Osteogenic Potential of Nano-Hydroxyapatite and Strontium-Substituted Nano-Hydroxyapatite.

Nanohydroxyapatite (nanoHA) is the major mineral component of bone. It is highly biocompatible, osteoconductive, and forms strong bonds with native bone, making it an excellent material for bone regeneration. However, enhanced mechanical properties and biological activity for nanoHA can be achieved through enrichment with strontium ions. Here, nanoHA and nanoHA with a substitution degree of 50 and 100% of calcium with strontium ions (Sr-nanoHA_50 and Sr-nanoHA_100, respectively) were produced via wet chemical precipitation using calcium, strontium, and phosphorous salts as starting materials. The materials were evaluated for their cytotoxicity and osteogenic potential in direct contact with MC3T3-E1 pre-osteoblastic cells. All three nanoHA-based materials were cytocompatible, featured needle-shaped nanocrystals, and had enhanced osteogenic activity in vitro. The Sr-nanoHA_100 indicated a significant increase in the alkaline phosphatase activity at day 14 compared to the control. All three compositions revealed significantly higher calcium and collagen production up to 21 days in culture compared to the control. Gene expression analysis exhibited, for all three nanoHA compositions, a significant upregulation of osteonectin and osteocalcin on day 14 and of osteopontin on day 7 compared to the control. The highest osteocalcin levels were found for both Sr-substituted compounds on day 14. These results demonstrate the great osteoinductive potential of the produced compounds, which can be exploited to treat bone disease.

Incorporation of strontium-containing bioactive particles into PEOT/PBT electrospun scaffolds for bone tissue regeneration.

The combination of biomaterials and bioactive particles has shown to be a successful strategy to fabricate electrospun scaffolds for bone tissue engineering. Among the range of bioactive particles, hydroxyapatite and mesoporous bioactive glasses (MBGs) have been widely used for their osteoconductive and osteoinductive properties. Yet, the comparison between the chemical and mechanical characteristics as well as the biological performances of these particle-containing scaffolds have been characterized to a limited extent. In this work, we fabricated PEOT/PBT-based composite scaffolds incorporating either nanohydroxyapatite (nHA), strontium-containing nanohydroxyapatite (nHA_Sr) or MBGs doped with strontium ions up to 15 wt./vol% and 12,5 wt./vol% for nHA and MBG, respectively. The composite scaffolds presented a homogeneous particle distribution. Morphological, chemical and mechanical analysis revealed that the introduction of particles into the electrospun meshes caused a decrease in the fiber diameter and mechanical properties, yet maintaining the hydrophilic nature of the scaffolds. The Sr2+ release profile differed according to the considered system, observing a 35-day slowly decreasing release from strontium-containing nHA scaffolds, whereas MBG-based scaffolds showed a strong burst release in the first week. In vitro, culture of human bone marrow-derived mesenchymal stromal cells (hMSCs) on composite scaffolds demonstrated excellent cell adhesion and proliferation. In maintenance and osteogenic media, all composite scaffolds showed high mineralization as well as expression of Col I and OCN compared to PEOT/PBT scaffolds, suggesting their ability to boost bone formation even without osteogenic factors. The presence of strontium led to an increase in collagen secretion and matrix mineralization in osteogenic medium, while gene expression analysis showed that hMSCs cultured on nHA-based scaffolds had a higher expression of OCN, ALP and RUNX2 compared to cells cultured on nHA_Sr scaffolds in osteogenic medium. Yet, cells cultured on MBGs-based scaffolds showed a higher gene expression of COL1, ALP, RUNX2 and BMP2 in osteogenic medium compared to nHA-based scaffolds, which is hypothesized to lead to high osteoinductivity in long term cultures.

Promotion of In Vitro Osteogenic Activity by Melt Extrusion-Based PLLA/PCL/PHBV Scaffolds Enriched with Nano-Hydroxyapatite and Strontium Substituted Nano-Hydroxyapatite.

Bone tissue engineering has emerged as a promising strategy to overcome the limitations of current treatments for bone-related disorders, but the trade-off between mechanical properties and bioactivity remains a concern for many polymeric materials. To address this need, novel polymeric blends of poly-L-lactic acid (PLLA), polycaprolactone (PCL) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) have been explored. Blend filaments comprising PLLA/PCL/PHBV at a ratio of 90/5/5 wt% have been prepared using twin-screw extrusion. The PLLA/PCL/PHBV blends were enriched with nano-hydroxyapatite (nano-HA) and strontium-substituted nano-HA (Sr-nano-HA) to produce composite filaments. Three-dimensional scaffolds were printed by fused deposition modelling from PLLA/PCL/PHBV blend and composite filaments and evaluated mechanically and biologically for their capacity to support bone formation in vitro. The composite scaffolds had a mean porosity of 40%, mean pores of 800 µm, and an average compressive modulus of 32 MPa. Polymer blend and enriched scaffolds supported cell attachment and proliferation. The alkaline phosphatase activity and calcium production were significantly higher in composite scaffolds compared to the blends. These findings demonstrate that thermoplastic polyesters (PLLA and PCL) can be combined with polymers produced via a bacterial route (PHBV) to produce polymer blends with excellent biocompatibility, providing additional options for polymer blend optimization. The enrichment of the blend with nano-HA and Sr-nano-HA powders enhanced the osteogenic potential in vitro.

