were collected at certain time. RESULTS: The age of all patients ranged from 5 to 13 years (average, 8.8 years). Operation time ranged from 120 to 190 min (average, 165 min). Intraoperative blood loss ranged from 200 to 800 ml (average, 460 ml). All patients were followed up for 24 to 57 months postoperatively (average, 33.5 months). The Cobb angle was changed significantly between preoperation and postoperation (P < 0.05), and there was no significant loss at the last follow-up. The preoperation ESR (62.5 ± 15.7) returned to normal (16.6 ± 8.1) within 3 months postoperatively in all patients (P < 0.05). Bone fusion was achieved within 3-5 months (average, 3.5 months). In the 13 cases, no postoperative severe complications occurred and neurologic function improved in various degrees. CONCLUSION: The outcomes of follow-up showed that one-stage posterior focus debridement, fusion, and instrumentation can be an effective treatment method for the lumbar spinal tuberculosis with kyphosis in children.

ERß induces the differentiation of cultured osteoblasts by both Wnt/ß-catenin signaling pathway and estrogen signaling pathways.

Although 17ß-estradial (E2) is known to stimulate bone formation, the underlying mechanisms are not fully understood. Recent studies have implicated the Wnt/ß-catenin pathway as a major signaling cascade in bone biology. The interactions between Wnt/ß-catenin signaling pathway and estrogen signaling pathways have been reported in many tissues. In this study, E2 significantly increased the expression of ß-catenin by inducing phosphorylations of GSK3ß at serine 9. ERß siRNAs were transfected into MC3T3-E1 cells and revealed that ERß involved E2-induced osteoblasts proliferation and differentiation via Wnt/ß-catenin signaling. The osteoblast differentiation genes (BGP, ALP and OPN) and proliferation related gene (cyclin D1) expression were significantly induced by E2-mediated ERß. Furthermore immunofluorescence and immunoprecipitation analysis demonstrated that E2 induced the accumulation of ß-catenin protein in the nucleus which leads to interaction with T-cell-specific transcription factor/lymphoid enhancer binding factor (TCF/LEF) transcription factors. Taken together, these findings suggest that E2 promotes osteoblastic proliferation and differentiation by inducing proliferation-related and differentiation-related gene expression via ERß/GSK-3ß-dependent Wnt/ß-catenin signaling pathway. Our findings provide novel insights into the mechanisms of action of E2 in osteoblastogenesis.

One-stage combined anterior-posterior approach treatment of multiple cervicothoracic spinal tuberculosis with kyphosis.

OBJECTIVE: The purpose of this study was to investigate the clinical efficacy and feasibility of one-stage posterior fixation, anterior debridement, bone grafting and anterior fixation for multiple cervicothoracic spinal tuberculosis with kyphosis. METHODS: From December 2006 to June 2011, fifteen patients (seven males, eight females) suffering from cervicothoracic spinal tuberculosis with kyphosis were admitted. The pathologic change regions were as follows: two in the C6-C7 segment, two in the C6-T1 segment, one in the C6-T2 segment, three in the C7-T1 segment, two in the T1-T2 segment, two in the C7-T2 segment, one in the T1-T3 segment, and two in the T2-T3 segment. All patients were treated with one-stage surgical treatment by posterior fixation, anterior debridement, bone grafting, and anterior fixation. Then, the clinical efficacy was evaluated using statistical analysis based on the materials about the Cobb angle of kyphosis, Frankel grade and erythrocyte sedimentation rate (ESR), which were collected at certain times. RESULTS: All patients ages ranged from 17 to 67 years (average, 40.9 years). Operation time ranged from 180 to 290 min (average, 226 minutes). Intra-operative blood loss ranged from 400 to 1000 ml (average, 650 ml). All patients were followed up for 18-46 months postoperatively (average, 27.7 months). The kyphosis angle was changed significantly between pre-operation and postoperation (P < 0.05), and there was no obvious loss at the last follow-up. The ESR recovered to normal within three months postoperatively in all patients (P < 0.05). Bone fusion was achieved within three to six months (average, 5.5 months). In the 15 cases, no postoperative severe complications occurred and neurologic function was improved in various degrees. CONCLUSION: The outcomes of follow-up showed that the one-stage combined anterior-posterior approach can be an effective treatment method for multiple cervicothoracic spinal tuberculosis with kyphosis.