Hyperbaric oxygen therapy enhances osteointegration of reimplanted cranial flap by regulating osteogenesis-angiogenesis coupling.

Craniectomy is a lifesaving procedure to alleviate dangerously high intracranial pressure by removing a bone flap from the calvarium. However, the osteointegration of reimplanted bone flap with the existing bone tissue is still a clinical challenge. Hyperbaric oxygen (HBO) therapy has shown efficacy in promoting bone repair and could be a promising treatment for accelerating postoperative recovery. However, the specific cell types that are responsive to HBO treatment are not well understood. In this study, we created a murine model of craniectomy, with reimplantation of the cranial flap after 1 week. The effects of HBO treatment on bone formation and blood vessel formation around reimplanted bone were examined by micro-computed tomography, histological staining, and immunofluorescence staining. Single-cell RNA sequencing (scRNAseq) was utilized to identify key cell subtypes and signaling pathways after HBO treatment. We found that HBO treatment increased bone volume around reimplanted cranial flaps. HBO also increased the volume of Osterix-expressing cells and type H vessels. scRNAseq data showed more mature osteoblasts and endothelial cells, with higher expressions of adhesion and migration-related genes after HBO treatment. Cell-cell interaction analysis revealed a higher expression level of genes between mature osteoblasts and endothelial cells from the angiopoietin 2-integrin α5ß1 pathway. Taken together, HBO therapy promotes the healing process of craniectomy by regulating the crosstalk between vascular endothelial cells and osteogenic cells. These findings provide evidence in a preclinical model that HBO therapy enhances osteointegration by regulating angiogenesis-osteogenesis coupling, providing a scientific basis for utilizing HBO therapy for accelerating postoperative recovery after craniectomy.

Human Infrapatellar Fat Pad Mesenchymal Stem Cell-derived Extracellular Vesicles Purified by Anion Exchange Chromatography Suppress Osteoarthritis Progression in a Mouse Model.

