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.

Long-term prevalence of taste and olfactory dysfunction in COVID-19 patients: A cross-sectional study.

OBJECTIVES: The objectives of the study were to investigate the long-term prevalence of taste disorder (TD) and olfactory disorder (OD) and associated risk factors in the non-hospitalized southeastern Brazil population of COVID-19 patients. METHODS: This cross-sectional open survey evaluated possible long-term OD and TD in non-hospitalized patients who had been diagnosed with COVID-19 for more than 30 days, through an online self-report questionnaire. Demographic data, comorbidities, symptoms, and the intensity of OD and TD at the time of diagnosis and at the time of completing the questionnaire were evaluated. RESULTS: Three hundred five responses were included. The reported prevalence of OD and TD was 72.9% and 67.4%, respectively, in the moment of diagnosis; after a mean follow-up period of 179 days, 45% and 50% still had some degree of the symptoms. There was a positive correlation between age and the high prevalence of OD (p = 0.02). However, there was no correlation between age and TD (p = 0.961) and weight in relation to OD/TD (p = 0.500 and p = 0.636, respectively). CONCLUSION: This study observed a high long-term prevalence of OD and TD associated with COVID-19, with a low recovery rate during the study period. There was a positive association between older participants and the prevalence of OD.

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 the biocompatibility and biodegradation of a new denatured plasma membrane combined with liquid PRF (Alb-PRF).

Guided bone regeneration (GBR) is a process that involves the regeneration of bone defects through the application of occlusive membranes that mechanically exclude the population of non-osteogenic cells from the surrounding soft tissue. Interestingly, platelet-rich fibrin (PRF) has previously been proposed as an autologous GBR membrane despite its short-term resorption period of 2-3 weeks. Recent clinical observations have demonstrated that, by heating a liquid platelet-poor plasma (PPP) layer and mixing the cell-rich buffy coat zone, the resorption properties of heated albumin gel with liquid-PRF (Alb-PRF) can be significantly improved. The aim of this study was to evaluate the inflammatory reaction, biocompatibility, and extended degradation properties of a new autologous Alb-PRF membrane in comparison to commonly utilized standard PRF after nude mice implantation, according to ISO 10993-6/2016. Two standard preparations of PRF (L-PRF and H-PRF) were compared to novel Alb-PRF following subcutaneous implantation at 7, 14, and 21 days. All groups demonstrated excellent biocompatibility owing to their autologous sources. However, it is worth noting that, while both L-PRF and H-PRF membranes demonstrated significant or complete resorption by 21 days, the Alb-PRF membrane remained volume-stable throughout the duration of the study. This study demonstrates-for the first time, to the best of our knowledge-a marked improvement in the membrane stability of Alb-PRF. This indicates its future potential for use as a biological barrier membrane for GBR procedures with a long-lasting half-life, or as a biological filler material in esthetic medicine applications. Thus, further studies are warranted to explore future clinical applications in various fields of medicine.

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.

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.

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.

Biological evaluation of zinc-containing calcium alginate-hydroxyapatite composite microspheres for bone regeneration.

Zinc is an important element for bone structure and metabolism. Its interaction with hydroxyapatite has been investigated for the improvement of bone repair. The objective of this study was to evaluate the in vitro and in vivo biological response to nanostructured calcium alginate-hydroxyapatite (HA) and zinc-containing HA (ZnHA). Cytocompatibility was evaluated by applying PrestoBlue reagent after exposing murine pre-osteoblast cells to extracts of each biomaterial microspheres. After physical and chemical characterization, the biomaterial microspheres were implanted in a critical size calvaria defect (8 mm) in Wistar rats (n = 30) that were randomly divided into the HA and ZnHA groups. Tissue samples were evaluated through histological and histomorphometric analyses after 1, 3, and 6 months (n = 5). The results showed cellular viability for both groups compared to the negative control, and no differences in metabolic activity were observed. The HA group presented a significant reduction of biomaterial compared with the ZnHA group in all experimental periods; however, a considerable amount of new bone formation was observed surrounding the ZnHA spheres at the 6-month time point compared with the HA group (p < .05). Both biomaterials were biocompatible, and the combination of zinc with hydroxyapatite was shown to improve bone repair.

