Bioglass/collagen scaffolds combined with bone marrow stromal cells on bone healing in an experimental model in cranial defects in rats.

This study aimed to develop bone regenerative therapeutic strategies, based on the addition of bone marrow stromal cells (BMSC) on bioglass/collagen (BG/COL) scaffolds. For this purpose, an in vivo study was conducted using tissue response of the BG/COL scaffolds combined with BMSC in a critical-size defects. Wistar rats were submitted to the surgical procedure to perform the cranial critical size bone defects and distributed in four groups (20 animals per group): Control Group (CG) (rats submitted to the cranial bone defect surgery without treatment), Bioglass Group (BG) (rats treated with BG), BG/COL Group (rats treated with BG/COL) and Bioglass/Collagen and BMSC Group (BG/COL/BMSC) (rats treated with BG/COL scaffolds enriched with BMSCs). Animals were euthanized 15 and 30 days after surgery. Scanning electron microscopy, histopathological and immunohistochemistry analysis were used. SEM analysis demonstrated that porous scaffolds were obtained, and Col fibers were successfully impregnated to BG matrices. The implantation of the BMSC on BG/COL based scaffolds was effective in stimulating newly bone formation and produced an increased immunoexpression of markers related to the bone repair. These results highlight the potential of BG/COL scaffolds and BMSCs to be used as a therapeutic approach for bone regeneration.

Marine collagen scaffolds and photobiomodulation on bone healing process in a model of calvaria defects.

INTRODUCTION: Collagen from marine esponges has been used as a promising material for tissue engineering proposals. Similarly, photobiomodulation (PBM) is able of modulating inflammatory processes after an injury, accelerating soft and hard tissue healing and stimulating neoangiogenesis. However, the effects of the associated treatments on bone tissue healing have not been studied yet. In this context, the present study aimed to evaluate the biological temporal modifications (using two experimental periods) of marine sponge collagen or sponging (SPG) based scaffold and PBM on newly formed bone using a calvaria bone defect model. MATERIAL AND METHODS: Wistar rats were distributed into two groups: SPG or SPG/PBM and euthanized into two different experimental periods (15 and 45 days post-surgery). A cranial critical bone defect was used to evaluate the effects of the treatments. Histology, histomorfometry and immunohistological analysis were performed. RESULTS: Histological findings demonstrated that SPG/PBM-treated animals, 45 days post-surgery, demonstrated a higher amount of connective and newly formed bone tissue at the region of the defect compared to CG. Notwithstanding, no difference among groups were observed in the histomorphometry. Interestingly, for both anti-transforming growth factor-beta (TGF-ß) and anti-vascular endothelial growth factor (VEGF) immunostaining, higher values for SPG/PBM, at 45 days post-surgery could be observed. CONCLUSION: It can be concluded that the associated treatment can be considered as a promising therapeutical intervention.

Influence of the incorporation of marine spongin into a Biosilicate®: an in vitro study.

The combination of different biomaterials can be a promising intervention for the composites manufacture, mainly by adding functional and structural characteristics of each material and guarantee the advantages of the use of these composites. In this context, the aim of this study was to develop and evaluated the influence of the incorporation of marine spongin (SPG) into Biosilicate® (BS) in different proportions be used during bone repair. For this purpose, it was to develop and investigate different BS/SPG formulations for physico-chemical and morphological characteristics by pH, loss mass, Fourier transform infrared spectrometer (FTIR) and scanning electron microscope (SEM) analysis. Additionally, the influence of these composites on cell viability, proliferation, and alkaline phosphatase (ALP) activity were investigated. The results revealed that the pH values of all BS groups (with or without SPG) increased over time. A significant mass loss was observed in all composites, mainly with higher SPG percentages. Additionaly, SEM micrographies demonstrated fibers of SPG into BS and material degradation over time. Moreover, FTIR spectral analysis revealed characteristic peaks of PMMA, BS, and SPG in BS/SPG composites. BS/SPG groups demonstrated a positive effect for fibroblast proliferation after 3 and 7 days of culture. Additionally, BS and BS/SPG formulations (at 10% and 20% of SPG) presented similar values of osteoblasts viability and proliferation after 7 days of culture. Furthermore, ALP activity demonstrated no significant difference between BS and BS/SPG scaffolds, at any composition. Based on the present in vitro results, it can be concluded that the incorporation of SPG into BS was possible and produced an improvement in the physical-chemical characteristics and in the biological performance of the graft especially the formulation with 80/20 and 90/10. Future research should focus on in vivo evaluations of this novel composite.

