In-vitroassessment of ß-tricalcium phosphate/bredigite-ciprofloxacin (CPFX) scaffolds for bone treatment applications.

In the present study, ß-tricalcium phosphate (ß-TCP) scaffolds with various amounts of bredigite (Bre) were fabricated by the space holder method. The effect of bredigite content on the structure, mechanical properties,in vitrobioactivity, and cell viability was investigated. The structural assessment of the composite scaffolds presented interconnected pores with diameter of 300-500 µm with around 78%-82% porosity. The results indicated that the compressive strength of the scaffolds with 20% bredigite (1.91 MPa) was improved in comparison with scaffolds with 10% bredigite (0.52 MPa), due to the reduction of the average pore and grain sizes. Also, the results showed that the bioactivity and biodegradability of ß-TCP/20Bre were better than that of ß-TCP/10Bre. Besides, in this study, the release kinetics of ciprofloxacin (CPFX) loaded ß-TCP/Bre composites as well as the ability of scaffolds to function as a sustained release drug carrier was investigated. Drug release pattern of ß-TCP/bredigite-5CPFX scaffolds exhibited the rapid burst release of 43% for 3 h along with sustained release (82%) for 32 h which is favorable for bone infection treatment. Antibacterial tests revealed that the antibacterial properties of ß-TCP/bredigite scaffolds are strongly related to the CPFX concentration, wherein the scaffold containing 5% CPFX showed the most significant zone of inhibition (33 ± 0.5 mm) againstStaphylococcus aureus. The higher specific surface areas of nanostructure ß-TCP/bredigite scaffolds containing CPFX lead to an initial rapid release followed by constant drug delivery. MTT assay showed that the cell viability of ß-TCP/bredigite scaffold loading with up to 1%-3% CPFX (95 ± 2%), is greater than for scaffolds containing 5% CPFX (84 ± 2%). In Overall, it may suggested that ß-TCP/bredigite containing 1%-3% CPFX possesses great cell viability and antibacterial activity and be employed as bactericidal biomaterials and bone infection treatment.

Mesoporous Silica-Bioglass Composite Pellets as Bone Drug Delivery System with Mineralization Potential.

For decades, local bone drug delivery systems have been investigated in terms of their application in regenerative medicine. Among them, inorganic polymers based on amorphous silica have been widely explored. In this work, we combined two types of amorphous silica: bioglass and doxycycline-loaded mesoporous silica MCM-41 into the form of spherical granules (pellets) as a bifunctional bone drug delivery system. Both types of silica were obtained in a sol-gel method. The drug adsorption onto the MCM-41 was performed via adsorption from concentrated doxycycline hydrochloride solution. Pellets were obtained on a laboratory scale using the wet granulation-extrusion-spheronization method and investigated in terms of physical properties, drug release, antimicrobial activity against Staphylococcus aureus, mineralization properties in simulated body fluid, and cytotoxicity towards human osteoblasts. The obtained pellets were characterized by satisfactory mechanical properties which eliminated the risk of pellets cracking during further investigations. The biphasic drug release from pellets was observed: burst stage (44% of adsorbed drug released within the first day) followed by prolonged release with zero-order kinetics (estimated time of complete drug release was 19 days) with maintained antimicrobial activity. The progressive biomimetic apatite formation on the surface of the pellets was observed. No cytotoxic effect of pellets towards human osteoblasts was noticed.

Zoledronic Acid-Loaded ß-TCP Inhibits Tumor Proliferation and Osteoclast Activation: Development of a Functional Bone Substitute for an Efficient Osteosarcoma Treatment.

Osteosarcoma has a poor survival rate due to relapse and metastasis. Zoledronic acid (ZOL), an anti-resorptive and anti-tumor agent, is used for treating osteosarcoma. Delivery of ZOL to the target region is difficult due to its high binding affinity to bone minerals. This study developed a novel treatment for osteosarcoma by delivering ZOL to the target region locally and sustainably. In this study, we fabricated a novel bone substitute by loading ZOL on ß-tricalcium phosphate (ß-TCP). The ZOL-loaded ß-TCP (ZOL/ß-TCP) would be expected to express the inhibitory effects via both bound-ZOL (bound to ß-TCP) and free-ZOL (release from ZOL/ß-TCP). To explore the ability to release ZOL from the ZOL/ß-TCP, the amount of released ZOL was measured. The released profile indicates that a small amount of ZOL was released, and most of it remained on the ß-TCP. Our data showed that ZOL/ß-TCP could successfully express the effects of ZOL via both bound-ZOL and free-ZOL. In addition, we examined the biological effects of bound/free-ZOL using osteosarcoma and osteoclasts (target cells). The results showed that two states of ZOL (bound/free) inhibit target cell activities. As a result, ZOL/ß-TCP is a promising candidate for application as a novel bone substitute.

