A Synergic Strategy: Adipose-Derived Stem Cell Spheroids Seeded on 3D-Printed PLA/CHA Scaffolds Implanted in a Bone Critical-Size Defect Model.

Bone critical-size defects and non-union fractures have no intrinsic capacity for self-healing. In this context, the emergence of bone engineering has allowed the development of functional alternatives. The aim of this study was to evaluate the capacity of ASC spheroids in bone regeneration using a synergic strategy with 3D-printed scaffolds made from poly (lactic acid) (PLA) and nanostructured hydroxyapatite doped with carbonate ions (CHA) in a rat model of cranial critical-size defect. In summary, a set of results suggests that ASC spheroidal constructs promoted bone regeneration. In vitro results showed that ASC spheroids were able to spread and interact with the 3D-printed scaffold, synthesizing crucial growth factors and cytokines for bone regeneration, such as VEGF. Histological results after 3 and 6 months of implantation showed the formation of new bone tissue in the PLA/CHA scaffolds that were seeded with ASC spheroids. In conclusion, the presence of ASC spheroids in the PLA/CHA 3D-printed scaffolds seems to successfully promote bone formation, which can be crucial for a significant clinical improvement in critical bone defect regeneration.

In Vivo Biological Evaluation of Biodegradable Nanofibrous Membranes Incorporated with Antibiofilm Compounds.

Guided bone regeneration involves excluding non-osteogenic cells from the surrounding soft tissues and allowing osteogenic cells originating from native bone to inhabit the defect. The aim of this work was to fabricate, analyze antibiofilm activity and evaluate in vivo biological response of poly (lactic-co-glycolic acid) (PLGA) electrospun membranes incorporated with tea tree oil and furan-2(5H)-one. Samples were exposed to Streptococcus mutans culture and after 48 h incubation, biofilm was evaluated by colony forming units (CFU/mL) followed by scanning electron microscopy. Additionally, seventy-five Balb-C mice were divided into five experimental groups for subcutaneous implantation: tea tree oil loaded PLGA electrospun fiber membrane, furanone loaded PLGA electrospun fiber membrane, neat PLGA electrospun fiber membrane, a commercially available PLGA membrane -Pratix® and Sham (no-membrane implantation). Post implantation period of each experimental group (1, 3 and 9 weeks), samples were collected and processed for by histological descriptive and semiquantitative evaluation. Results showed a significant reduction of bacterial attachment on tea tree oil and furan-2(5H)-one incorporated membranes. Macrophage counts were significant found in all the materials implanted, although giant cells were predominantly associated with electrospun fiber membranes. The incorporation of antibiofilm compounds in nanofibers membranes did not incite inflammatory response significantly different in comparison with pure PLGA electrospun membranes, indicating its potential for development of novel functionalized membranes targeting the inhibition of bacterial biofilms on membrane-grafting materials.

In Vitro and In Vivo Evaluation of Nanostructured Biphasic Calcium Phosphate in Granules and Putty Configurations.

Synthetic biphasic calcium phosphate (BCP) granules and powder are biocompatible biomaterials with a well-known capacity for osteoconduction, presenting very satisfactory clinical and histological results. It remains unanswered if the putty configuration impacts the biological response to the material. In this study, we aimed to compare the cytocompatibility and biocompatibility of nanostructured BCP in the putty configuration (moldable nanostructured calcium phosphate, MnCaP) on the healing of critical-sized bone defects (8 mm) in rat calvaria. Cytocompatibility was determined through the viability of fibroblast cells (V-79) to the extracts of different concentrations of MnCaP. Forty-five Wistar rats were randomly divided into three groups (n = 15)-clot, MnCaP, and commercial biphasic calcium phosphate in granules configurations (Nanosynt®)-and subdivided into three experimental periods (1, 3, and 6 months). Histological, histomorphometric, and microtomographic analyses allowed the evaluation of newly formed bone, residual biomaterial, and connective tissue. The in vitro evaluation showed that MnCaP was cytocompatible. The histomorphometric results showed that the Nanosynt® group granted the highest new-formed bone values at six months (p < 0.05), although the biomaterial volume did not differ between groups. The putty configuration was easier to handle, and both configurations were biocompatible and osteoconductive, presented similar biosorption rates, and preserved the calvaria architecture.