BACKGROUND: Extracellular vesicles derived from mesenchymal stem cells (MSCs) show great promise in treating osteoarthritis (OA). However, studies from the perspective of clinical feasibility that consider an accessible cell source and a scalable preparation method for MSC-extracellular vesicles are lacking. QUESTIONS/PURPOSES: (1) Does an infrapatellar fat pad obtained from patients undergoing TKA provide a suitable source to provide MSC-extracellular vesicles purified by anion exchange chromatography? Using an in vivo mouse model for OA in the knee, (2) how does injection of the infrapatellar fat pad-derived MSC-extracellular vesicles alter gait, cartilage structure and composition, protein expression (Type II collagen, MMP13, and ADAMTS5), subchondral bone remodeling and osteophytes, and synovial inflammation? METHODS: The infrapatellar fat pad was collected from three patients (all female; 62, 74, 77 years) during TKA for infrapatellar fat pad-derived MSC culturing. Patients with infection, rheumatic arthritis, and age > 80 years were excluded. MSC-extracellular vesicles were purified by anion exchange chromatography. For the animal study, we used 30 male C57BL/6 mice aged 10 weeks, divided into six groups. MSC-extracellular vesicles were injected weekly into the joint of an OA mouse model during ACL transection (ACLT). To answer our first research question, we characterized MSCs based on their proliferative potential, differentiation capacity, and surface antigen expression, and we characterized MSC-extracellular vesicles by size, morphology, protein marker expression, and miRNA profile. To answer our second research question, we evaluated the effects of MSC-extracellular vesicles in the OA mouse model with quantitative gait analysis (mean pressure, footprint area, stride length, and propulsion time), histology (Osteoarthritis Research Society International Score based on histologic analysis [0 = normal to 24 = very severe degeneration]), immunohistochemistry staining of joint sections (protein expression of Type II collagen, MMP13, and ADAMTS5), and micro-CT of subchondral bone (BV/TV and Tb.Pf) and osteophyte formation. We also examined the mechanism of action of MSC-extracellular vesicles by immunofluorescent staining of the synovium membrane (number of M1 and M2 macrophage cells) and by analyzing their influence on the expression of inflammatory factors (relative mRNA level and protein expression of IL-1ß, IL-6, and TNF-α) in lipopolysaccharide-induced macrophages. RESULTS: Infrapatellar fat pads obtained from patients undergoing TKA provide a suitable cell source for producing MSC-extracellular vesicles, and anion exchange chromatography is applicable for isolating MSC-extracellular vesicles. Cultured MSCs were spindle-shaped, proliferative at Passage 4 (doubling time of 42.75 ± 1.35 hours), had trilineage differentiation capacity, positively expressed stem cell surface markers (CD44, CD73, CD90, and CD105), and negatively expressed hematopoietic markers (CD34 and CD45). MSC-extracellular vesicles purified by anion exchange chromatography had diameters between 30 and 200 nm and a typical cup shape, positively expressed exosomal marker proteins (CD63, CD81, CD9, Alix, and TSG101), and carried plentiful miRNA. Compared with the ACLT group, the ACLT + extracellular vesicle group showed alleviation of pain 8 weeks after the injection, indicated by increased area (0.67 ± 0.15 cm2 versus 0.20 ± 0.03 cm2, -0.05 [95% confidence interval -0.09 to -0.01]; p = 0.01) and stride length (5.08 ± 0.53 cm versus 6.20 ± 0.33 cm, -1.12 [95% CI -1.86 to -0.37]; p = 0.005) and decreased propulsion time (0.22 ± 0.06 s versus 0.11 ± 0.04 s, 0.11 [95% CI 0.03 to 0.19]; p = 0.007) in the affected hindlimb. Compared with the ACLT group, the ACLT + extracellular vesicles group had lower Osteoarthritis Research Society International scores after 4 weeks (8.80 ± 2.28 versus 4.80 ± 2.28, 4.00 [95% CI 0.68 to 7.32]; p = 0.02) and 8 weeks (16.00 ± 3.16 versus 9.60 ± 2.51, 6.40 [95% CI 2.14 to 10.66]; p = 0.005). In the ACLT + extracellular vesicles group, there was more-severe OA at 8 weeks than at 4 weeks (9.60 ± 2.51 versus 4.80 ± 2.28, 4.80 [95% CI 0.82 to 8.78]; p = 0.02), indicating MSC-extracellular vesicles could only delay but not fully suppress OA progression. Compared with the ACLT group, the injection of MSC-extracellular vesicles increased Type II collagen expression, decreased MMP13 expression, and decreased ADAMTS5 expression at 4 and 8 weeks. Compared with the ACLT group, MSC-extracellular vesicle injection alleviated osteophyte formation at 8 weeks and inhibited bone loss at 4 weeks. MSC-extracellular vesicle injection suppressed inflammation; the ACLT + extracellular vesicles group had fewer M1 type macrophages than the ACLT group. Compared with lipopolysaccharide-treated cells, MSC-extracellular vesicles reduced mRNA expression and inhibited IL-1ß, IL-6, and TNF-α in cells. CONCLUSION: Using an OA mouse model, we found that infrapatellar fat pad-derived MSC-extracellular vesicles could delay OA progression via alleviating pain and suppressing cartilage degeneration, osteophyte formation, and synovial inflammation. The autologous origin of extracellular vesicles and scalable purification method make our strategy potentially viable for clinical translation. CLINICAL RELEVANCE: Infrapatellar fat pad-derived MSC-extracellular vesicles isolated by anion exchange chromatography can suppress OA progression in a mouse model. Further studies with large-animal models, larger animal groups, and subsequent clinical trials are necessary to confirm the feasibility of this technique for clinical OA treatment.

Bone disease imaging through the near-infrared-II window.

Skeletal disorders are commonly diagnosed by X-ray imaging, but the radiation limits its use. Optical imaging through the near-infrared-II window (NIR-II, 1000-1700 nm) can penetrate deep tissues without radiation risk, but the targeting of contrast agent is non-specific. Here, we report that lanthanide-doped nanocrystals can passively target the bone marrow, which can be effective for over two months. We therefore develop the high-resolution NIR-II imaging method for bone disease diagnosis, including the 3D bone imaging instrumentation to show the intravital bone morphology. We demonstrate the monitoring of 1 mm bone defects with spatial resolution comparable to the X-ray imaging result. Moreover, NIR-II imaging can reveal the early onset inflammation as the synovitis in the early stage of rheumatoid arthritis, comparable to micro computed tomography (µCT) in diagnosis of osteoarthritis, including the symptoms of osteophyte and hyperostosis in the knee joint.

Axial Compressive Loading Attenuates Early Osteoarthritis by Reducing Subchondral Bone Remodeling.