Long-term in vitro degradation and in vivo evaluation of resorbable bioceramics.

An essential criterion for the selection of resorbable bioceramics is their ability to degrade inside human body within a reasonable time frame. Furthermore, if the bioceramic can release beneficial ions, such as strontium, as it degrades, recovery time might be shortened. The present study demonstrates that strontium-containing calcium sulfate (Sr,Ca)SO4 can fulfill these criteria. A long-term in vitro degradation analysis for 12 weeks using sintered (Sr,Ca)SO4 discs in phosphate buffered solution (PBS) was conducted. The sintered (Sr,Ca)SO4 disc was then implanted into defects in the distal femur of rats. The degradation rate of (Sr,Ca)SO4 discs showed a strong dependence on the Sr content. Similar results were observed between the long-term in vitro degradation analysis and the in vivo evaluation. The sintered (3.8%Sr,Ca)SO4 disc lost more than 80% of its initial weight after soaking in PBS with shaking at 37 °C for 12 weeks. After 12 weeks in vivo, the remaining volume of the (3.8%Sr,Ca)SO4 disc within the bone defect was ~25%. Over the same time period, new bone was formed at a relative volume of 40%. This study demonstrates the potential of (Sr,Ca)SO4 bioceramic, and the benefits of using a long-term degradation test during the evaluation of resorbable bioceramics.

Mechanical properties and biodegradability of Mg-Zn-Ca alloys: homogenization heat treatment and hot rolling.

In this study, Mg was alloyed with Zn and Ca to produce six different Mg-Zn-Ca alloys (designated as ZX alloys) by the gravity die casting method. Zn contents of the alloys were 1 wt., 3 wt., and 5 wt.% and Ca contents of the alloys were 0.2 wt. and 1.8 wt.%. Homogenization heat treatment was applied to all cast alloys. After that, a part of each homogenization heat-treated alloys was hot-rolled. Microstructure, mechanical properties, electrochemical and immersion corrosion behaviors at simulated physiological conditions of the heat-treated and hot-rolled alloys were compared. Increasing the amount of alloying elements (Zn and Ca) in Mg reduces grain size and improves the hardness. It was seen that the microstructure consisted of α-Mg as a matrix phase and intermetallic phases: Mg2Ca phase for the alloy having Zn/Ca = 0.37 (ZX12) and Ca2Mg6Zn3 phase for the other alloys. When the mechanical properties and corrosion rates of homogenized and hot-rolled alloys were compared, it was seen that hot-rolled ZX10-h (Mg-0.94Zn-0.16Ca) alloy can be considered as a fracture bone fixation plate material with its acceptable properties: 121 ± 2.1 MPa yield strength, 226 ± 3.7 MPa tensile strength, % 4.1 ± 0.2 elongation, and 0.062 mm/year immersion corrosion rate.

Release kinetics of the model protein FITC-BSA from different polymer-coated bovine bone substitutes.