In Vivo Evaluation of Bovine Xenograft Associated With Oxygen Therapy in Alveolar Bone Repair.

To preserve alveolar bone thickness and width after extraction, clinical strategies have been adopted to reduce or eliminate the need for future surgical interventions to increase the alveolar ridge. The use of xenogeneic biomaterials has been increasing for such application. The association of bone substitutes with active oxygen-based materials, which is essential in the wound-healing process, could accelerate bone repair, optimizing the maintenance of alveolar architecture after extraction. However, the truth of this hypothesis is not clear. The present study aimed to compare the biological response to inorganic bovine bone graft Bonefill (BF), associated or not with active oxygen-based oral gel Bluem (BF+BM), in alveolar bone repair. Twenty female Wistar rats were randomly allocated. The left upper central incisor was extracted, and the dental sockets were filled with BF in the control group (n = 10) and with BF+BM in the experimental group (n = 10). The animals were euthanized at 7 and 42 days after implantation (n = 5), and the samples were processed for descriptive histological and histomorphometric evaluations. The results showed no significant difference between the groups (P > .05). Both groups presented a time-dependent increase in newly formed bone and biosorption biomaterial (P = .0001). The association between active oxygen-based gel and inorganic bovine bone graft did not interfere with or improve bone repair during the experimental periods of alveolar bone repair in rats.

Biomimetic Mineralization on 3D Printed PLA Scaffolds: On the Response of Human Primary Osteoblasts Spheroids and In Vivo Implantation.

This study aimed to assess the response of 3D printed polylactic acid (PLA) scaffolds biomimetically coated with apatite on human primary osteoblast (HOb) spheroids and evaluate the biological response to its association with Bone Morphogenetic Protein 2 (rhBMP-2) in rat calvaria. PLA scaffolds were produced via 3D printing, soaked in simulated body fluid (SBF) solution to promote apatite deposition, and characterized by physical-chemical, morphological, and mechanical properties. PLA-CaP scaffolds with interconnected porous and mechanical properties suitable for bone repairing were produced with reproducibility. The in vitro biological response was assessed with human primary osteoblast spheroids. Increased cell adhesion and the rise of in vitro release of growth factors (Platelet-Derived Growth Factor (PDGF), Basic Fibroblast Growth Factor (bFGF), Vascular Endothelial Growth Factor (VEGF) was observed for PLA-CaP scaffolds, when pre-treated with fetal bovine serum (FBS). This pre-treatment with FBS was done in a way to enhance the adsorption of serum proteins, increasing the number of bioactive sites on the surface of scaffolds, and to partially mimic in vivo interactions. The in vivo analysis was conducted through the implantation of 3D printed PLA scaffolds either alone, coated with apatite (PLA-CaP) or PLA-CaP loaded with rhBMP-2 on critical-sized defects (8 mm) of rat calvaria. PLA-CaP+rhBMP2 presented higher values of newly formed bone (NFB) than other groups at all in vivo experimental periods (p < 0.05), attaining 44.85% of NFB after six months. These findings indicated two new potential candidates as alternatives to autogenous bone grafts for long-term treatment: (i) 3D-printed PLA-CaP scaffold associated with spheroids, since it can reduce the time of repair in situ by expression of biomolecules and growth factors; and (ii) 3D-printed PLA-CaP functionalized rhBMP2 scaffold, a biocompatible, bioactive biomaterial, with osteoconductivity and osteoinductivity.

The role of apoptosis associated speck-like protein containing a caspase-1 recruitment domain (ASC) in response to bone substitutes.

