3D-printed polyether-ether-ketone/n-TiO2 composite enhances the cytocompatibility and osteogenic differentiation of MC3T3-E1 cells by downregulating miR-154-5p.

The object was to enhance the bioactivity of pure polyether-ether-ketone (PEEK) by incorporating nano-TiO2 (n-TiO2) and investigate its potential mechanism. PEEK/n-TiO2 composite was manufactured using a 3D PEEK printer and characterized by scanning electron microscopy (SEM), 3D profiler, energy-dispersive spectroscopy, and Fourier-transform infrared (FT-IR) analyses. Cytocompatibility was tested using SEM, fluorescence, and cell counting kit-8 assays. Osteogenic differentiation was evaluated by osteogenic gene and mineralized nodule levels. The expression of the candidate miRNAs were detected in composite group, and its role in osteogenic differentiation was studied. As a results the 3D-printed PEEK/n-TiO2 composite (Φ = 25 mm, H = 2 mm) was successfully fabricated, and the TiO2 nanoparticles were well distributed and retained the nanoscale size of the powder. The Ra value of the composite surface was 2.69 ± 0.29, and Ti accounted for 22.29 ± 12.09% (in weight), and FT-IR analysis confirmed the characteristic peaks of TiO2. The cells in the composite group possessed better proliferation and osteogenic differentiation abilities than those in the PEEK group. miR-154-5p expression was decreased in the composite group, and the inhibition of miR-154-5p significantly enhanced the proliferation and osteogenic differentiation abilities. In conclusion, 3D-printed PEEK/n-TiO2 composite enhanced cytocompatibility and osteogenic induction ability by downregulating miR-154-5p, which provides a promising solution for improving the osteointegration of PEEK.

Platelet-rich plasma has beneficial effects in mice with osteonecrosis of the femoral head by promoting angiogenesis.

Platelet-rich plasma (PRP) is autologous and multifunctional. Platelet concentrate from blood contains highly concentrated platelets and various types of cells, including growth factors. PRP promotes the recovery of cell proliferation and differentiation. Osteonecrosis of the femoral head is a disease caused by femoral head damage or an insufficient blood supply, which leads to the death of bone cells and abnormal bone marrow composition. The subsequent repair of bone cells may result in changes to the structure of femoral head, femoral head collapse and joint dysfunction. PRP may promote the repair of injured articular cartilage in patients with joint diseases through the removal of harmful inflammatory factors. In the present study, the therapeutic effects and primary mechanism of PRP action were investigated using a glucocorticoid-induced femoral head osteonecrosis mouse model. Dexamethasone (DEX) and phosphate-buffered saline were used as controls. The therapeutic efficacy of PRP to treat osteonecrosis in murine femoral heads was evaluated by assessing clinical arthritis scores. The present study indicated that mice with osteonecrosis of the femoral head treated with PRP exhibited downregulated expression of interleukin (IL)-17A, IL-1ß, tumor necrosis factor-α, receptor activator of nuclear factor κ-B ligand, IL-6 and interferon-γ in the inflammatory tissue. In addition, the levels of hepatocyte growth factor, intercellular adhesion molecule-1, osteopontin, platelet-derived endothelial cell growth factor, vascular endothelial growth factor, platelet-derived growth factor, insulin-like growth factor-1 and transforming growth factor-ß were increased following treatment with PRP. Joint tissue histological staining demonstrated that PRP alleviated osteonecrosis of the femoral head and reduced humoral and cellular immune responses that promoted beneficial effects on the histological parameters. Furthermore, the concentration of glucocorticoids were significantly decreased in the serum of PRP-treated mice with osteonecrosis compared with the DEX group (P<0.01). Notably, PRP promoted beneficial effects in mice with osteonecrosis of the femoral head by stimulating angiogenesis. Therefore, the present study indicated that treatment with PRP promotes beneficial effects by preventing joint inflammation, cartilage destruction and bone damage, and stimulating the repair of joint tissue in mice with osteonecrosis of the femoral head. These preclinical data suggest that PRP may be developed as a novel method of treating osteonecrosis of the femoral head.

