Effects of La2O3 contents on microstructure and properties of laser-cladded 5wt%CaB6/HA bioceramic coating

In the present work, 5wt%CaB6/HA bioceramic coatings with different La2O3contents (0-0.6wt%) have been fabricated by a laser cladding technique on Ti-6Al-4V. The effects of La2O3 contents on microstructure and properties of the laser-cladded 5wt%CaB6/HA coatings have been carefully investigated. The results show that the microstructure is obviously refined, and the structure is relatively uniform after doping 0.2-0.4 wt% La2O3. As the La2O3 content increases, the corrosion resistance are found to increase firstly and then gradually decrease. The XRD analysis confirms that the amount of HA and TCP in the coating reaches maximum after doping 0.2wt% La2O3. The La2O3-doped coatings show a significantly higher bone-like apatite precipitation after immersion in SBF compared with La2O3-free coating. In vitro experiment also shows that 5wt%CaB6/HA bioceramic coatings with 0.2-0.4wt% La2O3 are more suitable for the attachment and proliferation of MG63 cells, exhibiting superior bioactivity and biocompatibility.

Severe cellular stress activates apoptosis independently of p53 in osteosarcoma

Apoptosis induced by doxorubicin, bortezomib, or paclitaxel, targeting DNA, 26S proteasome, and microtubules respectively, was assessed in two osteosarcoma cells, p53 wild-type U2OS and p53-null MG63 cells. Doxorubicin-induced apoptosis only occurred in U2OS, not in MG63. In contrast, bortezomib and paclitaxel could drive U2OS or MG63 toward apoptosis effectively, suggesting that apoptosis induced by bortezomib or paclitaxel is p53-independent. The expressions of Bcl2 family members such as Bcl2, Bcl-xl, and Puma could be seen in U2OS and MG63 cells with or without doxorubicin, bortezomib, or paclitaxel treatment. In contrast, another member, Bim, only could be observed in U2OS, not in MG63, under the same conditions. Bim knockdown did not affect the doxorubicin-induced apoptosis in U2OS, suggested that a BH3-only protein other than Bim might participate in apoptosis induced by doxorubicin. Using a BH3-mimetic, ABT-263, to inhibit Bcl2 or Bcl-xl produced a limited apoptotic response in U2OS and MG63 cells, suggesting that this BH3-mimetic cannot activate the Bax/Bak pathway efficiently. Significantly, ABT-263 enhanced doxorubicin- and bortezomib-induced apoptosis synergistically in U2OS and MG63 cells. These results implied that the severe cellular stress caused by doxorubicin or bortezomib might be mediated through a dual process to control apoptosis. Respectively, doxorubicin or bortezomib activates a BH3-only protein in one way and corresponding unknown factors in another way to affect mitochondrial outer membrane permeability, resulting in apoptosis. The combination of doxorubicin with ABT-263 could produce synergistic apoptosis in MG63 cells, which lack p53, suggesting that p53 has no role in doxorubicin-induced apoptosis in osteosarcoma. In addition, ABT-263 enhanced paclitaxel to induce moderate levels of apoptosis.

Chitosan-TiO2 nanotubes scaffolds for proliferation and early differentiation of MG63 by functionalization with fetal bovine serum

Scaffolds have been used as alternative biomaterials to overcome physiological bone disorders. Production of scaffolds has been challenging to fulfil the following criteria: biodegradability, mechanical sustainability, and biocompatibility. For cellular interaction, protein adsorbed on scaffold surface is important for osteoblastic activities. This study aimed to functionalize chitosan-TiO2 nanotubes scaffolds with fetal bovine serum and investigate in vitro efficacy of such scaffolds with fetal bovine serum. Chitosan-TiO2 nanotubes scaffolds were prepared via direct blending and lyophilization. They were then functionalized with fetal bovine serum via adsorption for 4, 8, 12 and 24 h. The in vitro efficacy of the functionalized scaffolds was evaluated using MG63 cells. The adsorption of fetal bovine serum onto the scaffolds was complex where saturation of adsorption was hardly attained. The in vitro efficacy of scaffolds with adsorbed fetal bovine serum was higher than that of those without fetal bovine serum by promoting better osteoblastic functions. Notably, the scaffolds functionalized for 4 h enhanced cell adhesion and proliferation on 7 day suggesting good regulation of osteoblastic binding and proliferation. ALP protein was expressed on 26 day in all functionalized scaffolds. Chitosan-TiO2 nanotubes scaffolds with adsorbed fetal bovine serum can be a potential regenerative material for bone regeneration.