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.

In vitro and in vivo evaluations of nanocrystalline Zn-doped carbonated hydroxyapatite/alginate microspheres: zinc and calcium bioavailability and bone regeneration.

Background: Zinc-doped hydroxyapatite has been proposed as a graft biomaterial for bone regeneration. However, the effect of zinc on osteoconductivity is still controversial, since the release and resorption of calcium, phosphorus, and zinc in graft-implanted defects have rarely been studied. Methods: Microspheres containing alginate and either non-doped carbonated hydroxyapatite (cHA) or nanocrystalline 3.2 wt% zinc-doped cHA (Zn-cHA) were implanted in critical-sized calvarial defects in Wistar rats for 1, 3, and 6 months. Histological and histomorphometric analyses were performed to evaluate the volume density of newly formed bone, residual biomaterial, and connective tissue formation. Biomaterial degradation was characterized by transmission electron microscopy (TEM) and synchrotron radiation-based X-ray microfluorescence (SR-µXRF), which enabled the elemental mapping of calcium, phosphorus, and zinc on the microsphere-implanted defects at 6 months post-implantation. Results: The bone repair was limited to regions close to the preexistent bone, whereas connective tissue occupied the major part of the defect. Moreover, no significant difference in the amount of new bone formed was found between the two microsphere groups. TEM analysis revealed the degradation of the outer microsphere surface with detachment of the nanoparticle aggregates. According to SR-µXRF, both types of microspheres released high amounts of calcium, phosphorus, and zinc, distributed throughout the defective region. The cHA microsphere surface strongly adsorbed the zinc from organic constituents of the biological fluid, and phosphorus was resorbed more quickly than calcium. In the Zn-cHA group, zinc and calcium had similar release profiles, indicating a stoichiometric dissolution of these elements and non-preferential zinc resorption. Conclusions: The nanometric size of cHA and Zn-cHA was a decisive factor in accelerating the in vivo availability of calcium and zinc. The high calcium and zinc accumulation in the defect, which was not cleared by the biological medium, played a critical role in inhibiting osteoconduction and thus impairing bone repair.

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.

In vitro and in vivo evaluation of strontium-containing nanostructured carbonated hydroxyapatite/sodium alginate for sinus lift in rabbits.

Various synthetic bone substitutes have been developed to reconstruct bone defects. One of the most prevalent ceramics in bone treatment is hydroxyapatite (HA) that is a useful material as bone substitute, however, with a low rate of biodegradation. Its structure allows isomorphic cationic and anionic substitutions to be easily introduced, which can alter the crystallinity, morphology, biocompatibility, and osteoconductivity. The objective of this study was to investigate the in vitro and in vivo biological responses to strontium-containing nanostructured carbonated HA/sodium alginate (SrCHA) spheres (425<ϕ <600 µm) that were used for sinus lifts in rabbits using nanostructured carbonated HA/sodium alginate (CHA) as a reference. Cytocompatibility was determined using a multiparametric assay after exposing murine preosteoblasts to the extracts of these materials. Twelve male and female rabbits underwent bilateral sinus lift procedures and were divided into two groups (CHA or SrCHA) and in two experimental periods (4 and 12 weeks), for microscopic and histomorphometric analyses. The in vitro test revealed the overall viability of the cells exposed to the CHA and SrCHA extracts; thus, these extracts were considered cytocompatible, which was confirmed by three different parameters in the in vitro tests. The histological analysis showed chronic inflammation with a prevalence of macrophages around the CHA spheres after 4 weeks, and this inflammation decreased after 12 weeks. Bone formation was observed in both groups, and smaller quantities of SrCHA spheres were observed after 12 weeks, indicating greater bioresorption of SrCHA than CHA. SrCHA spheres are biocompatible and osteoconductive and undergo bioresorption earlier than CHA spheres.

Chlorhexidine-loaded hydroxyapatite microspheres as an antimicrobial delivery system and its effect on in vivo osteo-conductive properties.