Low-1 level mechanical vibration improves bone microstructure, tissue mechanical properties and porous titanium implant osseointegration by promoting anabolic response in type 1 diabetic rabbits.

Type 1 diabetes mellitus (T1DM) is associated with reduced bone mass, increased fracture risk, and impaired bone defect regeneration potential. These skeletal complications are becoming important clinical challenges due to the rapidly increasing T1DM population, which necessitates developing effective treatment for T1DM-associated osteopenia/osteoporosis and bone trauma. This study aims to investigate the effects of whole-body vibration (WBV), an easy and non-invasive biophysical method, on bone microstructure, tissue-level mechanical properties and porous titanium (pTi) osseointegration in alloxan-diabetic rabbits. Six non-diabetic and twelve alloxan-treated diabetic rabbits were equally assigned to the Control, DM, and DM with WBV stimulation (WBV) groups. A cylindrical drill-hole defect was established on the left femoral lateral condyle of all rabbits and filled with a novel non-toxic Ti2448 pTi. Rabbits in the WBV group were exposed to 1h/day WBV (0.3g, 30Hz) for 8weeks. After sacrifice, the left femoral condyles were harvested for histological, histomorphometric and nanoindentation analyses. The femoral sample with 2-cm height above the defect was used for qRT-PCR analysis. The right distal femora were scanned with µCT. We found that all alloxan-treated rabbits exhibited hyperglycemia throughout the experimental period. WBV inhibited the deterioration of cancellous and cortical bone architecture and tissue-level mechanical properties via µCT, histological and nanoindentation examinations. T1DM-induced reduction of bone formation was inhibited by WBV, as evidenced by elevated serum OCN and increased mineral apposition rate (MAR), whereas no alteration was observed in bone resorption marker TRACP5b. WBV also stimulated more adequate ingrowths of mineralized bone tissue into pTi pore spaces, and improved peri-implant bone tissue-level mechanical properties and MAR in T1DM bone defects. WBV mitigated the reductions in femoral BMP2, OCN, Wnt3a, Lrp6, and ß-catenin and inhibited Sost mRNA expression but did not alter RANKL or RANK gene expression in T1DM rabbits. Our findings demonstrated that WBV improved bone architecture, tissue-level mechanical properties, and pTi osseointegration by promoting canonical Wnt signaling-mediated skeletal anabolic response. This study not only advances our understanding of T1DM skeletal sensitivity in response to external mechanical cues but also offers new treatment alternatives for T1DM-associated osteopenia/osteoporosis and osseous defects in an economic and highly efficient manner.

Platelet-rich plasma exhibits beneficial effects for rheumatoid arthritis mice by suppressing inflammatory factors.

Platelet-rich plasma (PRP) is a multifunctional blood product containing highly concentrated platelets, and various cell growth factors which promote cell proliferation and differentiation. PRP exhibited benefits in injurious articular cartilage repair and the removal of inflammatory factors in clinical studies. Rheumatoid arthritis (RA) is an autoimmune disease manifesting primarily as inflammatory arthritis, which is associated with notable morbidity in humans. In the present study, the therapeutic effects and primary mechanism of PRP on a type II collagen­induced arthritis (CIA) mouse model was investigated. Inflammatory factors interleukin (IL)­6, IL­8, IL­17, IL­1ß, tumor necrosis factor (TNF)­α and interferon (IFN)­Î³ were analyzed in PRP and PBS­treated groups. Vascular endothelial growth factor (VEGF), platelet­derived growth factor (PDGF), insulin­like growth factor (IGF)­1 and transforming growth factor (TGF)­ß expression in peripheral whole blood was additionally analyzed. The therapeutic efficacy of PRP for RA mice was evaluated using clinical arthritis scores. The results of the present study demonstrated that treatment with PRP alleviated arthritis, and reduced humoral and cellular immune responses, leading to beneficial effects on histological parameters as observed using joint tissue histological staining. CIA mice treated with PRP exhibited downregulated expression of IL­6, IL­8, IL­17A, IL­1ß, TNF­α, receptor activator for nuclear factor­κB and IFN­Î³ in inflammatory tissue. In addition, VEGF, PDGF, IGF­1 and TGF­ß expression in peripheral whole blood was increased following treatment with PRP. The serum concentration of anti­collagen antibody was decreased in PRP­treated CIA mice. In conclusion, CIA mice treated with PRP exhibited beneficial effects, including decreased joint inflammation, cartilage destruction and bone damage, and increased repair of joint tissue. The results of the present study suggested that PRP may be an effective therapeutic agent for RA.