In Vitro Biocompatibility Assessment of Nano-Hydroxyapatite.

Hydroxyapatite (HA) is an important component of the bone mineral phase. It has been used in several applications, such as bone regenerative medicine, tooth implants, drug delivery and oral care cosmetics. In the present study, three different batches of a commercial nanohydroxyapatite (nHA) material were physicochemically-characterized and biologically-evaluated by means of cytotoxicity and genotoxicity using appropriate cell lines based on well-established guidelines (ISO10993-5 and OECD 487). The nHAs were characterized for their size and morphology by dynamic light scattering (DLS) and transmission electron microscopy (TEM) and were found to have a rod-like shape with an average length of approximately 20 to 40 nm. The nanoparticles were cytocompatible according to ISO 10993-5, and the in vitro micronucleus assay showed no genotoxicity to cells. Internalization by MC3T3-E1 cells was observed by TEM images, with nHA identified only in the cytoplasm and extracellular space. This result also validates the genotoxicity since nHA was not observed in the nucleus. The internalization of nHA by the cells did not seem to affect normal cell behavior, since the results showed good biocompatibility of these nHA nanoparticles. Therefore, this work is a relevant contribution for the safety assessment of this nHA material.

The antibacterial and angiogenic effect of magnesium oxide in a hydroxyapatite bone substitute.

Bone graft infections are serious complications in orthopaedics and the growing resistance to antibiotics is increasing the need for antibacterial strategies. The use of magnesium oxide (MgO) is an interesting alternative since it possesses broad-spectrum antibacterial activity. Additionally, magnesium ions also play a role in bone regeneration, which makes MgO more appealing than other metal oxides. Therefore, a bone substitute composed of hydroxyapatite and MgO (HAp/MgO) spherical granules was developed using different sintering heat-treatment cycles to optimize its features. Depending on the sintering temperature, HAp/MgO spherical granules exhibited distinct surface topographies, mechanical strength and degradation profiles, that influenced the in vitro antibacterial activity and cytocompatibility. A proper balance between antibacterial activity and cytocompatibility was achieved with HAp/MgO spherical granules sintered at 1100 ºC. The presence of MgO in these granules was able to significantly reduce bacterial proliferation and simultaneously provide a suitable environment for osteoblasts growth. The angiogenic and inflammation potentials were also assessed using the in vivo chicken embryo chorioallantoic membrane (CAM) model and the spherical granules containing MgO stimulated angiogenesis without increasing inflammation. The outcomes of this study evidence a dual effect of MgO for bone regenerative applications making this material a promising antibacterial bone substitute.

Nano-hydroxyapatite in oral care cosmetics: characterization and cytotoxicity assessment.

Nano-hydroxyapatite has been used as an oral care ingredient, being incorporated in several products for the treatment of dental hypersensitivity and enamel remineralisation. Despite its promising results, regulatory and safety concerns have been discussed and questioned by the European Scientific Committee on Consumer Safety (SCCS) regarding the usage of hydroxyapatite nanoparticles in oral care products. In this work, a commercially available nano-hydroxyapatite was characterized and its cytocompatibility towards human gingival fibroblasts was evaluated, as well as its irritation potential using the in vitro HET-CAM assay. All the conditions chosen in this study tried to simulate the tooth brushing procedure and the hydroxyapatite nanoparticles levels normally incorporated in oral care products. The commercial hydroxyapatite nanoparticles used in this study exhibited a rod-like morphology and the expected chemical and phase composition. The set of in vitro cytotoxicity parameters accessed showed that these nanoparticles are highly cytocompatible towards human gingival fibroblasts. Additionally, these nanoparticles did not possess any irritation potential on HET-CAM assay. This study clarifies the issues raised by SCCS and it concludes that this specific nano-hydroxyapatite is cytocompatible, as these nanoparticles did not alter the normal behaviour of the cells. Therefore, they are safe to be used in oral care products.

Antibacterial bone substitute of hydroxyapatite and magnesium oxide to prevent dental and orthopaedic infections.