BACKGROUND: Controlled release of proteins bound to conventional bone substitutes is still insufficient. Therefore, this study evaluates in-vitro release kinetics of the model protein FITC-BSA (fluorescein conjugated bovine serum albumine) from insoluble bovine collagenous bone matrices (ICBM) with different polymer coatings. Analyzes aim at comparing FITC-BSA release from uncoated versus coated ICBM over time to find bone substitute coatings with consistent release profiles. METHODS: Release kinetics of FITC-BSA from uncoated as well as coated ICBM with five different polymers (RESOMER R 203 H, RG 503 H, RG 504 H, RG 505, L 206 S) were measured over a period of 11 days (d). Measurements were conducted after 6 h (h), 12 h, 24 h, 3 d, 5 d, 7 d, 9 d and 11 d with six samples for each coated ICBM. Two groups were formed (1) with and (2) without medium change at times of measurement. For each group ANOVA with post-hoc Bonferroni testing was used. Scanning electron microscopy assessed morphologic differences between ICBM coating. RESULTS: In group 1 approx. 70% of FITC-BSA release from uncoated ICBM occurred after 6 h compared to approx. 50% in group 2. Only polymers with medium inherent viscosity, i.e. RESOMER RG 503 H, constantly showed significantly more FITC-BSA release throughout 11 d than uncoated ICBM (p = 0.007). The same was found for group 2 (p = 0.005). No significant differences between PLA and PLGA polymers were found. Scanning electron microscopy results indicate a weak adhesion of polymer coatings to ICBM explaining its rather weak retentive effect on overall FITC-BSA release. CONCLUSIONS: Medium molecular size polymers reduce the overall released FITC-BSA from ICBM over time. In clinical practice these polymers may prove ideal for bone substitute materials.

Resultados del uso de la fibrina rica en plaquetas y rellenos óseos en la regeneración ósea guiada. Revisión sistemática / Results of platelet rich fibrin and bone grafts in guided bone regeneration. A systematic review

OBJETIVO: El propósito de esta revisión fue evaluar sistemáticamente la literatura científica sobre los resultados que se obtienen al combinar la fibrina rica en plaquetas (PRF) y rellenos óseos en la regeneración ósea guiada. MATERIALES Y MÉTODOS: Búsqueda detallada en las bases de datos PubMed, Scopus, Web of Science, ScienceDirect, Cochrane y SciELO para obtener la información más actualizada de los resultados (grado de relleno óseo, éxito de la cirugía, movilidad del implante, complicaciones posquirúrgicas, supervivencia del implante) entre los casos tratados con PRF y los casos donde se combinó PRF con algún tipo de relleno óseo. RESULTADOS: De las 965 publicaciones identificadas inicialmente, se excluyeron reportes clínicos, revisiones, estudios observacionales, etc. Se incluyeron 12 ensayos clínicos que contrastaron las variables entre la técnica con PRF solo y la combinación con un relleno óseo. CONCLUSIÓN: La combinación entre PRF más rellenos óseos promueve la neoformación ósea, aumenta el trabeculado y mejora los tiempos de cicatrización; sin embargo, al cabo de 6 meses de control los resultados no son diferentes significativamente de los de los grupos que no utilizaron PRF en el procedimiento de levantamiento de piso de seno maxilar con técnica de ventana lateral. Respecto a la preservación de reborde alveolar, los distintos estudios no son concluyentes: algunos indican que la mezcla de PRF con un relleno óseo parece mejorar las proporciones volumétricas; sin embargo, otros refirieron pérdidas óseas en anchura e incluso mayor grado de inflamación

OBJECTIVE: To evaluate systematically the results obtained by combining platelet rich fibrin (PRF) and bone fill in guided bone regeneration, according to scientific literature. MATERIALS AND METHOD: Detailed search of database from PubMed, Scopus, Web of Science, ScienceDirect, Cochrane and SciELO to obtain the most updated information of clinical results (bone filling degree, surgical success, implant mobility, post-surgical complications, and implant survival) of the cases treated with PRF and the cases where the platelet rich fibrin was combined with a bone filler. Those results registered and compared. RESULTS: From the 965 files initially identified, were excluded clinical reports, reviews, observational studies, comments, studies with pediatric patients and so on. There were included 12 clinical essays where the variables of the technique with PRF and the technique where the PRF was combined with a bone filler were contrasted. CONCLUSION: The combination of PRF plus bone filler promotes bone neoformation, increased trabecular bones, and improved healing times; however, after 6 months of monitoring, the results were not significantly different with the groups that did not use the PRF in the procedure of maxillary sinus floor lift with lateral window technique. Regarding the preservation of alveolar ridge the different studies are not conclusive: some indicate that the mixture of a concentrate with a bone filling seems to improve the volumetric proportions; however, other studies report bone loss in width and even greater degree of inflammation

Resorption of the calcium phosphate layer on S53P4 bioactive glass by osteoclasts.