Platelet-rich plasma inhibits inflammatory factors and represses rheumatoid fibroblast-like synoviocytes in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic disease affecting daily life of numerous patients, and uncontrolled proliferation of synovial fibroblasts plays vital role during the pathology of RA. Platelet-rich plasma (PRP), widely used in tissue regeneration and pain management, is rarely studied in RA. This study aims to investigate the effect of PRP on synovial fibroblasts during RA. Rheumatoid fibroblast-like synoviocyte MH7A cells were stimulated by lipopolysaccharide (LPS) to simulate RA conditions and treated with PRP, after that the concentration of inflammatory factors interleukin (IL) 1ß, tumor necrosis factor alpha (TNFα) and IL6 in the supernatant of culture medium was quantified by ELISA. MTT assay, flow cytometry and tube formation assay were performed to assess changes in cell viability, apoptosis and effect on angiogenesis in vitro, respectively. Besides, the expression levels of main factors in the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signal pathway were examined. Results showed that PRP markedly inhibited the production of IL1ß, TNFα and IL6 (P < 0.05) that was stimulated by LPS. LPS promoted MH7A cell viability, inhibited apoptosis and accelerated angiogenesis in vitro, while PRP could markedly relieve these effects (P < 0.05). The mRNA and protein levels of AKT1, PI3K (p58) and nuclear factor κ beta were elevated by LPS and then suppressed by PRP (P < 0.01). This study uncovered the potential of PRP in inhibiting inflammation, repressing synovial fibroblasts and regulating the PI3K/AKT signaling, providing basic proof for future application of PRP in managing RA. Further investigation is necessary to reveal detailed mechanism of PRP.

Pulsed electromagnetic fields promote osteogenesis and osseointegration of porous titanium implants in bone defect repair through a Wnt/ß-catenin signaling-associated mechanism.

Treatment of osseous defects remains a formidable clinical challenge. Porous titanium alloys (pTi) have been emerging as ideal endosseous implants due to the excellent biocompatibility and structural properties, whereas inadequate osseointegration poses risks for unreliable long-term implant stability. Substantial evidence indicates that pulsed electromagnetic fields (PEMF), as a safe noninvasive method, inhibit osteopenia/osteoporosis experimentally and clinically. We herein investigated the efficiency and potential mechanisms of PEMF on osteogenesis and osseointegration of pTi in vitro and in vivo. We demonstrate that PEMF enhanced cellular attachment and proliferation, and induced well-organized cytoskeleton for in vitro osteoblasts seeded in pTi. PEMF promoted gene expressions in Runx2, OSX, COL-1 and Wnt/ß-catenin signaling. PEMF-stimulated group exhibited higher Runx2, Wnt1, Lrp6 and ß-catenin protein expressions. In vivo results via µCT and histomorphometry show that 6-week and 12-week PEMF promoted osteogenesis, bone ingrowth and bone formation rate of pTi in rabbit femoral bone defect. PEMF promoted femoral gene expressions of Runx2, BMP2, OCN and Wnt/ß-catenin signaling. Together, we demonstrate that PEMF improve osteogenesis and osseointegration of pTi by promoting skeletal anabolic activities through a Wnt/ß-catenin signaling-associated mechanism. PEMF might become a promising biophysical modality for enhancing the repair efficiency and quality of pTi in bone defect.