The apoptosis-associated Speck-like protein containing a caspase-1 recruitment domain (ASC), present in inflammasomes, regulates inflammation events and is involved in osteogenic phenotype. Nevertheless, its function in bone repair induced by bone substitute biomaterials is unclear. This study aimed to unveil the role of ASC on osteoprogenitor and tissue response to stoichiometric-hydroxyapatite (HA), nanostructured carbonated-hydroxyapatite (CHA), and CHA containing 5% Strontium (SrCHA), characterized previously by XRD, uXRF-SR, and FTIR spectroscopy implants. Thereafter, conditioned media by the biomaterials were used later to treat pre-osteoblasts and an osteogenic stimulus was shown in response to the materials, with higher expression of Runx2, Osterix, ALP, and Collagen 1a1 genes, with significant involvement of inflammatory-related genes. Thus, to better address the involvement of inflammasome, primary cells obtained from both genotypes [Wild-Type (WT) and ASC Knockout (ASC-KO) mice] were subjected to conditioned media up to 7 days, and our data reinforces both HA and CHA induces lower levels of alkaline phosphatase (ALP) than SrCHA, considering both genotypes (p < 0.01), and ASC seems contribute with osteogenic stimulus promoted by SrCHA. Complimentarily, the biomaterials were implanted into both subcutaneous and bone defects in tibia. Histological analysis on 28 days after implantation of biomaterials into mice's subcutaneous tissue revealed moderate inflammatory response to them. Both histomorphometry and µCT analysis of tibias indicated that the biomaterials did not reverse the delay in bone repair of ASC KO, reinforcing the involvement of ASC on bone regeneration and bone de novo deposition. Also, the bone density in CHA was >2-fold higher in WT than ASC-KO samples. HA was virtually not resorbed throughout the experimental periods, in opposition to CHA in the WT group. CHA reduced to half-area after 28 days, and the bone deposition was higher in CHA for WT mice than HA. Taken together, our results show that biomaterials did not interfere with the healing pattern of the ASC KO, but CHA promoted higher bone deposition in the WT group, probably due to its greater biodegradability. These results reinforce the importance of ASC during bone de novo deposition and healing.

Randomized Controlled Clinical Trial of Nanostructured Carbonated Hydroxyapatite for Alveolar Bone Repair.

The properties of the biodegradation of bone substitutes in the dental socket after extraction is one of the goals of regenerative medicine. This double-blind, randomized, controlled clinical trial aimed to compare the effects of a new bioabsorbable nanostructured carbonated hydroxyapatite (CHA) with a commercially available bovine xenograft (Bio-Oss®) and clot (control group) in alveolar preservation. Thirty participants who required tooth extraction and implant placement were enrolled in this study. After 90 days, a sample of the grafted area was obtained for histological and histomorphometric evaluation and an implant was installed at the site. All surgical procedures were successfully carried out without complications and none of the patients were excluded. The samples revealed a statistically significant increase of new bone formation (NFB) in the CHA group compared with Bio-Oss® after 90 days from surgery (p < 0.05). However, the clot group presented no differences of NFB compared to CHA and Bio-Oss®. The CHA group presented less amount of reminiscent biomaterial compared to Bio-Oss®. Both biomaterials were considered osteoconductors, easy to handle, biocompatible, and suitable for alveolar filling. Nanostructured carbonated hydroxyapatite spheres promoted a higher biodegradation rate and is a promising biomaterial for alveolar socket preservation before implant treatment.

Hydrophilic surface of Ti6Al4V-ELI alloy improves the early bone apposition of sheep tibia.

OBJECTIVE: The aim of this preclinical study was to compare histologically and histomorphometrically both sandblasted/acid-etched implant surfaces with or without maintained in an isotonic solution of 0.9% sodium chloride in early stages of osseointegration. MATERIAL AND METHODS: Both implant surfaces were composed of a titanium/aluminum/vanadium alloy (Ti6Al4V-ELI), but they had different surface chemistries: sandblasted/acid-etched titanium surface (FN) or sandblasted/acid-etched surface maintained in an isotonic solution of 0.9% sodium chloride (FA). The surface morphology, topography and chemistry were evaluated by scanning electron microscopy (SEM), confocal microscopy (CM) and X-ray photoelectron spectroscopy (XPS), respectively. Dynamic contact angle (DCA) was employed for wettability evaluation. One implant from each group was placed in the left tibia of twenty healthy, skeletally mature Santa Ines sheep (n = 5). Bone area (BA) and bone-to-implant contact (BIC) were performed on thin sections (30 µm) at 7, 14, 21 and 28 days after implant installation. RESULTS: Despite the roughness and morphology similarities between the groups, at the XPS evaluation, the FA group presented 2.3 times less carbon on the surface (FN: 27.3% and FA: 11.6%), sharply enhanced hydrophilicity and significantly enhanced BA and BIC at 14, 21 and 28 days of healing (P < 0.05) compared with the FN. CONCLUSION: The data suggest that the hydrophilic FA accelerates the BA apposition and BIC interface around the implants during early stages of bone formation, providing highest degree of osseointegration.