Clinically, S53P4 bioactive glass (BAG) has shown very promising results in bone infection treatment, but it is also known to degrade very slowly in vivo. To evaluate which mechanisms (cellular or dissolution) can play a role in the degradation of S53P4 BAG and S53P4 BAG putty, in vitro degradation experiments at different pH (7.4 and 4.6) were performed. Micro computed tomography showed a rapid dissolution of the synthetic binder in the putty formulation, within 12 h is simulated body fluid (pH = 7.4), leaving behind only loose granules. Therefore the degradation of the loose granules was investigated further. Significant weight loss was observed and ion chromatography showed that Ca2+, Na+ and PO43- ions were released from S54P4 BAG granules in the two fluids. It was observed that the weight loss and ion release were increased when the pH of the fluid was decreased to 4.6. Osteoclasts are known to create such a low pH when resorbing bone and therefore their capacity to degrade S53P4 surfaces were studied as well. Scanning electron microscopy and energy-dispersive X-ray spectroscopy confirmed that osteoclasts were able to create resorption pits in the calcium phosphate layer on S53P4 BAG surfaces. The silica of the BAG, located underneath the calcium phosphate, seemed to hinder further osteclastic resorption of the material. To our knowledge we were the first to observe actively resorbing osteoclasts on S53P4 bioactive glass surfaces, in vitro. Future research is needed to define the specific role osteoclasts play in the degradation of BAG in vivo.

In vivo and in vitro bioactivity of a "precursor of apatite" treatment on polyetheretherketone.

We recently developed a surface treatment, "precursor of apatite" (PrA), for polyetheretherketone (PrA-PEEK) via a simple, low-temperature process aiming to achieve stronger and faster adhesion to bone. The treatment involves three steps: H2SO4 immersion, exposure to O2 plasma discharge, and alkaline simulated body fluid (alkaline SBF) treatment. This method produces homogeneous fine particles of amorphous calcium phosphate on the PEEK, and we confirmed that PrA-PEEK had excellent apatite formation ability in an SBF immersion test. In the present study using PEEK implants in rabbit tibia, mechanical tests, and histological and radiological analyses revealed that PrA provided the PEEK substrate with excellent bone-bonding properties and osteo-conductivity at early stages (4 and 8 weeks), extending to 16 weeks. In vitro study using MC3T3-E1 cells revealed via XTT assay that PrA on the PEEK substrate resulted in no cytotoxicity, though PrA treatment seemed to suppress gene expression of integrin ß-1 and Alp after 7-day incubation as shown by real-time PCR. On the whole, PrA treatment succeeded in giving in vivo bone-bonding properties to the PEEK substrate, and the treatment is a safe and promising method that can be applied in clinical settings. There was an inconsistency between in vivo and in vitro bioactivity, and this discrepancy indicated that apatite formation does not always need activation of osteoblasts at very early stage and that optimal conditions at cell and organism level may be different. STATEMENT OF SIGNIFICANCE: Polyetheretherketone (PEEK) is an attractive engineering polymer used for spine and dental surgery. To further improve clinical outcome of PEEK-based materials, we developed "Precursor of apatite" (PrA) treatment on the PEEK surface to confer bone-bonding properties. The advantages of this treatment are that it does not require high-temperature processing or special chemicals, and it is inexpensive. The present study clarified excellent in vivo bone-bonding property of PrA treatment. In addition, the results revealed important insights indicating that optimal conditions, especially wettability and crystallinity in calcium phosphate, differ at cell and organism levels. Moreover, our results indicated that prediction of in vivo bioactivity should be done in combination with multiple in vitro tests.

Strontium-releasing fluorapatite glass-ceramic scaffolds: Structural characterization and in vivo performance.