BACKGROUND: Mechanical loading and alendronate (ALN) can be used as noninvasive physical therapy methods for osteoarthritis (OA). However, the timing and efficacy for treatments are unknown. PURPOSE: To determine whether the timing of mechanical loading and ALN influences the pathobiological changes of OA. STUDY DESIGN: Controlled laboratory study. METHODS: Mice with OA induced by anterior cruciate ligament transection were subjected to early (1-3 weeks) or late (5-7 weeks) axial compressive dynamic load or intraperitoneal injection of ALN. Changes in gait were analyzed using gait analysis system, pathobiological changes in subchondral bone, cartilage, osteophyte, and synovitis were assessed using micro-computed tomography, tartrate-resistant acid phosphatase staining, pathologic section staining, and immunohistochemistry at 1, 2, 4, and 8 weeks. RESULTS: At 1, 2, and 4 weeks, the OA limb had lower mean footprint pressure intensity, lower bone volume per tissue volume (BV/TV) in the subchondral bone, and more osteoclasts. At 4 weeks, the early loading, ALN, and load + ALN treatments induced less cartilage destruction, with a corresponding reduction in Osteoarthritis Research Society International score and increased hyaline cartilage thickness. The treatments also resulted in fewer osteoclasts and higher BV/TV and bone mineral density of subchondral bone and suppressed inflammation and interleukin 1ß- and tumor necrosis factor α-positive cells in synovium. At 8 weeks, early loading or load + ALN improved the mean footprint pressure intensity and knee flexion. At 8 weeks, early load + ALN had a synergistic effect on protecting hyaline cartilage and proteoglycans. Footprint pressure intensity and cartilage destruction were worse in late loading limbs, and no differences in BV/TV, bone mineral density, osteophyte formation, and synovium inflammation were found between the late load, ALN, and load + ALN groups and the anterior cruciate ligament transection group. CONCLUSION: Dynamic axial mechanical loading or ALN in the early stages of knee trauma protected against OA by suppressing subchondral bone remodeling. However, late loading promoted cartilage degeneration in advanced OA, indicating that reduced loading should be performed in the late stages of OA to avoid the acceleration of OA. CLINICAL RELEVANCE: Early low-level functional exercise or antiosteoporotic drugs could clearly slow or prevent the progression of early OA. For patients with mild to severe OA, loading reduction via brace protection or maintenance of joint stability via early ligament reconstruction surgery may ameliorate OA exacerbation.

Antibiotic-impregnated calcium sulfate for the treatment of pediatric hematogenous osteomyelitis.

BACKGROUND: Antibiotic-impregnated calcium sulfate has excellent curative efficacy in chronic osteomyelitis. However, its curative efficacy in pediatric hematogenous osteomyelitis has not been sufficiently studied. The purpose of this study was to evaluate the curative effects of antibiotic-impregnated calcium sulfate in the treatment of pediatric hematogenous osteomyelitis. METHODS: Overall, twenty-one pediatric patients with hematogenous osteomyelitis treated at our hospital between 2013 and 2018 were included for assessment. The clinical history, clinical manifestation, infection recurrence rate, sinus leakage, incision leakage, pathological fractures, bone growth and surgical procedures were analyzed. RESULTS: The infection recurrence rate was 0% (0/21) at a minimum of 31 months (range 31 to 91 months) of follow-up. Postoperative incision leakage was found in one pediatric patient. Osteolysis was found in one pediatric patient. Acceleration of bone growth occurred in one pediatric patient. Retardation of bone growth occurred in one pediatric patient. Genu valgus deformity occurred in one pediatric patient. CONCLUSIONS: Although noninfectious complications occurred, the curative effect of antibiotic-impregnated calcium sulfate in pediatric hematogenous osteomyelitis was satisfactory.

Metallic Scaffold with Micron-Scale Geometrical Cues Promotes Osteogenesis and Angiogenesis via the ROCK/Myosin/YAP Pathway.

The advent of precision manufacturing has enabled the creation of pores in metallic scaffolds with feature size in the range of single microns. In orthopedic implants, pore geometries at the micron scale could regulate bone formation by stimulating osteogenic differentiation and the coupling of osteogenesis and angiogenesis. However, the biological response to pore geometry at the cellular level is not clear. As cells are sensitive to curvature of the pore boundary, this study aimed to investigate osteogenesis in high- vs low-curvature environments by utilizing computer numerical control laser cutting to generate triangular and circular precision manufactured micropores (PMpores). We fabricated PMpores on 100 µm-thick stainless-steel discs. Triangular PMpores had a 30° vertex angle and a 300 µm base, and circular PMpores had a 300 µm diameter. We found triangular PMpores significantly enhanced the elastic modulus, proliferation, migration, and osteogenic differentiation of MC3T3-E1 preosteoblasts through Yes-associated protein (YAP) nuclear translocation. Inhibition of Rho-associated kinase (ROCK) and Myosin II abolished YAP translocation in all pore types and controls. Inhibition of YAP transcriptional activity reduced the proliferation, pore closure, collagen secretion, alkaline phosphatase (ALP), and Alizarin Red staining in MC3T3-E1 cultures. In C166 vascular endothelial cells, PMpores increased the VEGFA mRNA expression even without an angiogenic differentiation medium and induced tubule formation and maintenance. In terms of osteogenesis-angiogenesis coupling, a conditioned medium from MC3T3-E1 cells in PMpores promoted the expression of angiogenic genes in C166 cells. A coculture with MC3T3-E1 induced tubule formation and maintenance in C166 cells and tubule alignment along the edges of pores. Together, curvature cues in micropores are important stimuli to regulate osteogenic differentiation and osteogenesis-angiogenesis coupling. This study uncovered key mechanotransduction signaling components activated by curvature differences in a metallic scaffold and contributed to the understanding of the interaction between orthopedic implants and cells.