Incorporation of Collagen from Marine Sponges (Spongin) into Hydroxyapatite Samples: Characterization and In Vitro Biological Evaluation.

Biomaterial-based bone grafts have an important role in the field of bone tissue engineering. One of the most promising classes of biomaterials is collagen, including the ones from marine biodiversity (in general, called spongin (SPG)). Also, hydroxyapatite (HA) has an important role in stimulating bone metabolism. Therefore, this work investigated the association of HA and SPG composites in order to evaluate their physico-chemical and morphological characteristics and their in vitro biological performance. For this, pre-set composite disks were evaluated by means of mass loss after incubation, pH, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and "in vitro" cell viability. pH measurements showed no statistical difference between groups. Moreover, a higher mass loss was observed for HA/SPG70/30 compared to the other groups for all experimental periods. Moreover, SEM representative micrographs showed the degradation of the samples with and without immersion. FTIR analysis demonstrated the absorption peaks for poly(methyl methacrylate) (PMMA), HA, and SPG. A higher L292 cell viability for control and PMMA was observed compared to HA and HA/SPG 90/10. Also, HA/SPG 70/30 showed higher cell viability compared to HA and HA/SPG 90/10 on days 3 and 7 days of culture. Furthermore, HA showed a significant lower MC3T3 cell viability compared to control and HA/SPG 70/30 on day 3 and no significant difference was observed between the composites in the last experimental period. Based on our investigations, it can be concluded that the mentioned composites were successfully obtained, presenting improved biological properties, especially the one mimicking the composition of bone (with 70% of HA and 30% of SPG). Consequently, these data highlight the potential of the introduction of SPG into HA to improve the performance of the graft for bone regeneration applications. Further long-term studies should be carried out to provide additional information concerning the late stages of material degradation and bone healing in the presence of HA/SPG.

Biosilicate/PLGA osteogenic effects modulated by laser therapy: In vitro and in vivo studies.

The main purpose of the present work was to evaluate if low laser level therapy (LLLT) can improve the effects of Biosilicate®/PLGA (BS/PLGA) composites on cell viability and bone consolidation using a tibial defects of rats. The composites were characterized by scanning electron microscope (SEM) and reflection Fourier transform infrared spectrometer (FTIR). For the in vitro study, fibroblast and osteoblast cells were seeded in the extract of the composites irradiated or not with LLLT (Ga-Al-As, 808nm, 10J/cm2) to assess cell viability after 24, 48 and 72h. For the in vivo study, 80 Wistar rats with tibial bone defects were distributed into 4 groups (BS; BS+LLLT; BS/PLGA and BS/PLGA+LLLT) and euthanized after 2 and 6weeks. Laser irradiation Ga-Al-As (808nm, 30J/cm2) in the rats was performed 3 times a week. The SEM and FTIR results revealed that PLGA were successfully inserted into BS and the microparticles degraded over time. The in vitro findings demonstrated higher fibroblast viability in both BS/PLGA groups after 24h and higher osteoblast viability in BS/PLGA+LLLT in all periods. As a conclusion, animals treated with BS/PLGA+LLLT demonstrated an improved material degradation and an increased amount of granulation tissue and newly formed bone.

Characterization and biological evaluation of the introduction of PLGA into biosilicate®.