Mechanical Vibration Mitigates the Decrease of Bone Quantity and Bone Quality of Leptin Receptor-Deficient Db/Db Mice by Promoting Bone Formation and Inhibiting Bone Resorption.

Leptin, a major hormonal product of adipocytes, is involved in regulating appetite and energy metabolism. Substantial studies have revealed the anabolic actions of leptin on skeletons and bone cells both in vivo and in vitro. Growing evidence has substantiated that leptin receptor-deficient db/db mice exhibit decreased bone mass and impaired bone microstructure despite several conflicting results previously reported. We herein systematically investigated bone microarchitecture, mechanical strength, bone turnover and its potential molecular mechanisms in db/db mice. More importantly, we also explored an effective approach for increasing bone mass in leptin receptor-deficient animals in an easy and noninvasive manner. Our results show that deterioration of trabecular and cortical bone microarchitecture and decreases of skeletal mechanical strength-including maximum load, yield load, stiffness, energy, tissue-level modulus and hardness-in db/db mice were significantly ameliorated by 12-week, whole-body vibration (WBV) with 0.5 g, 45 Hz via micro-computed tomography (µCT), three-point bending, and nanoindentation examinations. Serum biochemical analysis shows that WBV significantly decreased serum tartrate-resistant acid phosphatase 5b (TRACP5b) and CTx-1 levels and also mitigated the reduction of serum osteocalcin (OCN) in db/db mice. Bone histomorphometric analysis confirmed that decreased bone formation-lower mineral apposition rate, bone formation rate, and osteoblast numbers in cancellous bone-in db/db mice were suppressed by WBV. Real-time PCR assays show that WBV mitigated the reductions of tibial alkaline phosphatase (ALP), OCN, Runt-related transcription factor 2 (RUNX2), type I collagen (COL1), BMP2, Wnt3a, Lrp6, and ß-catenin mRNA expression, and prevented the increases of tibial sclerostin (SOST), RANK, RANKL, RANL/osteoprotegerin (OPG) gene levels in db/db mice. Our results show that WBV promoted bone quantity and quality in db/db mice with obvious anabolic and anticatabolic effects. This study not only enriches our basic knowledge about bone quality and bone turnover mechanisms in leptin receptor-deficient animals, but also advances our understanding of the skeletal sensitivity of leptin-resistant db/db mice in response to external mechanical stimulation. © 2016 American Society for Bone and Mineral Research.

Pulsed electromagnetic fields promote in vitro osteoblastogenesis through a Wnt/ß-catenin signaling-associated mechanism.

Substantial evidence indicates that pulsed electromagnetic fields (PEMF) could accelerate fracture healing and enhance bone mass, whereas the unclear mechanism by which PEMF stimulation promotes osteogenesis limits its extensive clinical application. In the present study, effects and potential molecular signaling mechanisms of PEMF on in vitro osteoblasts were systematically investigated. Osteoblast-like MC3T3-E1 cells were exposed to PEMF burst (0.5, 1, 2, or 6 h/day) with 15.38 Hz at various intensities (5 Gs (0.5 mT), 10 Gs (1 mT), or 20 Gs (2 mT)) for 3 consecutive days. PEMF stimulation at 20 Gs (2 mT) for 2 h/day exhibited most prominent promotive effects on osteoblastic proliferation via Cell Counting kit-8 analyses. PEMF exposure induced well-organized cytoskeleton, and promoted formation of extracellular matrix mineralization nodules. Significantly increased proliferation-related gene expressions at the proliferation phase were observed after PEMF stimulation, including Ccnd 1 and Ccne 1. PEMF resulted in significantly increased gene and protein expressions of alkaline phosphatase and osteocalcin at the differentiation phase of osteoblasts rather than the proliferation phase via quantitative reverse transcription polymerase chain reaction and Western blotting analyses. Moreover, PEMF upregulated gene and protein expressions of collagen type 1, Runt-related transcription factor 2 and Wnt/ß-catenin signaling (Wnt1, Lrp6, and ß-catenin) at proliferation and differentiation phases. Together, our present findings highlight that PEMF stimulated osteoblastic functions through a Wnt/ß-catenin signaling-associated mechanism and, hence, regulates downstream osteogenesis-associated gene/protein expressions. Bioelectromagnetics. 37:152-162, 2016. © 2016 Wiley Periodicals, Inc.