EMMPRIN expression is associated with metastatic progression in osteosarcoma

Background Extracellular matrix metalloproteinase inducer (EMMPRIN), a cell-surface glycoprotein, is overexpressed in several cancer types. EMMPRIN induces a metastatic phenotype by triggering the production of matrix metalloproteinase proteins (MMPs) such as MMP1 and MMP2, and vascular endothelial growth factor (VEGF) in cancer cells and the surrounding stromal cells. The purpose of this study was to investigate the expression and role of EMMPRIN in osteosarcoma. Methods The level of EMMPRIN expression was evaluated using reverse transcriptase polymerase chain reaction (RT-PCR) in 6 tumor-derived osteosarcoma cell lines and compared with that in normal osteoblasts. To study the prognostic significance of EMMPRIN expression, immunohistochemistry was carried out in prechemotherapy biopsies of 54 patients. siRNA knockdown of EMMPRIN in SaOS-2 cells was conducted to explore the role of EMMPRIN. To study the role of EMMPRIN in tumor-stromal interaction in MMP production and invasion, co-culture of SaOS-2 cells with osteoblasts and fibroblasts was performed. Osteosarcoma 143B cells were injected into the tail vein of BALB/c mice and lung metastasis was analyzed. Results EMMRIN mRNA expression was significantly higher in 5 of 6 (83%) tumor-derived cells than in MG63 cells. 90% of specimens (50/54) stained positive for EMMPRIN by immunohistochemistry, and higher expression of EMMPRIN was associated with shorter metastasis-free survival (p = 0.023). Co-culture of SaOS-2 with osteoblasts resulted in increased production of pro-MMP2 and VEGF expression, which was inhibited by EMMPRIN-targeting siRNA. siRNA knockdown of EMMPRIN resulted in decreased invasion. EMMPRIN shRNA-transfected 143B cells showed decreased lung metastasis in vivo. Conclusions Our data suggest that EMMPRIN acts as a mediator of osteosarcoma metastasis by regulating MMP and VEGF production in cancer cells as well as stromal cells. EMMPRIN could serve as a therapeutic target in osteosarcoma.

Anti-Osteoporotic Activity of Pueraria lobata Fermented with Lactobacillus paracasei JS1 by Regulation of Osteoblast Differentiation and Protection against Bone Loss in Ovariectomized Mice

Osteoporosis is the most common bone disease associated with low bone mineral density. It is the process of bone loss and is most commonly caused by decreased estrogen production in women, particularly after menopause. Pueraria lobata, which contains various metabolites, especially isoflavone, is widely known as regulator for bone mineral contents. In this study, the effects of the P. lobata extract (PE) with or without fermentation with Lactobacillus paracasei JS1 (FPE) on osteoporosis were investigated in vitro and in vivo. The effects of PE and FPE on human osteoblastic MG63 cells, RAW 264.7 cells, and ovariectomized (OVX)-induced model mice were analyzed at various ratios. We found that FPE increased calcium deposition and inhibited bone resorption by in vitro assay. Furthermore, treatment with PE and FPE has significantly restored destroyed trabecular bone in the OVX-induced bone loss mouse model. Overall, FPE demonstrated bioactivity to prevent bone loss by decreasing bone turnover.

