In Vivo Effects of Biosilica and Spongin-Like Collagen Scaffolds on the Healing Process in Osteoporotic Rats.

Due to bioactive properties, introducing spongin-like collagen (SPG) into the biosilica (BS) extracted from marine sponges would present an enhanced biological material for improving osteoporotic fracture healing by increasing bone formation rate. Our aim was to characterize the morphology of the BS/SPG scaffolds by scanning electron microscopy (SEM), the chemical bonds of the material by Fourier transform infrared spectroscopy (FTIR), and evaluating the orthotopic in vivo response of BS/SPG scaffolds in tibial defects of osteoporotic fractures in rats (histology, histomorphometry, and immunohistochemistry) in two experimental periods (15 and 30 days). SEM showed that scaffolds were porous, showing the spicules of BS and fibrous aspect of SPG. FTIR showed characteristic peaks of BS and SPG. For the in vivo studies, after 30 days, BS and BS/SPG showed a higher amount of newly formed bone compared to the first experimental period, observed both in the periphery and in the central region of the bone defect. For histomorphometry, BS/SPG presented higher %BV/TV compared to the other experimental groups. After 15 days, BS presented higher volumes of collagen type I. After 30 days, all groups demonstrated higher volumes of collagen type III compared to volumes at 15 days. After 30 days, BS/SPG presented higher immunostaining of osteoprotegerin compared to the other experimental groups at the same experimental period. The results showed that BS and BS/SPG scaffolds were able to improve bone healing. Future research should focus on the effects of BS/SPG on longer periods in vivo studies.

3D printed scaffolds of biosilica and spongin from marine sponges: analysis of genotoxicity and cytotoxicity for bone tissue repair.

Biosilica (BS) and spongin (SPG) from marine sponges are highlighted for their potential to promote bone regeneration. Moreover, 3D printing is introduced as a technology for producing bone grafts with optimized porous structures, allowing for better cell attachment, proliferation, and differentiation. Thus, this study aimed to characterize the BS and BS/SPG 3D printed scaffolds and to evaluate the biological effects in vitro. The scaffolds were printed using an ink containing 4 wt.% of sodium alginate. The physicochemical characteristics of BS and BS/SPG 3D printed scaffolds were analyzed by SEM, EDS, FTIR, porosity, evaluation of mass loss, and pH measurement. For in vitro analysis, the cellular viability of the MC3T3-E1 cell lineage was assessed using the AlamarBlue® assay and confocal microscopy, while genotoxicity and mineralization potential were evaluated through the micronucleus assay and Alizarin Red S, respectively. SEM analysis revealed spicules in BS, the fibrillar structure of SPG, and material degradation over the immersion period. FTIR indicated peaks corresponding to silicon oxide in BS samples and carbon oxide and amine in SPG samples. BS-SPG scaffolds exhibited higher porosity, while BS scaffolds displayed greater mass loss. pH measurements indicated a significant decrease induced by BS, which was mitigated by SPG over the experimental periods. In vitro studies demonstrated the biocompatibility and non-cytotoxicity of scaffold extracts. .Also, the scaffolds promoted cellular differentiation. The micronucleus test further confirmed the absence of genotoxicity. These findings suggest that 3D printed BS and BS/SPG scaffolds may possess desirable morphological and physicochemical properties, indicating in vitro biocompatibility.

Comparison of Different Methods for Spongin-like Collagen Extraction from Marine Sponges (Chondrilla caribensis and Aplysina fulva): Physicochemical Properties and In Vitro Biological Analysis.

This study aimed to compare different protocols (Protocol 1: P1; Protocol 2: P2; Protocol 3: P3; Protocol 4: P4) for the extraction of spongin-like collagen (SC) from marine sponges. The SEM micrographs demonstrated a fibrillar structure for the extracts from Chondrilla caribensis and the nodular/particulate aggregates for Aplysina fulva. FTIR showed for all samples peaks similar to collagen for both species. For C. caribensis, the extracts obtained using P2, P3, and P4 protocols presented higher values of extraction yield, TPQ, and GAGs. P2 and P4 showed higher values of SC concentration and for antioxidant analysis. For A. fulva, P2, P3, and P4 provided a higher extraction yield besides an increase in the antioxidant assay. For both species, no difference was observed for Col quantification and TPQ analysis; also, higher values of GAGs were found using P2 and P4. Fibroblast proliferation observed for C. caribensis was lower for P1 on day 1 and for P2 and P3 on day 3 (for 50%) compared to the control group. There was a significant reduction in fibroblast cell proliferation for all A. fulva extracts evaluated. It can be concluded that protocols P2 and P4 were more efficient for extracting SC from C. caribensis.