There is increasing interest in biodegradable ceramic scaffolds for bone tissue engineering capable of in situ delivery of ionic species favoring bone formation. Strontium has been shown to be osteogenic, but strontium-containing drugs such as strontium ranelate, used in Europe for the treatment of osteoporosis, are now restricted due to clinical evidence of systemic effects. By doping fluorapatite-based glasses with strontium, we developed ceramic scaffolds with fully interconnected macroporosity and cell size similar to that of cancellous bone, that are also capable of releasing strontium. The crystallization behavior, investigated by XRD and SEM, revealed the formation of akermanite and fluorapatite at the surface of strontium-free glass-ceramic scaffolds, and strontium-substituted fluorapatite at the surface of the strontium-doped scaffolds. At 8 weeks after implantation in a rat calvarial critical size defect, scaffolds doped with the highest amount of strontium led to the highest mineral apposition rate. A significantly higher amount of newly-formed bone was found with the strontium-free glass-ceramic scaffold, and possibly linked to the presence of akermanite at the scaffold surface. We demonstrate by energy dispersive XRF analyses of skull sections that strontium was present in newly formed bone with the strontium-doped scaffolds, while a significant amount of fluorine was incorporated in newly formed bone, regardless of composition or crystallization state. STATEMENT OF SIGNIFICANCE: The present work demonstrates the in vivo action of strontium-containing glass-ceramic scaffolds. These bone graft substitutes are targeted at non load-bearing bone defects. Results show that strontium is successfully incorporated in newly formed bone. This is associated with a significantly higher Mineral Apposition Rate. The benefits of in situ release of strontium are demonstrated. The broader scientific impact of this works builds on the concept of resorbable ceramic scaffolds as reservoirs of ionic species capable of enhancing bone regeneration.

Limitation of the antibiotic-eluting bone graft substitute: An example of gentamycin-impregnated calcium sulfate.

Patients with inadequate volume of alveolar processes or bone defects commonly require graft substitutes in oral, maxillofacial or orthopedic surgery. Ridge augmentation and reconstruction of facial bony defects with bone graft materials achieve better outcomes in functional and aesthetic rehabilitation. The injectable calcium sulfate filler is used widely in intra-operative applications. Calcium sulfate bone filler has been shown to upregulate bone formation-related mRNA genes in vitro and improve osseointegration in vivo. In addition, the bone graft substitute can be used as a drug delivery system for antibiotics to treat or prevent infections based on the clinical experiences. However, the influences of antibiotics addition on the calcium sulfate are not fully understood. In this study, calcium sulfate impregnated with gentamycin in different weight ratios was characterized. The results showed that gentamycin prolonged the hydration process and extended initial/final setting times of calcium sulfate. The addition of gentamycin slowed the conversion from calcium sulfate hemihydrate to dihydrate and changed the crystalline phase and microstructure. Higher amounts of gentamycin added resulted in faster degradation and lower mechanical strength of calcium sulfate. This study reveals that the extended setting time, decreased compressive strength, and the accelerated degradation of the gentamycin-impregnated calcium sulfate bone graft substitutes should be considered during intra-operative applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 80-87, 2018.

Osteoinductive porous biphasic calcium phosphate ceramic as an alternative to autogenous bone grafting in the treatment of mandibular bone critical-size defects.

The bone-induction capacity of a porous biphasic calcium phosphate (pBCP) using heterotopic implantation in mouse (mHI-model) and its efficacy as substitute for autograft in mandibular critical-size defect in rabbit (rabMCSD-model) was investigated. In mHI-model, pBCP was implanted into the thigh muscles and bone formation was histomorphometrically and immunohistochemically evaluated. In rabMCSD-model, 13 mm bone defects were treated with pBCP or autograft and bone repair comparatively evaluated by radiographic and histomorphometric methods. In mHI-model, formed bone and immunolabeling for bone morphogenetic protein-2 and osteopontin were observed in 90% of pBCP implanted samples after 12 weeks. In rabMCSD-model neither statistically significant difference was found in newly formed bone between pBCP and autograft groups at 4 weeks (18.8 ± 5.5% vs 27.1 ± 5.6%), 8 weeks (22.3 ± 2.7% vs 26.2 ± 5.1), and 12 weeks (19.6 ± 4.7% vs 19.6 ± 2.3%). At 12 weeks, the stability and contour of the mandible were restored in both treatments. Near tooth remaining, pBCP particles were covered by small amount of mineralized tissue exhibiting perpendicular attachments of collagen fiber bundles with histological characteristic of acellular cementum. Within the limitations of this study, it was concluded that pBCP is osteoinductive and able to stimulate the new formation of bone and cementum-like tissues in rabMCSD-model, suggesting that it may be an alternative to treatment of large bone defect and in periodontal regenerative therapy. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1546-1557, 2018.

