Murilo C. Crovace

Effect of 830 nm Laser Phototherapy on Osteoblasts Grown In Vitro on Biosilicate® Scaffolds

OBJECTIVE The purpose of this study was (i) to develop a method for successfully seeding osteoblasts onto a glass-ceramic scaffold designed for use in clinical settings, and (ii) to determine whether the application of laser phototherapy at 830 nm would result in osteoblast proliferation on the glass-ceramic scaffold. BACKGROUND The use of bioscaffolds is considered a promising strategy for a number of clinical applications where tissue healing is sub-optimal. As in vitro osteoblast growth is a slow process, laser phototherapy could be used to stimulate osteoblast proliferation on bioscaffolds. METHODS A methodology was developed to seed an osteoblastic (MC3T3) cell line onto a novel glass-ceramic scaffold. Seeded scaffolds were irradiated with a single exposure of 830 nm laser at 10 J/cm(2) (at diode). Non-irradiated seeded scaffolds acted as negative controls. Cell proliferation was assessed seven days after irradiation. RESULTS Osteoblastic MC3T3 cells were successfully grown on discs composed of a glass-ceramic composite. Laser irradiation produced a 13% decrease in MC3T3 cell proliferation on glass-ceramic discs (mean +/- SD = 0.192 +/- 0.002) compared with control (non-irradiated) discs (mean +/-SD = 0.22 +/- 0.002). CONCLUSIONS Despite successful seeding of bioscaffolds with osteoblasts, laser phototherapy resulted in a reduction in cell growth compared to non-irradiated controls. Future research combining laser phototherapy and glass-ceramic scaffolds should take into account possible interactions of the laser with matrix compounds.

Incorporation of bioactive glass in calcium phosphate cement: An evaluation.

Bioactive glasses (BGs) are known for their unique ability to bond to living bone. Consequently, the incorporation of BGs into calcium phosphate cement (CPC) was hypothesized to be a feasible approach to improve the biological performance of CPC. Previously, it has been demonstrated that BGs can successfully be introduced into CPC, with or without poly(d,l-lactic-co-glycolic) acid (PLGA) microparticles. Although an in vitro physicochemical study on the introduction of BG into CPC was encouraging, the biocompatibility and in vivo bone response to these formulations are still unknown. Therefore, the present study aimed to evaluate the in vivo performance of BG supplemented CPC, either pure or supplemented with PLGA microparticles, via both ectopic and orthotopic implantation models in rats. Pre-set scaffolds in four different formulations (1: CPC; 2: CPC/BG; 3: CPC/PLGA; and 4: CPC/PLGA/BG) were implanted subcutaneously and into femoral condyle defects of rats for 2 and 6 weeks. Upon ectopic implantation, incorporation of BG into CPC improved the soft tissue response by improving capsule and interface quality. Additionally, the incorporation of BG into CPC and CPC/PLGA showed 1.8- and 4.7-fold higher degradation and 2.2- and 1.3-fold higher bone formation in a femoral condyle defect in rats compared to pure CPC and CPC/PLGA, respectively. Consequently, these results highlight the potential of BG to be used as an additive to CPC to improve the biological performance for bone regeneration applications. Nevertheless, further confirmation is necessary regarding long-term in vivo studies, which also have to be performed under compromised wound-healing conditions.

Biosilicate®–gelatine bone scaffolds by the foam replica technique: development and characterization

Abstract The development of bioactive glass-ceramic materials has been a topic of great interest aiming at enhancing the mechanical strength of traditional bioactive scaffolds. In the present study, we test and demonstrate the use of Biosilicate® glass-ceramic powder to fabricate bone scaffolds by the foam replica method. Scaffolds possessing the main requirements for use in bone tissue engineering (95% porosity, 200–500 μm pore size) were successfully produced. Gelatine coating was investigated as a simple approach to increase the mechanical competence of the scaffolds. The gelatine coating did not affect the interconnectivity of the pores and did not significantly affect the bioactivity of the Biosilicate® scaffold. The gelatine coating significantly improved the compressive strength (i.e. 0.80 ± 0.05 MPa of coated versus 0.06 ± 0.01 MPa of uncoated scaffolds) of the Biosilicate® scaffold. The combination of Biosilicate® glass-ceramic and gelatine is attractive for producing novel scaffolds for bone tissue engineering.