Metabolomic Profiling of Fresh Goji (Lycium barbarum L.) Berries from Two Cultivars Grown in Central Italy: A Multi-Methodological Approach

The metabolite profile of fresh Goji berries from two cultivars, namely Big Lifeberry (BL) and Sweet Lifeberry (SL), grown in the Lazio region (Central Italy) and harvested at two different periods, August and October, corresponding at the beginning and the end of the maturation, was characterized by means of nuclear magnetic resonance (NMR) and electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR MS) methodologies. Several classes of compounds such as sugars, amino acids, organic acids, fatty acids, polyphenols, and terpenes were identified and quantified in hydroalcoholic and organic Bligh-Dyer extracts. Sweet Lifeberry extracts were characterized by a higher content of sucrose with respect to the Big Lifeberry ones and high levels of amino acids (glycine, betaine, proline) were observed in SL berries harvested in October. Spectrophotometric analysis of chlorophylls and total carotenoids was also carried out, showing a decrease of carotenoids during the time. These results can be useful not only to valorize local products but also to suggest the best harvesting period to obtain a product with a chemical composition suitable for specific industrial use. Finally, preliminary studies regarding both the chemical characterization of Goji leaves generally considered a waste product, and the biological activity of Big Lifeberry berries extracts was also investigated. Goji leaves showed a chemical profile rich in healthy compounds (polyphenols, flavonoids, etc.) confirming their promising use in the supplements/nutraceutical/cosmetic field. MG63 cells treated with Big Lifeberry berries extracts showed a decrease of iNOS, COX-2, IL-6, and IL-8 expression indicating their significant biological activity.

Octacalcium Phosphate Bone Substitute (Bontree®): From Basic Research to Clinical Case Study

Bone grafts used in alveolar bone regeneration can be categorized into autografts, allografts, xenografts, and synthetic bones, depending on their origin. The purpose of this study was to evaluate the effect of a commercialized octacalcium phosphate (OCP)-based synthetic bone substitute material (Bontree®) in vitro, in vivo, and in clinical cases. Material characterization of Bontree® granules (0.5 mm and 1.0 mm) using scanning electron microscopy and X-ray diffraction showed that both 0.5 mm and 1.0 mm Bontree® granules were uniformly composed mainly of OCP. The receptor activator of NF-κB ligand (RANKL) and alkaline phosphatase (ALP) activities of MG63 cells were assessed and used to compare Bontree® with a commercial biphasic calcium phosphate ceramic (MBCP+TM). Compared with MBCP+TM, Bontree® suppressed RANKL and increased ALP activity. A rabbit tibia model used to examine the effects of granule size of Bontree® grafts showed that 1.0 mm Bontree® granules had a higher new bone formation ability than 0.5 mm Bontree® granules. Three clinical cases using Bontree® for ridge or sinus augmentation are described. All eight implants in the three patients showed a 100% success rate after 1 year of functional loading. This basic research and clinical application demonstrated the safety and efficacy of Bontree® for bone regeneration.

Effect of Allium fistulosum Extracts on the Stimulation of Longitudinal Bone Growth in Animal Modeling Diet-Induced Calcium and Vitamin D Deficiencies

Allium fistulosum is a perennial plant species grown worldwide belonging to the family Liliaceae. In Korean medicine, it is referred to as Chongbaek (CB), and it is prescribed for symptoms associated with the common cold due to its antipyretic properties. This study examined the effects of aqueous (CBW) and 30% ethanol (CBE) extracts on bone growth using a calcium- and vitamin D-deficient animal model. In an in vitro experiment, the alkaline phosphate activities of the extracts were examined using MC3T3-E1 and MG63 cells, and both the aqueous and ethanolic extracts had significant alkaline phosphate activities. In vivo, a serum analysis indicated that the CB extracts promoted bone growth based on the osteogenic markers ALP, calcium, osteocalcin, and collagen type 1 and increased the bone mineral content (BMC), bone mineral density (BMD), and growth plate length. Overall, our results indicate that both CBW and CBE of A. fistulosum can be utilized to facilitate bone growth and increase BMD in children and adolescents by lengthening the growth plate without adverse side effects, such as metabolic disorders or the release of obesity-inducing hormones.