Hydroxyapatite (HA) has been investigated as a delivery system for antimicrobial and antibacterial agents to simultaneously stimulate bone regeneration and prevent infection. Despite evidence supporting the bactericidal efficiency of these HA carriers, few studies have focused on the effect of this association on bone regeneration. In this work, we evaluated the physico-chemical properties of hydroxyapatite microspheres loaded with chlorhexidine (CHX) at two different concentrations, 0.9 and 9.1 µgCHX/cm2 HA, and characterized their effects on in vitro osteoblast viability and bone regeneration. Ultraviolet-visible spectroscopy, scanning and transmission electron microscopy associated with energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy were used to characterize the association of CHX and HA nanoparticles. The high CHX loading dose induced formation of organic CHX plate-like aggregates on the HA surface, whereas a Langmuir film was formed at the low CHX surface concentration. Quantitative evaluation of murine osteoblast viability parameters, including adhesion, mitochondrial activity and membrane integrity of cells exposed to HA/CHX extracts, revealed a cytotoxic effect for both loading concentrations. Histomorphological analysis upon implantation into the dorsal connective tissues and calvaria of rats for 7 and 42 days showed that the high CHX concentration induced the infiltration of inflammatory cells, resulting in retarded bone growth. Despite a strong decrease in in vitro cell viability, the low CHX loading dose did not impair the biocompatibility and osteoconductivity of HA during bone repair. These results indicate that high antimicrobial doses may activate a strong local inflammatory response and disrupt the long-term osteoconductive properties of CHX-HA delivery systems.

Long-term biocompatibility evaluation of 0.5 % zinc containing hydroxyapatite in rabbits.

This study investigates the long-term biocompatibility of 0.5 % zinc-containing hydroxyapatite compared with hydroxyapatite. Spheres (425 < ∅ < 550) of both materials were produced by extrusion of ceramic slurry in calcium chloride and characterized by FTIR, XRD, XRF and SEM. Fifteen White New Zealand rabbits were submitted to general anesthesia, and an perforation (2 mm), was made in each tibia, one for zinc-containing hydroxyapatite sphere implantation and one for hydroxyapatite sphere implantation. After 26, 52 and 78 weeks, the animals were euthanized, and the fragment containing the biomaterial was harvested. A 30-50 µm section was obtained for histological analysis in bright field and polarized light. SEM images revealed similar morphologies between the tested biomaterials. Histological analysis showed that there was no difference between the test groups. The morphometric analysis, however, indicates that there was a greater absorption. The materials are biocompatible, promote osteogenesis and that the zinc-containing hydroxyapatite microspheres were absorbed more quickly.

Effect of surface modifications on early bone healing around plateau root form implants: an experimental study in rabbits.

PURPOSE: The objective of the present study was to evaluate the biomechanical fixation and bone-to-implant contact (BIC) of plateau root form implants of varied surfaces. MATERIALS AND METHODS: Plateau root form implants, 3.5 mm in diameter, 8 mm in length, with 4 surfaces (n = 16 each)--machined, alumina-blasted/acid-etched, alumina-blasted/acid-etched plus nanothickness bioceramic coating, and plasma-sprayed calcium-phosphate--were used. They were bilaterally placed at the distal femur of 16 New Zealand rabbits and remained in place for 2 and 4 weeks in vivo. After euthanizing the rabbits, the implants were subjected to torque to interface fracture and were subsequently processed as nondecalcified approximately 30-microm-thickness slides for histomorphologic analysis and BIC determination. Statistical analysis was performed using analysis of variance at the 95% level of significance, considering implantation time and implant surface as independent variables and the torque-to-interface fracture and BIC as dependent variables. RESULTS: The torque-to-interface fracture was significantly affected by the implant surface (P < .001) but was not affected by the implantation time (P > .20). The implantation time and implant surface had significant effects on the BIC (P < .04 and P < .001, respectively). The greatest torque-to-interface fracture and BIC was observed for the plasma-sprayed calcium-phosphate. CONCLUSION: The implant surface significantly influenced early bone healing around plateau root form implants.