Bone substitutes market is growing due to the great demand for bone regenerative therapies. However, most of the actual products available in the market are incapable of inhibiting bacterial colonization, which can lead to tissue infection and possible implant failure. Some bone substitutes are combined with antibiotics to avoid the development of implant-associated infections, but the growing bacterial resistance to antibiotics often makes these products ineffective. Therefore, it is mandatory to develop new and alternative approaches. In the present work, a granular bone substitute of hydroxyapatite was produced, where different percentages of magnesium oxide were introduced. The antibacterial activity and biofilm formation was evaluated towards Staphylococcus aureus and Escherichia coli. The inclusion of magnesium oxide particles reduced bacterial growth and biofilm formation in a concentration-dependent manner, when compared with pure hydroxyapatite materials. Superior antibacterial activity and inhibition of biofilm formation was observed for Staphylococcus aureus with complete eradication when magnesium oxide percentages were equal or above 3 wt%. The materials cytotoxicity was assessed under ISO 10993-5:2009 guidance and through Live/Dead cell marking and none of the produced granules was cytotoxic. In addition, reactive oxygen species production was also evaluated and the results revealed that the exposure to the materials extracts did not induce the formation of reactive oxygen species by cells.

Lontra longicaudis infected with canine parvovirus and parasitized by Dioctophyma renale / Lontra longicaudis de vida livre infectada por parvovirus canino e parasitada por Dioctophyma renale

This study describes a case of parvovirus infection in a river otter (Lontra longicaudis) assisted at the Wildlife Rehabilitation Center and Wildlife Screening Center, Federal University of Pelotas (UFPel), Rio Grande do Sul state, Brazil. Clinical signs included apathy, dark and fetid diarrhea, and crusted lesions on the palmar pads of the fore and hind limbs. The animal died after undergoing support treatment with antibiotics, anti-inflammatory, and fluid therapy. At necropsy, the intestines were reddened and edematous and the right kidney was diminished by one third of its normal size and covered with whitish, spongy material. A female Dioctophyma renale was found free in the abdominal cavity. Histologically, dilatation of the intestinal crypts and fusion and blunting of the intestinal villi were observed. In addition, moderate, multifocal lymphocytic enteritis with lymphoid depletion in Peyer's patches and mesenteric lymph nodes were present. Immunohistochemistry with anti-canine parvovirus monoclonal antibody (anti-CPV) was strongly positive in the bone marrow cells and enterocytes of the intestinal crypts, confirming the diagnosis of parvovirus infection. The peritoneum on the right kidney was expanded with a cuboidal cell border, forming multiple papillary projections associated with eggs of D. renale and severe inflammatory infiltrate (giant cells, macrophages, lymphocytes, eosinophils, and plasma cells). Areas of necrosis and mineralization were also observed. Due to fragmentation and degradation of its natural habitat, the otter approached the urban area and was contaminated with the virus, which is hosted and disseminated by domestic animals. Infection with D. renale can be associated with the large population of parasitized domestic animals, which eliminate the helminth eggs through urine, contaminating the environment where the parasite intermediate and paratenic hosts co-inhabit. The diseases of these animals can be a decline factor of wild populations that inhabit the region and are an alert to spillover risk.(AU)

Descreve-se um caso de parvovirose em uma lontra (Lontra longicaudis) enviada ao Núcleo de Reabilitação da Fauna Silvestre e Centro de Triagem de Animais Silvestres da Universidade Federal de Pelotas, Rio Grande do Sul, Brasil. O animal estava debilitado, apático, apresentava diarreia escura e fétida e lesões crostosas nos coxins palmares dos membros torácicos e pélvicos, morrendo após tratamento de suporte com antibiótico, anti-inflamatório e fluidoterapia. Na necropsia os intestinos estavam edematosos e avermelhados e o rim direito estava recoberto de material brancacento e esponjoso, com comprometimento de cerca de um terço do órgão. Foi observado, também, um exemplar de Dioctophyma renale, fêmea, livre na cavidade abdominal. Histologicamente havia fusionamento das vilosidades, dilatação das criptas intestinais com enterite linfocítica moderada multifocal e depleção linfoide nos linfonodos mesentéricos. Na técnica de imuno-histoquímica (IHQ) com anticorpo monoclonal anti-Parvovírus canino (Anti-CPV) houve marcação positiva nos enterócitos da base das vilosidades intestinais e na medula óssea, confirmando o diagnóstico de parvovirose. O peritônio sobre o rim direito estava espessado e revestido por células cuboides, formando múltiplas projeções papilares, nas quais observava-se acentuado infiltrado de células gigantes, macrófagos, linfócitos, eosinófilos e plasmócitos. Entre as projeções papilares havia ovos de Dioctophyma renale, áreas de necrose, calcificação e células gigantes. Conclui-se que a lontra, em função da fragmentação e degradação de seu habitat natural, aproximou-se do centro urbano e contaminou-se com o vírus, o qual é mantido e disseminado por animais domésticos. Por sua vez, a infecção por D. renale pode estar relacionada com a presença de animais domésticos parasitados, os quais eliminam ovos do helminto através da urina contaminando o ambiente, onde coabitam hospedeiros intermediários e paratênicos do parasito. As doenças desses animais podem ser um fator de declínio das populações de animais silvestres e alerta para o risco de spill-over na região.(AU)