Fabrication and Evaluation of Alginate/Bacterial Cellulose Nanocrystals–Chitosan–Gelatin Composite Scaffolds

It is common knowledge that pure alginate hydrogel is more likely to have weak mechanical strength, a lack of cell recognition sites, extensive swelling and uncontrolled degradation, and thus be unable to satisfy the demands of the ideal scaffold. To address these problems, we attempted to fabricate alginate/bacterial cellulose nanocrystals-chitosan-gelatin (Alg/BCNs-CS-GT) composite scaffolds using the combined method involving the incorporation of BCNs in the alginate matrix, internal gelation through the hydroxyapatite-d-glucono-δ-lactone (HAP-GDL) complex, and layer-by-layer (LBL) electrostatic assembly of polyelectrolytes. Meanwhile, the effect of various contents of BCNs on the scaffold morphology, porosity, mechanical properties, and swelling and degradation behavior was investigated. The experimental results showed that the fabricated Alg/BCNs-CS-GT composite scaffolds exhibited regular 3D morphologies and well-developed pore structures. With the increase in BCNs content, the pore size of Alg/BCNs-CS-GT composite scaffolds was gradually reduced from 200 μm to 70 μm. Furthermore, BCNs were fully embedded in the alginate matrix through the intermolecular hydrogen bond with alginate. Moreover, the addition of BCNs could effectively control the swelling and biodegradation of the Alg/BCNs-CS-GT composite scaffolds. Furthermore, the in vitro cytotoxicity studies indicated that the porous fiber network of BCNs could fully mimic the extracellular matrix structure, which promoted the adhesion and spreading of MG63 cells and MC3T3-E1 cells on the Alg/BCNs-CS-GT composite scaffolds. In addition, these cells could grow in the 3D-porous structure of composite scaffolds, which exhibited good proliferative viability. Based on the effect of BCNs on the cytocompatibility of composite scaffolds, the optimum BCNs content for the Alg/BCNs-CS-GT composite scaffolds was 0.2% (w/v). On the basis of good merits, such as regular 3D morphology, well-developed pore structure, controlled swelling and biodegradation behavior, and good cytocompatibility, the Alg/BCNs-CS-GT composite scaffolds may exhibit great potential as the ideal scaffold in the bone tissue engineering field.

Bioactivity and Bone Cell Formation with Poly-ε-Caprolactone/Bioceramic 3D Porous Scaffolds

This study applied poly-ε-caprolactone (PCL), a biomedical ceramic powder as an additive (nano-hydroxyapatite (nHA) or β-tricalcium diphosphate (β-TCP)), and sodium chloride (NaCl) and ammonium bicarbonate ((NH4)HCO3) as porogens; these stuffs were used as scaffold materials. An improved solvent-casting/particulate-leaching method was utilized to fabricate 3D porous scaffolds. In this study we examined the physical properties (elastic modulus, porosity, and contact angle) and degradation properties (weight loss and pH value) of the 3D porous scaffolds. Both nHA and β-TCP improved the mechanical properties (elastic modulus) of the 3D porous scaffolds. The elastic modulus (0.15~1.865 GPa) of the various composite scaffolds matched that of human cancellous bone (0.1~4.5 GPa). Osteoblast-like (MG63) cells were cultured, a microculture tetrazolium test (MTT) was conducted and alkaline phosphatase (ALP) activity of the 3D porous scaffolds was determined. Experimental results indicated that both nHA and β-TCP powder improved the hydrophilic properties of the scaffolds. The degradation rate of the scaffolds was accelerated by adding nHA or β-TCP. The MTT and ALP activity tests indicated that the scaffolds with a high ratio of nHA or β-TCP had excellent properties of in vitro biocompatibility (cell attachment and proliferation).