Characterization and Biological Performance of Marine Sponge Collagen

Abstract This study characterized the morphological aspects of marine collagen - spongin (SPG) extract from marine sponges, as well as, evaluating its in vitro and in vivo biological performance. Aplysina fulva marine sponge was used for the SPG extraction. It was investigated the physicochemical and morphological properties of SPG by using scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction and compared to PMMA and bovine collagen. Additionally, the SPG cytotoxicity and its influence on cell proliferation, through in vitro tests. Moreover, the in vivo biological response was investigated using an experimental model of tibial bone defect. The results demonstrated that SPG presented an irregular granular aspect, with a composition of OH, C=O, NH, CN and an amorphous profile. Also, in vitro viability results for the L929 and MC3T3 cell lines cultured with SPG extracts demonstrated normal growth in comparison to controls, except for MC3T3 viability at day 3. For in vivo analysis, using tibial bone defects in rats, SPG treated animals presented an increased rate of material resorption and higher granulation and bone formation deposition in the region of the defect, mainly after 45 days. As a conclusion, SPG was successfully extracted. The in vitro and in vivo studies pointed out that SPG samples produced an increase in L929 and MC3T3 viability and improved the performance in tibial bone defects. It can be concluded that SPG can be used as a bone graft for bone regeneration.

Marine spongin incorporation into Biosilicate® for tissue engineering applications: An in vivo study.

Biomaterials and bone grafts, with the ability of stimulating tissue growth and bone consolidation, have been emerging as very promising strategies to treat bone fractures. Despite its well-known positive effects of biosilicate (BS) on osteogenesis, its use as bone grafts in critical situations such as bone defects of high dimensions or in non-consolidated fractures may not be sufficient to stimulate tissue repair. Consequently, several approaches have been explored to improve the bioactivity of BS. A promising strategy to reach this aim is the inclusion of an organic part, such as collagen, in order to mimic bone structure. Thus, the present study investigated the biological effects of marine spongin (SPG)-enriched BS composites on the process of healing, using a critical experimental model of cranial bone defect in rats. Histopathological and immunohistochemistry analyzes were performed after two and six weeks of implantation to investigate the effects of the material on bone repair (supplemental material-graphical abstract). Histological analysis demonstrated that for both BS and BS/SPG, similar findings were observed, with signs of material degradation, the presence of granulation tissue along the defect area and newly formed bone into the area of the defect. Additionally, histomorphometry showed that the control group presented higher values for Ob.S/BS (%) and for N.Ob/T.Ar (mm2) (six weeks post-surgery) compared to BS/SPG and higher values of N.Ob/T.Ar (mm2) compared to BS (two weeks post-surgery). Moreover, BS showed higher values for OV/TV (%) compared to BS/SPG (six weeks post-surgery). Also, VEGF immunohistochemistry was increased for BS (two weeks post-surgery) and for BS/SPG (six weeks) compared to CG. TGFb immunostaining was higher for BS compared to CG. The results of this study demonstrated that the BS and BS/SPG scaffolds were biocompatible and able to support bone formation in a critical bone defect in rats. Moreover, an increased VEGF immunostaining was observed in BS/SPG.

Evaluation of the In Vivo Biological Effects of Marine Collagen and Hydroxyapatite Composite in a Tibial Bone Defect Model in Rats.

One of the most promising strategies to improve the biological performance of bone grafts is the combination of different biomaterials. In this context, the aim of this study was to evaluate the effects of the incorporation of marine spongin (SPG) into Hydroxyapatite (HA) for bone tissue engineering proposals. The hypothesis of the current study is that SPG into HA would improve the biocompatibility of material and would have a positive stimulus into bone formation. Thus, HA and HA/SPG materials were produced and scanning electron microscopy (SEM) analysis was performed to characterize the samples. Also, in order to evaluate the in vivo tissue response, samples were implanted into a tibial bone defect in rats. Histopathological, immunohistochemistry, and biomechanical analyses were performed after 2 and 6 weeks of implantation to investigate the effects of the material on bone repair. The histological analysis demonstrated that composite presented an accelerated material degradation and enhanced newly bone formation. Additionally, histomorphometry analysis showed higher values of %BV/TV and N.Ob/T.Ar for HA/SPG. Runx-2 immunolabeling was higher for the composite group and no difference was found for VEGF. Moreover, the biomechanical analysis demonstrated similar values for all groups. These results indicated the potential of SPG to be used as an additive to HA to improve the biological performance for bone regeneration applications. However, further long-term studies should be carried out to provide additional information regarding the material degradation and bone regeneration.