Understanding long-term silver release from surface modified porous titanium implants.

Prevention of orthopedic device related infection (ODRI) using antibiotics has met with limited amount of success and is still a big concern during post-surgery. As an alternative, use of silver as an antibiotic treatment to prevent surgical infections is being used due to the well-established antimicrobial properties of silver. However, in most cases silver is used in particulate form with wound dressings or with short-term devices such as catheters but not with load-bearing implants. We hypothesize that strongly adherent silver to load-bearing implants can offer longer term solution to infection in vivo. Keeping that in mind, the focus of this study was to understand the long term release study of silver ions for a period of minimum 6months from silver coated surface modified porous titanium implants. Implants were fabricated using a LENS™ system, a powder based additive manufacturing technique, with at least 25% volume porosity, with and without TiO2 nanotubes in phosphate buffer saline (pH 7.4) to see if the total release of silver ions is within the toxic limit for human cells. Considering the fact that infection sites may reduce the local pH, silver release was also studied in acetate buffer (pH 5.0) for a period of 4weeks. Along with that, the osseointegrative properties as well as cytotoxicity of porous titanium implants were assessed in vivo for a period of 12weeks using a rat distal femur model. In vivo results indicate that porous titanium implants with silver coating show comparable, if not better, biocompatibility and bonding at the bone-implant interface negating any concerns related to toxicity related to silver to normal cells. The current research is based on our recently patented technology, however focused on understanding longer-term silver release to mitigate infection related problems in load-bearing implants that can even arise several months after the surgery. STATEMENT OF SIGNIFICANCE: Prevention of orthopedic device related infection using antibiotics has met with limited success and is still a big concern during post-surgery. Use of silver as an antibiotic treatment to prevent surgical infections is being explored due to the well-established antimicrobial properties of silver. However, in most cases silver is used in particulate form with wound dressings or with short-term devices such as catheters but not with load-bearing implants. We hypothesize that strongly adherent silver to load-bearing implants can offer longer-term solution towards infection in vivo. Keeping that in mind, the focus of this study was to understand the long-term release of silver ions, for a period of minimum 6months, from silver coated surface modified porous titanium implants.

Difference in release kinetics of unwashed and washed platelet-released supernatants from bone substitute materials: the impact of platelet preparation modalities.

BACKGROUND AND OBJECTIVE: In regenerative dentistry, platelet preparations are applied to stimulate bone healing and periodontal regeneration. Here, we pursue a strategy where bone substitutes are used as carriers for platelet-released supernatants. The mitogenic capacity and release kinetics of loaded bone substitutes were assessed. MATERIAL AND METHODS: Platelet-released supernatants of washed platelets (washed PRS) and platelet-released supernatants of unwashed platelets (unwashed PRS) were lyophilized onto the bone substitutes deproteinized bovine bone mineral, hydroxyapatite and ß-tricalcium phosphate. Scanning electron microscopy images were taken. Supernatants of bone substitutes were collected at hours 1, 3, 6, 24, and 48 and medium was replaced. We evaluated the protein content with the bicinchoninic acid assay and the effect on proliferation using bioassays with human periodontal fibroblasts. Release of growth factors from the loaded bone substitutes was measured based on the platelet-derived growth factor isoform (PDGF-BB) and thrombin immunoassays. Furthermore, we assessed DNA and RNA content of washed PRS and unwashed PRS. RESULTS: Unwashed PRS showed higher total protein concentrations than washed PRS, while the concentration of PDGF-BB, thrombin, DNA, RNA and their mitogenic effect was not significantly different. The bone substitute materials adsorbed protein over time but no significant changes in overall appearance was found. Supernatants collected from unwashed PRS-loaded bone substitute after 1 h induced a potent mitogenic response in periodontal fibroblasts. This pro-mitogenic capacity of the supernatants decreased over the observation period. Supernatants of washed PRS-loaded bone substitutes did not induce a substantial mitogenic effect. Levels of PDGF-BB, thrombin and protein were higher in supernatants of unwashed PRS-loaded bone substitutes than of washed PRS-loaded bone substitutes. CONCLUSION: Bone substitutes loaded with unwashed PRS, but not bone substitutes loaded with washed PRS show continuously declining release kinetics. These data suggest that plasma components in platelet preparations can modify the release kinetics profile.