Incorporation of bioactive glass in calcium phosphate cement:material characterization and in vitro degradation

Calcium phosphate cements (CPCs) have been widely used as an alternative to biological grafts due to their excellent osteoconductive properties. Although degradation has been improved by using poly(D,L-lactic-co-glycolic) acid (PLGA) microspheres as porogens, the biological performance of CPC/PLGA composites is insufficient to stimulate bone healing in large bone defects. In this context, the aim of this study was to investigate the effect of incorporating osteopromotive bioactive glass (BG; up to 50 wt %) on setting properties, in vitro degradation behavior and morphological characteristics of CPC/BG and CPC/PLGA/BG. The results revealed that the initial and final setting time of the composites increased with increasing amounts of incorporated BG. The degradation test showed a BG-dependent increasing effect on pH of CPC/BG and CPC/PLGA/BG pre-set scaffolds immersed in PBS compared to CPC and CPC/PLGA equivalents. Whereas no effects on mass loss were observed for CPC and CPC/BG pre-set scaffolds, CPC/PLGA/BG pre-set scaffolds showed an accelerated mass loss compared with CPC/PLGA equivalents. Morphologically, no changes were observed for CPC and CPC/BG pre-set scaffolds. In contrast, CPC/PLGA and CPC/PLGA/BG showed apparent degradation of PLGA microspheres and faster loss of integrity for CPC/PLGA/BG pre-set scaffolds compared with CPC/PLGA equivalents. Based on the present in vitro results, it can be concluded that BG can be successfully introduced into CPC and CPC/PLGA without exceeding the setting time beyond clinically acceptable values. All injectable composites containing BG had suitable handling properties and specifically CPC/PLGA/BG showed an increased rate of mass loss. Future investigations should focus on translating these findings to in vivo applications.

Characterization and In Vivo Biological Performance of Biosilicate

After an introduction showing the growing interest in glasses and glass-ceramics as biomaterials used for bone healing, we describe a new biomaterial named Biosilicate. Biosilicate is the designation of a group of fully crystallized glass-ceramics of the Na2O-CaO-SiO2-P2O5 system. Several in vitro tests have shown that Biosilicate is a very active biomaterial and that the HCA layer is formed in less than 24 hours of exposure to “simulated body fluid” (SBF) solution. Also, in vitro studies with osteoblastic cells have shown that Biosilicate disks supported significantly larger areas of calcified matrix compared to 45S5 Bioglass, indicating that this bioactive glass-ceramic may promote enhancement of in vitro bone-like tissue formation in osteogenic cell cultures. Finally, due to its special characteristics, Biosilicate has also been successfully tested in several in vivo studies. These studies revealed that the material is biocompatible, presents excellent bioactive properties, and is effective to stimulate the deposition of newly formed bone in animal models. All these data highlight the huge potential of Biosilicate to be used in bone regeneration applications.

Effects of biosilicate(®) scaffolds and low-level laser therapy on the process of bone healing.

OBJECTIVE This study aimed to investigate the in vivo tissue performance of the association of Biosilicate(®) scaffolds and low-level laser therapy (LLLT) in a tibial bone defects model in rats. BACKGROUND DATA Many studies have been demonstrating the osteogenic potential of Biosilicate and LLLT. However, there is a need to investigate the effects of both treatments for bone consolidation. METHODS The animals were divided into control group (CG), Biosilicate scaffold group (BG), and Biosilicate scaffolds plus LLLT group (BLG). Animals were euthanized after 15, 30, and 45 days post-injury. RESULTS The histological analysis revealed that all the experimental groups showed inflammatory infiltrate and granulation tissue, at the area of the defect at day 15. After 30 days, CG still showed granulation tissue and bone ingrowth. Both Biosilicate groups presented newly formed bone and interconected trabeculae. At 45 days, CG showed immature newly formed bone. A more mature newly formed bone was observed in BG and BLG. On day 15, BG demonstrated a statistically higher expression of cyclooxygenase (COX)-2 compared with CG and BLG. No statistically significant difference was observed in COX-2 immunoexpression among the groups at 30 and 45 days. Similar expression of bone morphogenetic protein (BMP)-9 was demonstrated for all experimental groups at 15 and 30 days. At 45 days, the BMP-9 immunoexpression was statistically upregulated in the BLG compared with the CG and BG. No statistically significant difference was observed in the receptor activator of nuclear factor kappa-B ligand (RANKL) immunoexpression among the groups in all periods evaluated. Biosilicate groups presented a decrease in biomechanical properties compared with CG at 30 and 45 days post-surgery. CONCLUSIONS Our findings suggest that Biosilicate presented osteogenic activity, accelerating bone repair. However, laser therapy was not able to enhance the bioactive properties of the Biosilicate.