High efficiency degradation of tetracycline by peroxymonosulfate activated with Fe/NC catalysts: Performance, intermediates, stability and mechanism.

Carbon-based catalysts have the advantages of biological cleaning, eco-friendly and cost-effective in water treatment. While, nitrogen doped biochar promotes the development of non-radical peroxymonosulfate (PMS) activation in environmental remediation. Thus, three-dimensional sponge-like porous Fe and N co-doped biochar (Fe/CN-30) with high catalytic activity for PMS activation was synthesized. In a wide pH range (1-11), the Fe/CN-30 catalyst can efficiently degrade tetracycline (TC) with a small amount of PMS. The non-radical pathways are prominent in the TC decomposition process according to the quenching experiments, electron paramagnetic resonance (EPR) and gas chromatograph-mass spectrometer (GC-MS) analysis, in which the contribution of high-valent iron-oxo species (Fe(IV) = O) was dominant. X-ray photoelectron spectroscopy and reaction kinetic experiments confirmed that the coordination sites of Fe and N in the Fe/CN-30 are the reactive centers for TC degradation. Moreover, the successive addition of low concentration PMS into the system was confirmed to favor the PMS utilization, and the high selectivity of the Fe/CN-30 was confirmed by the analysis of pollutant structure. Furthermore, by-products of TC degradation in the Fe/CN-30/PMS system and the possible TC degradation pathways were proposed via liquid chromatography-mass spectrometry (LC-MS). Therefore, this study dedicates to providing new insights into the non-radical pathway-catalyzed AOPs.

[Application of picture archiving and communication systems in lateral wedge osteotomy for cubitus varus deformity in teenagers].

OBJECTIVE: To investigate effectiveness of picture archiving and communication systems (PACS) in lateral wedge osteotomy for cubitus varus deformity in teenagers. METHODS: A clinical data of 16 teenagers with cubitus varus deformity between July 2014 and July 2016 was retrospectively analyzed. All patients were treated with lateral wedge osteotomy and fixed with plate. Before operation, the osteotomy design (the osteotomy angle and length) was done in the PACS, including the carrying angle of healthy limb and the varus angle of affected side. There were 10 males and 6 females, with an average age of 11.4 years (range, 10-17 years). The disease duration ranged from 2 to 10 years (mean, 5.6 years). The preoperative X-ray film showed that the supracondylar fractures of the humerus had all healed, and 9 cases had internal rotation deformity; the varus angle of the affected side was 19.5°-33.5°. After operation, the fracture healing and cubitus varus deformity correction were observed by X-ray films, the elbow function was evaluated by Mayo scoring, and the elbow range of motion was detected. RESULTS: There was no significant difference between the actual intraoperative osteotomy angle and length and the preoperative design ( P>0.05). The hospital stay was 2-8 days, with an average of 4.5 days. No complication such as incision infection or ulnar nerve injury occurred. All 16 cases were followed up 12-18 months, with an average of 14 months. X-ray films showed that the osteotomy healed at 2-7 months after operation, with an average of 2.5 months. The internal fixators were removed within 8-14 months after operation (mean, 12.0 months). X-ray films measurement showed that the carrying angle of the affected side recovered to (10.3±2.0)° at 1 day after operation, which was not significantly different from that of the healthy side [(10.6±1.5)°] before operation ( t=0.480, P=0.637). The carrying angle of the affected side was (9.8±2.6)° at 1 year after operation, which was not significantly different from that of the healthy side [(10.4±1.6)°] at the same time point ( t=0.789, P=0.438). At 1 year after operation, the ranges of flexion and extension of affected side were (131.6±8.4)° and (6.4±2.6)°, respectively; and the ranges of flexion and extension of healthy side were (134.2±6.3)° and (5.9±2.2)°, respectively. There was no significant difference between the healthy and affected sides ( t=1.143, P=0.262; t=0.587, P=0.561). The elbow joint function at 1 year after operation evaluated by Mayo scoring standard rated as excellent in 9 cases, good in 6 cases, and fair in 1 case, and the excellent and good rate was 93.7%. CONCLUSION: Before lateral wedge osteotomy, the PACS is used to design the osteotomy angle and length, which can guide the operation and make the osteotomy more accurate and simple.