The aims of this study were to characterize different BS/PLGA composites for their physicochemical and morphological characteristics and evaluate the in vitro and in vivo biological performance. The physicochemical and morphological modifications were analyzed by pH, mass loss, XRD, setting time, and SEM. For in vitro analysis, the osteoblast and fibroblast viability was evaluated. For in vivo evaluations, histopathology and immunohistochemistry were performed in a tibial defect in rats. After incubation, all composites presented lower values in pH and mass loss over time. Moreover, XRD and SEM analysis confirmed that the composites degraded over time. Additionally, pore formation was observed by SEM analysis after incubation mainly in BS/PLGA groups. BS/PLGA showed significantly increased in osteoblast viability 24 h. Moreover, BS/PLGA composites demonstrated an increase in fibroblast viability in all periods analyzed when compared to BS. In the in vivo study, after 2 and 6 weeks of implantation of biomaterials, histopathological findings revealed that the BS/PLGA composites degrades over time, mainly at periphery. Moreover, can be observed the presence of granulation tissue, bone formation, Runx-2, and RANKL immunoexpression in all groups. In conclusion, BS/PLGA composites present appropriate physicochemical characteristics, stimulate the cellular viability, and enhance the bone repair in vivo. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1063-1074, 2017.

Characterization and biocompatibility of a fibrous glassy scaffold.

Bioactive glasses (BGs) are known for their ability to bond to living bone and cartilage. In general, they are readily available in powder and monolithic forms, which are not ideal for the optimal filling of bone defects with irregular shapes. In this context, the development of BG-based scaffolds containing flexible fibres is a relevant approach to improve the performance of BGs. This study is aimed at characterizing a new, highly porous, fibrous glassy scaffold and evaluating its in vitro and in vivo biocompatibility. The developed scaffolds were characterized in terms of porosity, mineralization and morphological features. Additionally, fibroblast and osteoblast cells were seeded in contact with extracts of the scaffolds to assess cell proliferation and genotoxicity after 24, 72 and 144 h. Finally, scaffolds were placed subcutaneously in rats for 15, 30 and 60 days. The scaffolds presented interconnected porous structures, and the precursor bioglass could mineralize a hydroxyapatite (HCA) layer in simulated body fluid (SBF) after only 12 h. The biomaterial elicited increased fibroblast and osteoblast cell proliferation, and no DNA damage was observed. The in vivo experiment showed degradation of the biomaterial over time, with soft tissue ingrowth into the degraded area and the presence of multinucleated giant cells around the implant. At day 60, the scaffolds were almost completely degraded and an organized granulation tissue filled the area. The results highlight the potential of this fibrous, glassy material for bone regeneration, due to its bioactive properties, non-cytotoxicity and biocompatibility. Future investigations should focus on translating these findings to orthotopic applications. Copyright © 2015 John Wiley & Sons, Ltd.

Aerobic exercise training and low-level laser therapy modulate inflammatory response and degenerative process in an experimental model of knee osteoarthritis in rats.

OBJECTIVE: The aim of this study was to evaluate the effects of an aerobic exercise training and low-level laser therapy (LLLT) (associated or not) on degenerative modifications and inflammatory mediators on the articular cartilage using an experimental model of knee OA. MATERIAL AND METHODS: Fifty male Wistar rats were randomly divided into five groups: control group (CG); knee OA control group (OAC); OA plus exercise training group (OAT); OA plus LLLT group (OAL); OA plus exercise training associated with LLLT group (OATL). The exercise training (treadmill; 16 m/min; 50 min/day) and the laser irradiation (two points-medial and lateral side of the left joint; 24 sessions) started 4 weeks after the surgery, 3 days/week for 8 weeks. RESULTS: The results showed that all treated groups showed (irradiated or not) a better pattern of tissue organization, with less fibrillation and irregularities along the articular surface and chondrocytes organization, a lower degenerative process measured by OARSI score and higher thickness values. Additionally, all treated group showed a reduced expression in IL-1ß, caspase-3 and MMP-13 compared to OAC. Moreover, a lower caspase-3 expression was observed in OATL compared to OAL and OAT. CONCLUSION: These results suggest that exercise training and LLLT were effective in preventing cartilage degeneration and modulating inflammatory process induced by knee OA.

Effect of a new bioactive fibrous glassy scaffold on bone repair.