Histopathological, cytotoxicity and genotoxicity evaluation of Biosilicate® glass–ceramic scaffolds

This study evaluated the biocompatibility of Biosilicate® scaffolds by means of histopathological, cytotoxicity, and genotoxicity analysis. The histopathologic analysis of the biomaterial was performed using 65 male rats, distributed into the groups: control and Biosilicate®, evaluated at 7, 15, 30, 45, and 60 days after implantation. The cytotoxicity analysis was performed by the methyl thiazolyl tetrazolium (MTT) assay, with various concentrations of extracts from the biomaterial in culture of osteoblasts and fibroblasts after 24, 72, and 120 h. The genotoxicity analysis (comet assay) was performed in osteoblasts and fibroblasts after contact with the biomaterial during 24, 72, and 96 h. In the histopathology analysis, we observed a foreign body reaction, characterized by the presence of granulation tissue after 7 days of implantation of the biomaterial, and fibrosis connective tissue and multinucleated giant cells for longer periods. In the cytotoxicity analysis, extracts from the biomaterial did not inhibit the proliferation of osteoblasts and fibroblasts, and relatively low concentrations (12.5% and 25%) stimulated the proliferation of both cell types after 72 and 120 h. The analysis of genotoxicity showed that Biosilicate® did not induce DNA damage in both lineages tested in all periods. The results showed that the Biosilicate® scaffolds present in vivo and in vitro biocompatibility.

Bone regeneration and gene expression in bone defects under healthy and osteoporotic bone conditions using two commercially available bone graft substitutes.

Biosilicate(®) and Bio-Oss(®) are two commercially available bone substitutes, however, little is known regarding their efficacy in osteoporotic conditions. The purpose of this study was to evaluate the osteogenic properties of both materials, at tissue and molecular level. Thirty-six Wistar rats were submitted to ovariectomy (OVX) for inducing osteoporotic conditions and sham surgery (SHAM) as a control. Bone defects were created in both femurs, which were filled with Biosilicate(®) or Bio-Oss(®), and empty defects were used as control. For the healthy condition both Biosilicate(®) and Bio-Oss(®) did not improve bone formation after 4 weeks. Histomorphometric evaluation of osteoporotic bone defects with bone substitutes showed more bone formation, significant for Bio-Oss(®). Molecular biological evaluation was performed by gene-expression analysis (Runx-2, ALP, OC, OPG, RANKL). The relative gene expression was increased with Biosilicate(®) for all genes in OVX rats and for Runx-2, ALP, OC and RANKL in SHAM rats. In contrast, with Bio-Oss(®), the relative gene expression of OVX rats was similar for all three groups. For SHAM rats it was increased for Runx-2, ALP, OC and RANKL. Since both materials improved bone regeneration in osteoporotic conditions, our results suggest that bone defects in osteoporotic conditions can be efficiently treated with these two bone substitutes.

Bioactive glass-based surfaces induce differential gene expression profiling of osteoblasts

The ability of Biosilicate® with two crystalline phases (BioS-2P) to drive osteoblast differentiation encourages the investigation of the cellular mechanisms involved in this process. Then, the aim of our study was to analyze the large-scale gene expression of osteoblasts grown on BioS-2P compared with Bioglass® 45S5 (45S5). Osteoblasts differentiated from rat bone marrow mesenchymal stem cells were cultured under osteogenic conditions on BioS-2P, 45S5 and polystyrene (control). After 10 days, the expression of 23,794 genes was analyzed using mRNA Sequencing and the data were validated by real-time PCR. The BioS-2P exhibited 5 genes upregulated and 3 downregulated compared with 45S5. Compared with control, BioS-2P upregulated 15 and downregulated 11 genes, while 45S5 upregulated 25 and downregulated 21 genes. Eight genes were commonly upregulated and 4 downregulated by both bioactive glasses. In conclusion, our results demonstrated that bioactive glasses affect the gene expression profiling of osteoblasts. Most of the regulated genes by both BioS-2P and 45S5 are associated with the process of mineralization highlighting their osteostimulation property that is, at least in part, derived from the ability to modulate the intracellular machinery to promote osteoblast genotype expression.

Putty-like bone fillers based on CaP ceramics or Biosilicate(R) combined with carboxymethylcellulose: Characterization, optimization, and evaluation

Calcium phosphates and bioactive glass ceramics have been considered promising biomaterials for use in surgeries. However, their moldability should be further enhanced. We here thereby report the handling, physicochemical features, and morphological characteristics of formulations consisting of carboxymethylcellulose–glycerol and hydroxyapatite-tricalcium phosphate or Biosilicate® particles. We hypothesized that combining either material with carboxymethylcellulose–glycerol would improve handling properties, retaining their bioactivity. In addition to scanning electron microscopy, cohesion, mineralization, pH, and viscoelastic properties of the novel formulations, cell culture experiments were performed to evaluate the cytotoxicity and cell proliferation. Putty-like formulations were obtained with improved cohesion and moldability. Remarkably, mineralization in simulated body fluid of hydroxyapatite-tricalcium phosphate/carboxymethylcellulose–glycerol formulations was enhanced compared to pure hydroxyapatite-tricalcium phosphate. Cell experiments showed that all formulations were noncytotoxic and that HA-TCP60 and BGC50 extracts led to an increased cell proliferation. We conclude that combining carboxymethylcellulose–glycerol with either hydroxyapatite-tricalcium phosphate or Biosilicate® allows for the generation of moldable putties, improves handling properties, and retains the ceramic bioactivity.