Mechanical and cytotoxicity evaluation of nanostructured hydroxyapatite-bredigite scaffolds for bone regeneration.

Despite the attractive characteristics of three-dimensional pure hydroxyapatite (HA) scaffolds, due to their weak mechanical properties, researches have focused on the development of composite scaffolds via introducing suitable secondary components. The aim of this study was to develop, for the first time, three-dimensional HA-bredigite (Ca7MgSi4O16) scaffolds containing various amounts of bredigite nanopowder (0, 5, 10 and 15wt.%) using space holder technique. Transmission electron microscopy, scanning electron microscopy, energy-dispersive X-ray spectroscopy and X-ray diffraction spectroscopy were applied in order to study the morphology, fracture surface and phase compositions of nanopowders and scaffolds. Furthermore, the effects of scaffold composition on the mechanical properties, bioactivity, biodegradability, and cytotoxicity were also evaluated. Results showed that the composite scaffolds with average pore size in the range of 220-310µm, appearance porosity of 63.1-75.9% and appearance density of 1.1±0.04g/cm(3) were successfully developed, depending on bredigite content. Indeed, the micropore size of the scaffolds reduced with increasing bredigite content confirming that the sinterability of the scaffolds was improved. Furthermore, the compression strength and modulus of the scaffolds significantly enhanced via incorporation of bredigite content from 0 to 15wt.%. The composite scaffolds revealed superior bioactivity and biodegradability with increasing bredigite content. Moreover, MTT assay confirmed that HA-15wt.% bredigite scaffold significantly promoted cell proliferation compared to tissue culture plate (control) and HA scaffold. Based on these results, three-dimensional HA-bredigite scaffolds could be promising replacements for HA scaffolds in bone regeneration.

Effect of copper-doped silicate 13-93 bioactive glass scaffolds on the response of MC3T3-E1 cells in vitro and on bone regeneration and angiogenesis in rat calvarial defects in vivo.

The release of inorganic ions from biomaterials could provide an alternative approach to the use of growth factors for improving tissue healing. In the present study, the release of copper (Cu) ions from bioactive silicate (13-93) glass scaffolds on the response of cells in vitro and on bone regeneration and angiogenesis in vivo was studied. Scaffolds doped with varying concentrations of Cu (0-2.0wt.% CuO) were created with a grid-like microstructure by robotic deposition. When immersed in simulated body fluid in vitro, the Cu-doped scaffolds released Cu ions into the medium in a dose-dependent manner and converted partially to hydroxyapatite. The proliferation and alkaline phosphatase activity of pre-osteoblastic MC3T3-E1 cells cultured on the scaffolds were not affected by 0.4 and 0.8wt.% CuO in the glass but they were significantly reduced by 2.0wt.% CuO. The percent new bone that infiltrated the scaffolds implanted for 6weeks in rat calvarial defects (46±8%) was not significantly affected by 0.4 or 0.8wt.% CuO in the glass whereas it was significantly inhibited (0.8±0.7%) in the scaffolds doped with 2.0wt.% CuO. The area of new blood vessels in the fibrous tissue that infiltrated the scaffolds increased with CuO content of the glass and was significantly higher for the scaffolds doped with 2.0wt.% CuO. Loading the scaffolds with bone morphogenetic protein-2 (1µg/defect) significantly enhanced bone infiltration and reduced fibrous tissue in the scaffolds. These results showed that doping the 13-93 glass scaffolds with up to 0.8wt.% CuO did not affect their biocompatibility whereas 2.0wt.% CuO was toxic to cells and detrimental to bone regeneration.

A biocompatible hybrid material with simultaneous calcium and strontium release capability for bone tissue repair.