Effect of low-level mechanical vibration on osteogenesis and osseointegration of porous titanium implants in the repair of long bone defects.

Emerging evidence substantiates the potential of porous titanium alloy (pTi) as an ideal bone-graft substitute because of its excellent biocompatibility and structural properties. However, it remains a major clinical concern for promoting high-efficiency and high-quality osseointegration of pTi, which is beneficial for securing long-term implant stability. Accumulating evidence demonstrates the capacity of low-amplitude whole-body vibration (WBV) in preventing osteopenia, whereas the effects and mechanisms of WBV on osteogenesis and osseointegration of pTi remain unclear. Our present study shows that WBV enhanced cellular attachment and proliferation, and induced well-organized cytoskeleton of primary osteoblasts in pTi. WBV upregulated osteogenesis-associated gene and protein expression in primary osteoblasts, including OCN, Runx2, Wnt3a, Lrp6 and ß-catenin. In vivo findings demonstrate that 6-week and 12-week WBV stimulated osseointegration, bone ingrowth and bone formation rate of pTi in rabbit femoral bone defects via µCT, histological and histomorphometric analyses. WBV induced higher ALP, OCN, Runx2, BMP2, Wnt3a, Lrp6 and ß-catenin, and lower Sost and RANKL/OPG gene expression in rabbit femora. Our findings demonstrate that WBV promotes osteogenesis and osseointegration of pTi via its anabolic effect and potential anti-catabolic activity, and imply the promising potential of WBV for enhancing the repair efficiency and quality of pTi in osseous defects.

Moderate-intensity rotating magnetic fields do not affect bone quality and bone remodeling in hindlimb suspended rats.

Abundant evidence has substantiated the positive effects of pulsed electromagnetic fields (PEMF) and static magnetic fields (SMF) on inhibiting osteopenia and promoting fracture healing. However, the osteogenic potential of rotating magnetic fields (RMF), another common electromagnetic application modality, remains poorly characterized thus far, although numerous commercial RMF treatment devices have been available on the market. Herein the impacts of RMF on osteoporotic bone microarchitecture, bone strength and bone metabolism were systematically investigated in hindlimb-unloaded (HU) rats. Thirty two 3-month-old male Sprague-Dawley rats were randomly assigned to the Control (n = 10), HU (n = 10) and HU with RMF exposure (HU+RMF, n = 12) groups. Rats in the HU+RMF group were subjected to daily 2-hour exposure to moderate-intensity RMF (ranging from 0.60 T to 0.38 T) at 7 Hz for 4 weeks. HU caused significant decreases in body mass and soleus muscle mass of rats, which were not obviously altered by RMF. Three-point bending test showed that the mechanical properties of femurs in HU rats, including maximum load, stiffness, energy absorption and elastic modulus were not markedly affected by RMF. µCT analysis demonstrated that 4-week RMF did not significantly prevent HU-induced deterioration of femoral trabecular and cortical bone microarchitecture. Serum biochemical analysis showed that RMF did not significantly change HU-induced decrease in serum bone formation markers and increase in bone resorption markers. Bone histomorphometric analysis further confirmed that RMF showed no impacts on bone remodeling in HU rats, as evidenced by unchanged mineral apposition rate, bone formation rate, osteoblast numbers and osteoclast numbers in cancellous bone. Together, our findings reveal that RMF do not significantly affect bone microstructure, bone mechanical strength and bone remodeling in HU-induced disuse osteoporotic rats. Our study indicates potentially obvious waveform-dependent effects of electromagnetic fields-stimulated osteogenesis, suggesting that RMF, at least in the present form, might not be an optimal modality for inhibiting disuse osteopenia/osteoporosis.