Carbon quantum dots decorated heteroatom co-doped core-shell Fe0@POCN for degradation of tetracycline via multiply synergistic mechanisms.

In this study, novel core-shell catalyst with a new ternary heterostructure was synthesized (Fe0@POCN/CQDs) for the degradation of tetracycline (TC). The TEM results showed that the Fe0 particles were wrapped in POCN material and many nano CQDs were uniformly dispersed in the material. The new ternary nanocomposite exhibits excellent photocatalytic activity for the removal of TC, which was approximately 4.76 times higher than that of GCN. The enhancement of photocatalytic activity was attributed to the effective heterojunction as well as the multiply synergistic effects of POCN combined with Fe0 and CQDs, which was beneficial for retardation of recombination rate of photogenerated electron-hole pairs and generation of more free radicals for the oxidation of TC. Besides, the reactive oxygen species (ROS) of h+, •O2- and •OH played pivotal roles in the degradation of TC by Fe0@POCN/CQDs during the photocatalytic reaction. At the same times, sulfate radical (SO4•-) and hydroxyl radical (•OH) highlighted the dominant role in the degradation process compared with other free radicals under persulfate hybrid mixture system (PS system), which was further confirmed by radical scavenger experiments and electron spin resonance (ESR) analysis. The response surface methodology (RSM) study indicated that the optimal removal parameters of tetracycline could reach 97.57% within 30 min under PS system. In addition, the possible degradation pathway intermediates of TC were studied by HPLC-MS and the reaction catalytic activity mechanism of Fe0@POCN/CQDs/persulfate system was discussed.

Mechanisms of action of neuropeptide Y on stem cells and its potential applications in orthopaedic disorders.

Musculoskeletal disorders are the leading causes of disability and result in reduced quality of life. The neuro-osteogenic network is one of the most promising fields in orthopaedic research. Neuropeptide Y (NPY) system has been reported to be involved in the regulations of bone metabolism and homeostasis, which also provide feedback to the central NPY system via NPY receptors. Currently, potential roles of peripheral NPY in bone metabolism remain unclear. Growing evidence suggests that NPY can regulate biological actions of bone marrow mesenchymal stem cells, hematopoietic stem cells, endothelial cells, and chondrocytes via a local autocrine or paracrine manner by different NPY receptors. The regulative activities of NPY may be achieved through the plasticity of NPY receptors, and interactions among the targeted cells as well. In general, NPY can influence proliferation, apoptosis, differentiation, migration, mobilization, and cytokine secretion of different types of cells, and play crucial roles in the development of bone delayed/non-union, osteoporosis, and osteoarthritis. Further basic research should clarify detailed mechanisms of action of NPY on stem cells, and clinical investigations are also necessary to comprehensively evaluate potential applications of NPY and its receptor-targeted drugs in management of musculoskeletal disorders.

Calcitonin gene-related peptide promotes proliferation and inhibits apoptosis in endothelial progenitor cells via inhibiting MAPK signaling.

BACKGROUND: Calcitonin gene-related peptide (CGRP) contributes to bone formation by stimulating bone marrow stromal cell (BMSC) proliferation and differentiation. However, the proliferative and apoptotic effects of CGRP on bone marrow-derived endothelial progenitor cells (EPCs) have not been investigated. METHODS: We tested the effects of CGRP on EPC proliferation and apoptosis by Cell Counting Kit-8, flow cytometry, and studied the effects of CGRP on the expression of proliferation- and apoptosis-related markers in EPCs and the underlying mitogen-activated protein kinase (MAPK) signalling pathway by quantitative polymerase chain reaction and western blotting. RESULTS: We detected EPC markers (CD34, CD133 and VEGFR-2) in 7-day cultures and found that CGRP (10- 10-10- 12 M) promoted the proliferation of cultured EPCs, with a peak increase of 30% at 10- 10 M CGRP. CGRP also upregulated the expression of proliferation-associated genes, including cyclin D1 and cyclin E, and increased the percentages of G2/M-phase and S-phase cells after incubation 72 h. CGRP inhibited serum deprivation (SD)-induced apoptosis in EPCs after 24 and 48 h and downregulated the expression of apoptosis-related genes, including caspase-3, caspase-8, caspase-9 and Bax. Phosphorylated (p-)ERK1/2, p-p38 and p-JNK protein levels in EPCs treated with CGRP were significantly lower than those in untreated EPCs. Pre-treatment with the calcitonin receptor-like receptor (CRLR) antagonist CGRP8-37 or a MAPK pathway inhibitor (PD98059, SB203580 or SP600125) completely or partially reversed the pro-proliferative and anti-apoptotic effects and the reduced p-ERK1/2, p-p38 and p-JNK expression induced by CGRP. CONCLUSION: Our results show that CGRP exerts pro-proliferative and anti-apoptotic effects on EPCs and may act by inhibiting MAPK pathways.