Researchers have investigated several therapeutic approaches to treat non-union fractures. Among these, bioactive glasses and glass ceramics have been widely used as grafts. This class of biomaterial has the ability to integrate with living bone. Nevertheless, bioglass and bioactive materials have been used mainly as powder and blocks, compromising the filling of irregular bone defects. Considering this matter, our research group has developed a new bioactive glass composition that can originate malleable fibers, which can offer a more suitable material to be used as bone graft substitutes. Thus, the aim of this study was to assess the morphological structure (via scanning electron microscope) of these fibers upon incubation in phosphate buffered saline (PBS) after 1, 7 and 14 days and, also, evaluate the in vivo tissue response to the new biomaterial using implantation in rat tibial defects. The histopathological, immunohistochemistry and biomechanical analyzes after 15, 30 and 60 days of implantation were performed to investigate the effects of the material on bone repair. The PBS incubation indicated that the fibers of the glassy scaffold degraded over time. The histological analysis revealed a progressive degradation of the material with increasing implantation time and also its substitution by granulation tissue and woven bone. Histomorphometry showed a higher amount of newly formed bone area in the control group (CG) compared to the biomaterial group (BG) 15 days post-surgery. After 30 and 60 days, CG and BG showed a similar amount of newly formed bone. The novel biomaterial enhanced the expression of RUNX-2 and RANK-L, and also improved the mechanical properties of the tibial callus at day 15 after surgery. These results indicated a promising use of the new biomaterial for bone engineering. However, further long-term studies should be carried out to provide additional information concerning the material degradation in the later stages and the bone regeneration induced by the fibrous material.

Molecular characterization of Hepatozoon sp. from Brazilian dogs and its phylogenetic relationship with other Hepatozoon spp.

To characterize phylogenetically the species which causes canine hepatozoonosis at two rural areas of Rio de Janeiro State, Brazil, we used universal or Hepatozoon spp. primer sets for the 18S SSU rRNA coding region. DNA extracts were obtained from blood samples of thirteen dogs naturally infected, from four experimentally infected, and from five puppies infected by vertical transmission from a dam, that was experimentally infected. DNA of sporozoites of Hepatozoon americanum was used as positive control. The amplification of DNA extracts from blood of dogs infected with sporozoites of Hepatozoon spp. was observed in the presence of primers to 18S SSU rRNA gene of Hepatozoon spp., whereas DNA of H. americanum sporozoites was amplified in the presence of either universal or Hepatozoon spp.-specific primer sets; the amplified products were approximately 600bp in size. Cloned PCR products obtained from DNA extracts of blood from two dogs experimentally infected with Hepatozoon sp. were sequenced. The consensus sequence, derived from six sequence data sets, were blasted against sequences of 18S SSU rRNA of Hepatozoon spp. available at GenBank and aligned to homologous sequences to perform the phylogenetic analysis. This analysis clearly showed that our sequence clustered, independently of H. americanum sequences, within a group comprising other Hepatozoon canis sequences. Our results confirmed the hypothesis that the agent causing hepatozoonosis in the areas studied in Brazil is H. canis, supporting previous reports that were based on morphological and morphometric analyses.

Diagnosis of Hepatozoon spp. in Amblyomma ovale and its experimental transmission in domestic dogs in Brazil.

Transmission of Hepatozoon spp. to dogs was investigated using four species of ixodid ticks: Rhipicephalus sanguineus, Amblyomma aureolatum, Amblyomma ovale and Amblyomma cajennense. We collected completely or partially engorged adult ticks of these species from dogs that were naturally infested and positive for Hepatozoon spp. We selected some of these ixodids and inoculated them orally in four negative dogs. The other ticks were dissected and examined for oocysts. Of all dogs inoculated orally with R. sanguineus, A. aureolatum, A. cajennense and A. ovale, only the animal that received the macerate of A. ovale was positive; evidence (gametocytes in peripheral blood) of infection was found 63 days after inoculation. Among all dissected ticks, we found only two oocysts; these were similar to those of Hepatozoon canis, and both were recovered from a single A. ovale specimen. We inoculated sporozoites recovered from the oocysts intraperitoneally into a Hepatozoon spp. negative dog, and circulating gametocytes were detected 84 days later. Our study demonstrated that A. ovale can be a vector of Hepatozoon spp. in Brazil.