Vascular biosafety of commercial hydroxyapatite particles: discrepancy between blood compatibility assays and endothelial cell behavior.

BACKGROUND: Vascular homeostasis is ensured by a dynamic interplay involving the endothelium, the platelets and the coagulation system. Thus, the vascular safety of particulate materials must address this integrated system, an approach that has been largely neglected. This work analysed the effects of commercial hydroxyapatite (HA) particles in blood compatibility and in endothelial cell behavior, due to their clinical relevance and scarcity of data on their vascular biosafety. RESULTS: Particles with similar chemical composition and distinct size and morphology were tested, i.e. rod-like, nano dimensions and low aspect ratio (HAp1) and needle-shape with wider size and aspect ratio (HAp2). HAp1 and HAp2, at 1 to 10 mg/mL, did not affect haemolysis, platelet adhesion, aggregation and activation, or the coagulation system (intrinsic and extrinsic pathways), although HAp2 exhibited a slight thrombogenic potential at 10 mg/mL. Notwithstanding, significantly lower levels presented dose-dependent toxicity on endothelial cells' behavior. HAp1 and HAp2 decreased cell viability at levels ≥ 250 and ≥ 50 µg/mL, respectively. At 10 and 50 µg/mL, HAp1 did not interfere with the F-actin cytoskeleton, apoptotic index, cell cycle progression, expression of vWF, VECad and CD31, and the ability to form a network of tubular-like structures. Comparatively, HAp2 caused dose-dependent toxic effects in these parameters in the same concentration range. CONCLUSION: The most relevant observation is the great discrepancy of HA particles' levels that interfere with the routine blood compatibility assays and the endothelial cell behavior. Further, this difference was also found to be dependent on the particles' size, morphology and aspect ratio, emphasizing the need of a complementary biological characterization, taking into consideration the endothelial cells' functionality, to establish the vascular safety of particulate HA.

Comparison of nanoparticular hydroxyapatite pastes of different particle content and size in a novel scapula defect model.

Nanocrystalline hydroxyapatite (HA) has good biocompatibility and the potential to support bone formation. It represents a promising alternative to autologous bone grafting, which is considered the current gold standard for the treatment of low weight bearing bone defects. The purpose of this study was to compare three bone substitute pastes of different HA content and particle size with autologous bone and empty defects, at two time points (6 and 12 months) in an ovine scapula drillhole model using micro-CT, histology and histomorphometry evaluation. The nHA-LC (38% HA content) paste supported bone formation with a high defect bridging-rate. Compared to nHA-LC, Ostim® (35% HA content) showed less and smaller particle agglomerates but also a reduced defect bridging-rate due to its fast degradation The highly concentrated nHA-HC paste (48% HA content) formed oversized particle agglomerates which supported the defect bridging but left little space for bone formation in the defect site. Interestingly, the gold standard treatment of the defect site with autologous bone tissue did not improve bone formation or defect bridging compared to the empty control. We concluded that the material resorption and bone formation was highly impacted by the particle-specific agglomeration behaviour in this study.

A highly reproducible continuous process for hydroxyapatite nanoparticles synthesis.

This work presents a continuous process for producing hydroxyapatite nanoparticles (NanoXIM) in a network reactor, NETmix, fed by a calcium solution, a phosphorus solution and an alkaline solution. Hydroxyapatite is considered a biomaterial, used as: food additives and nutritional supplements; bone graft for bone replacement, growth and repair; biocements and coating of metallic implant. Some of the most recent applications include their use in cosmetics, toothpaste and in esthetical treatments for diminishing wrinkles by stimulating conjunctive tissue formation. The proposed process enables the micromixing control, which is essential to form nanometric structures, but it is also a determining factor in the crystals purity, crystallinity and morphology. The reactants distribution scheme at the inlet of the reactor and along the reactor, performed continuously or varying in time, is also a crucial factor to programme the properties of reactant media along the reactor, such as the pH, the supersaturation degree, the Ca/P molar ratio, and the temperature. The calcium phosphate nanoparticles suspension that exits the reactor is submitted to further aging, separation, drying, sintering and milling processes.