[Neuropeptide Y Y1 receptor antagonist PD160170 promotes osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro and femoral defect repair in rats].

OBJECTIVE: To investigate the effects of neuropeptide Y (NPY) Y1 receptor antagonist PD160170 in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and accelerating healing of femoral defect in rats. METHODS: The third generation of rat BMSCs were treated with PBS (control) or 10-6, 10-7, or 10-8 mol/L NPY Y1 receptor antagonist PD160170. After 7 and 14 days of treatment, the cells were examined for osteogenic differentiation with alkaline phosphatase (ALP) and alizarin red staining. At 7 and 21 days of treatment, the mRNA and protein expressions of collagen type I (COLI), osteocalcin (OCN) and Runt-related transcription factor 2 (Runx2) in the cells were detected using q-PCR and Westem Blotting. In a male SD rat model (body weight 300∓20 g) of bilateral femoral condyle defects (2.5 mm in diameter), the effect of daily local injection of 0.2 mL PD160170 (10-6 and 10-8 mol/L, for 28 consecutive days) in promoting bone defect repair was evaluated with micro-CT scans. RESULTS: ALP and alizarin red staining showed that the BMSCs treated with PD160170, at the optimal concentration of 10-8 mol/L, contained more intracellular cytoplasmic brown particles and mineralized nodules in extracellular matrix than PBS-treated cells. PD160170 (10-8 mol/L) significantly up-regulated the mRNA and protein expressions of COLI at day 7 and those of OCN and Runx2 at day 21 (P<0.05). In the rat models of femoral bone defect, the volume/tissue volume ratio, bone mineral density and the number of bone trabeculae were significantly greater in 10-6 mol/L PD160170 group than in the control group (P<0.05), but the bone trabecular thickness (P=0.07) and bone volume (P=0.35) were similar between the two groups. CONCLUSION: NPY Y1 receptor antagonist PD160170 can promote osteogenic differentiation of BMSCs and healing of femoral defects in rats, suggesting the potential of therapeutic strategies targeting NPY Y1 receptor signaling in the prevention and treatment of bone fracture and osteoporosis.

Repair of Osteochondral Defects in a Rabbit Model Using Bilayer Poly(Lactide-co-Glycolide) Scaffolds Loaded with Autologous Platelet-Rich Plasma.

BACKGROUND To examine the effects of the addition of autologous platelet-rich plasma (PRP) into bilayer poly(lactide-co-glycolide) (PLGA) scaffolds on the reconstruction of osteochondral defects in a rabbit model. MATERIAL AND METHODS Porous PLGA scaffolds were prepared in a bilayered manner to reflect the structure of chondral and subchondral bone. Bone defects, measuring 4 mm in diameter and 4 mm in thickness, were created in both knee joints in 18 healthy New Zealand white rabbits, aged between 120-180 days old. Rabbits were randomly divided into three groups: rabbits with bone defects implanted with bilayer PLGA scaffolds (PLGA group) (N=6); or with bilayer PLGA and autologous PRP (PLGA/PRP group) (N=6); and the untreated group (control group) (N=6). The gross morphology, histology, and immunohistochemistry for the expression of collagen type II and aggrecan were observed at 12 weeks after surgery and compared using a scoring system. Micro-computed tomography (CT) imaging and relative expression of specific genes were also assessed. RESULTS The platelet concentrations in the PRP samples were found to be 4.9 times greater than that of whole blood samples. The total score on gross appearance and histology was greatest in the PLGA/PRP group, as was the expression of collagen II and aggrecan of the neo-tissue. Micro-CT imaging showed that more subchondral bone was formed in the PLGA/PRP group. CONCLUSIONS Bilayer PLGA scaffolds loaded with autologous PRP improve the reconstruction of osteochondral defects in the rabbit model.

Neuropeptide Y enhances proliferation and prevents apoptosis in rat bone marrow stromal cells in association with activation of the Wnt/ß-catenin pathway in vitro.

