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.

Determining the setting of root canal sealers using an in vivo animal experimental model.

OBJECTIVES: To present and explore the potential of an animal-based experimental model developed to determine the set of root canal sealers in vivo. The setting of AH Plus, BioC Sealer, TotalFill BC Sealer, and Sealapex was determined using either ISO 6876 or the novel in vivo method proposed in this study. MATERIAL AND METHODS: The in vitro setting time of the sealers tested was determined in accordance with ISO 6876:2012. In determining the in vivo set, 24 adult Wistar rats were followed up for two evaluation periods: 1 and 4 weeks. Their upper-right incisor was extracted, and its pulp tissue was removed. The root canal was then filled from retrograde with one of the 4 sealers, and the tooth was re-implanted and fixed with a layer of a flowable composite resin. After 1 or 4 weeks of the surgical procedures, the animals were euthanized, and their incisors were extracted. Two-mm-thick slices of the middle third of the tooth root were obtained and assessed with a Gillmore device, to determine whether or not the sealer had set. RESULTS: The following in vitro results were obtained by using ISO 6876 methodology: AH Plus set after a mean time of 423 ± 20 min and 476 ± 35 min, in metal and plaster molds, respectively. BioC Sealer set after 7 days (in dental plaster molds), whereas TotalFill BC Sealer and Sealapex did not set even after 25 days in both tested conditions (metal or dental plaster molds). Using the novel in vivo methodology, AH Plus, BioC Sealer, and TotalFill BC Sealer set after both 7 and 30 days. In contrast, Sealapex did not set at either time point. CONCLUSIONS: AH Plus and BioC Sealer set under both in vitro and in vivo test conditions. TotalFill BC Sealer did not set under in vitro conditions but did after 1 week under in vivo conditions. Sealapex did not set under either in vitro or in vivo conditions. CLINICAL RELEVANCE: The influence of the testing conditions on the setting results is a clear indication that new in vivo experimental models should be useful in future studies on Bioceramics root canal sealers.

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.

Evaluation of the In Vivo Biocompatibility of Amorphous Calcium Phosphate-Containing Metals.

Among the biomaterials based on calcium phosphate, hydroxyapatite has been widely used due to its biocompatibility and osteoconduction. The substitution of the phosphate group by the carbonate group associated with the absence of heat treatment and low synthesis temperature leads to the formation of carbonated hydroxyapatite (CHA). The association of CHA with other metals (strontium, zinc, magnesium, iron, and manganese) produces amorphous calcium phosphate-containing metals (ACPMetals), which can optimize their properties and mimic biological apatite. This study aimed to evaluate the biocompatibility and biodegradation of ACPMetals in mice subcutaneous tissue. The materials were physicochemically characterized with Fourier Transform InfraRed (FTIR), X-Ray Diffraction (XRD), and Atomic Absorption Spectrometry (AAS). Balb-C mice (n = 45) were randomly divided into three groups: carbonated hydroxyapatite, CHA (n = 15), ACPMetals (n = 15), and without implantation of material (SHAM, n = 15). The groups were subdivided into three experimental periods (1, 3, and 9 weeks). The samples were processed histologically for descriptive and semiquantitative evaluation of the biological effect of biomaterials according to ISO 10993-6:2016. The ACPMetals group was partially biodegradable; however, it presented a severe irritating reaction after 1 and 3 weeks and moderately irritating after nine weeks. Future studies with other concentrations and other metals should be carried out to mimic biological apatite.

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.