Neuropeptide Y (NPY) exhibits a critical but poorly understood regulatory signaling function and has been shown to promote proliferation, vascularization and migration in several types of cells and tissues. However, little is known about the specific role of NPY in the proliferation and apoptosis of bone marrow stromal cells (also known as bone marrow-derived mesenchymal stem cells, BMSCs), which contain a subpopulation of multipotent skeletal stem cells. Based on BrdU incorporation tests, Cell Counting Kit-8, flow cytometry, quantitative polymerase chain reaction and western blotting, we showed that NPY significantly promoted the proliferation of BMSCs in a concentration-dependent manner, with a maximal effect observed at a concentration of 10-10M for pro-proliferative and 10-12M for anti-apoptotic activities. Furthermore, NPY significantly increased the percentage of cells in S and G2/M phases. In addition, NPY exhibited a protective effect after 24h of serum starvation as illustrated by a reduction in the apoptosis rate, degree of nuclear condensation, and expression of apoptosis markers, including caspase-3, caspase-9 and Bax mRNA expression. NPY also increased the mRNA and protein expression levels of canonical Wnt signaling pathway proteins, including ß-catenin and c-myc, during the induced proliferative and anti-apoptotic processes. However, the proliferative and anti-apoptotic activities of NPY were partially blocked by both PD160170 (1µM) and DKK1 (0.2µg/mL). These compounds also blocked the mRNA and protein expression of ß-catenin, p-GSK-3ß and c-myc. Therefore, the results of the present study demonstrated that NPY exerts a proliferative and protective effect on BMSCs in a dose- and time-dependent manner in vitro, and importantly, these effects may be mediated via its Y1 receptor and involved in activation of the canonical Wnt signaling pathway.

Safe Zone of Posterior Screw Insertion for Talar Neck Fractures on 3-Dimensional Reconstruction Model.

OBJECTIVE: To investigate the optimal posterior screw placement and the geometry of safe zones for screw insertion in the talar neck. METHODS: Computed tomography data for 15 normal feet were imported into Mimics 10.01 software for 3-dimensional reconstruction; 4.0-mm-diameter screws were simulated from the lateral tubercle of the posterior process of the talus to the talar head. The range of screw paths trajectories and screw lengths at nine locations that did not breach the cortex of the talus were evaluated. In addition, the farthest (point a) and nearest point (point b) of the safe zone to the subtalar joint at each location, the anteversion angle (angle A), which is parallel to the sagittal plane, and the horizontal angle (angle B), which is perpendicular to the sagittal plane, were measured. RESULTS: The safe zone was mainly between the 30% location and the 60% location; the width of each safe zone was 13.6° ± 1.4°; the maximum height of each safe zone was 7.8° ± 1.2°. The height of the safe zone was lowest at the 30% location (4.5°) and highest at the 50% location (7.3°). The mixed safe zone of all tali was between the 50% location and the 60% location. When a screw was inserted at point a, the safe entry distance (screw length) ranged from 48.8 to 49.5 mm, and when inserted to point b, the distance ranged from 48.2 to 48.9 mm. And inserting a 48.7 mm screw, 5.6° laterally and 7.4° superiorly, from the lateral tubercle of the posterior process of the talus towards the talar head is safest. CONCLUSION: The safe zone of posterior screw fixation have been defined applying to most talus, assuming the fractures are well reduced, this may strengthen the stability, shorten the operation time and reduce the incidence of surgical complications.

The fast track to canonical Wnt signaling in MC3T3-E1 cells protected by substance P against serum deprivation-induced apoptosis.

The canonical Wnt pathway is vital to bone physiology by increasing bone mass through elevated osteoblast survival. Although investigated extensively in stem cells, its role in osteoblastic MC3T3-E1 cells has not been completely determined. To explore how this pathway is regulated by different conditions, we assessed the anti-apoptotic effects of substance P on the canonical Wnt pathway in MC3T3-E1 cells by treating cells with serum deprivation or serum starving with "substance P," a neuropeptide demonstrated to promote bone growth and stimulate Wnt signaling. The results showed that serum deprivation both induced apoptosis and activated Wnt signal transduction while substance P further stimulated the Wnt pathway via the NK-1 receptor but protected the cells from apoptotic death. Fast-tracking of Wnt signaling by substance P was also noted. These results indicate that nutritional deprivation and substance P synergistically activated the canonical Wnt pathway, a finding that helps to reveal the role of Wnt signaling in bone physiology affected by nutritional deprivation and neuropeptide substance P.

Neuropeptide Y stimulates osteoblastic differentiation and VEGF expression of bone marrow mesenchymal stem cells related to canonical Wnt signaling activating in vitro.

Neuropeptide Y (NPY) is a neuropeptide secreted by sensory nerve fibers distributed in the marrow and vascular canals of bone tissue. However, the effect of NPY on the osteogenic ability of bone marrow mesenchymal stem cells (BMSCs) remains controversial and has not been thoroughly investigated. To explore the osteogenic activity and the migration and VEGF expression capabilities of BMSCs affected by NPY, as well as the underlying mechanisms, we investigated the potential relationships among NPY, osteoblastic differentiation, angiogenesis and canonical Wnt signaling in BMSCs. NPY was observed to regulate osteoblastic differentiation at concentrations ranging from 10(-8) to 10(-12)mol/L, and the effects of NPY on the levels of Wnt signaling proteins were detected using Western blotting. To unravel the underlying mechanism, BMSCs were treated with NPY after pretreatment with the NPY-1R antagonist PD160170 or the Wnt pathway antagonist DKK1, and gene expression levels of Wnt signaling molecules and osteoblastic markers were determined by qPCR. Our results indicated that NPY significantly promoted osteoblastic differentiation of BMSCs in a concentration-dependent manner and up-regulated the expression levels of proteins including ß-catenin and p-GSK-3ß and the mRNA level of ß-catenin. Moreover, NPY promoted the translocation of ß-catenin into nucleus. The effects of NPY were inhibited by PD160170 or DKK1. Additionally, NPY enhanced the ability of BMSCs to migrate and promoted the expression of vascular endothelial growth factor (VEGF) as measured by immunocytochemical staining, qPCR and Western blot. These results suggested that NPY may stimulate osteoblastic differentiation via activating canonical Wnt signaling and enhance the angiogenic capacity of BMSCs.

Protective Effect of Neuropeptide Substance P on Bone Marrow Mesenchymal Stem Cells against Apoptosis Induced by Serum Deprivation.

Substance P (SP) contributes to bone formation by stimulating the proliferation and differentiation of bone marrow stromal cells (BMSCs); however, the possible involved effect of SP on apoptosis induced by serum deprivation (SD) in BMSCs is unclear. To explore the potential protective effect of SP and its mechanism, we investigated the relationships among SP, apoptosis induced by SD, and Wnt signaling in BMSCs. SP exhibited a protective effect, as indicated by a reduction in the apoptotic rate, nuclear condensation, caspase-3 and caspase-9 activation, and the ratio of Bax/Bcl-2 that was observed after 24 h of SD. This protective effect was blocked by the inhibition of Wnt signaling or antagonism of the NK-1 receptor. Moreover, SP promoted the mRNA and protein expression of Wnt signaling molecules such as ß-catenin, p-GSK-3ß, c-myc, and cyclin D1 in addition to the nuclear translocation of ß-catenin, indicating that active Wnt signaling is involved in SP inhibition of apoptosis. Our results revealed that mediated by the NK-1 receptor, SP exerts an inhibitory effect on serum deprivation induced apoptosis in BMSCs that is related to the activation of canonical Wnt signaling.

Involvement of group I mGluRs in LTP induced by strong high frequency stimulation in the dentate gyrus in vitro.

The involvement of group I metabotropic glutamate receptors (mGluRs) and ryanodine receptors was investigated in the induction of LTP induced either by application of one standard high frequency stimulation (HFS) or by strong multiple HFS in the medial perforant path to granule cell synapse of the rat dentate gyrus. Whilst a standard brief HFS induced LTP close to 50%, strong stimulation consisting of multiple HFS induced a much larger LTP. mGluR5 was found to be partially involved in the induction of the enhanced LTP induced by the strong HFS but not in the standard LTP induced by the brief HFS. Thus the mGluR5 antagonists LY341495 and MPEP partially inhibited the induction of LTP induced by strong HFS but did not inhibit LTP induced by a standard HFS. Ryanodine was found to partially inhibit LTP induced by the strong HFS but not to inhibit the standard LTP induced by the brief HFS, demonstrating the involvement of Ca-induced Ca release from ryanodine-sensitive Ca stores in the former. These studies demonstrate that the large amplitude LTP induced by strong stimulation involves additional mechanisms to the LTP induced by brief HFS, in particular involving activation of mGluR5 and RyR-sensitive Ca stores.

Long-term potentiation is mediated by multiple kinase cascades involving CaMKII or either PKA or p42/44 MAPK in the adult rat dentate gyrus in vitro.

The induction of NMDA-receptor-dependent long-term potentiation (LTP) in adult CA1 is contingent on activation of Ca/calmodulin-dependent protein kinase II (CaMKII). However, little is known about kinase mediation of LTP in the dentate gyrus. In the present study, the involvement of the kinases CaMKII, PKA, and MAPK in the induction of LTP was studied in the dentate gyrus of adult rats. Individual application of selective inhibitors of CaMKII, MEK, or PKA did not inhibit induction of LTP. In contrast, coapplication of a CaMKII inhibitor with either a PKA or MEK inhibitor resulted in a strong block of LTP. Induction of LTP was blocked by the coapplication of the inhibitors CaMKII and PKA or MEK, both when they were applied 1 h before the induction stimulus and also when they were applied after the induction stimulus. Thus LTP is mediated by either of two parallel cascades, one involving CaMKII and the other PKA or MAPK. Moreover, these cascades are active for a certain period after the induction stimulus.