The increasing interest in the effect of strontium in bone tissue repair has promoted the development of bioactive materials with strontium release capability. According to literature, hybrid materials based on the system PDMS-SiO2 have been considered a plausible alternative as they present a mechanical behavior similar to the one of the human bone. The main purpose of this study was to obtain a biocompatible hybrid material with simultaneous calcium and strontium release capability. A hybrid material, in the system PDMS-SiO2-CaO-SrO, was prepared with the incorporation of 0.05 mol of titanium per mol of SiO2. Calcium and strontium were added using the respective acetates as sources, following a sol-gel technique previously developed by the present authors. The obtained samples were characterized by FT-IR, solid-state NMR, and SAXS, and surface roughness was analyzed by 3D optical profilometry. In vitro studies were performed by immersion of the samples in Kokubo's SBF for different periods of time, in order to determine the bioactive potential of these hybrids. Surfaces of the immersed samples were observed by SEM, EDS and PIXE, showing the formation of calcium phosphate precipitates. Supernatants were analyzed by ICP, revealing the capability of the material to simultaneously fix phosphorus ions and to release calcium and strontium, in a concentration range within the values reported as suitable for the induction of the bone tissue repair. The material demonstrated to be cytocompatible when tested with MG63 osteoblastic cells, exhibiting an inductive effect on cell proliferation and alkaline phosphatase activity.

Combination of simvastatin, calcium silicate/gypsum, and gelatin and bone regeneration in rabbit calvarial defects.

The present study was performed to determine whether simvastatin improves bone regeneration when combined with calcium silicate/gypsum and gelatin (CS-GEL). The surface morphology was determined using field-emission scanning electron microscopy (FSEM). Degradation in vitro was evaluated by monitoring the weight change of the composites soaked in phosphate buffered saline (PBS). Drug release was evaluated using high-performance liquid chromatography (HPLC). Cytotoxicity testing was performed to assess the biocompatibility of composites. Four 5 mm-diameter bone defects were created in rabbit calvaria. Three sites were filled with CS-GEL, 0.5 mg simvastatin-loaded CS-GEL (SIM-0.5) and 1.0 mg simvastatin-loaded CS-GEL (SIM-1.0), respectively, and the fourth was left empty as the control group. Micro-computed tomography (micro-CT) and histological analysis were carried out at 4 and 12 weeks postoperatively. The composites all exhibited three-dimensional structures and showed the residue with nearly 80% after 4 weeks of immersion. Drug release was explosive on the first day and then the release rate remained stable. The composites did not induce any cytotoxicity. The results in vivo demonstrated that the new bone formation and the expressions of BMP-2, OC and type I collagen were improved in the simvastatin-loaded CS-GEL group. It was concluded that the simvastatin-loaded CS-GEL may improve bone regeneration.

Novel nanocomposite biomaterials with controlled copper/calcium release capability for bone tissue engineering multifunctional scaffolds.

This work aimed to develop novel composite biomaterials for bone tissue engineering (BTE) made of bioactive glass nanoparticles (Nbg) and alginate cross-linked with Cu(2+) or Ca(2+) (AlgNbgCu, AlgNbgCa, respectively). Two-dimensional scaffolds were prepared and the nanocomposite biomaterials were characterized in terms of morphology, mechanical strength, bioactivity, biodegradability, swelling capacity, release profile of the cross-linking cations and angiogenic properties. It was found that both Cu(2+) and Ca(2+) are released in a controlled and sustained manner with no burst release observed. Finally, in vitro results indicated that the bioactive ions released from both nanocomposite biomaterials were able to stimulate the differentiation of rat bone marrow-derived mesenchymal stem cells towards the osteogenic lineage. In addition, the typical endothelial cell property of forming tubes in Matrigel was observed for human umbilical vein endothelial cells when in contact with the novel biomaterials, particularly AlgNbgCu, which indicates their angiogenic properties. Hence, novel nanocomposite biomaterials made of Nbg and alginate cross-linked with Cu(2+) or Ca(2+) were developed with potential applications for preparation of multifunctional scaffolds for BTE.

BMP2-loaded hollow hydroxyapatite microspheres exhibit enhanced osteoinduction and osteogenicity in large bone defects.

The regeneration of large bone defects is an osteoinductive, osteoconductive, and osteogenic process that often requires a bone graft for support. Limitations associated with naturally autogenic or allogenic bone grafts have demonstrated the need for synthetic substitutes. The present study investigates the feasibility of using novel hollow hydroxyapatite microspheres as an osteoconductive matrix and a carrier for controlled local delivery of bone morphogenetic protein 2 (BMP2), a potent osteogenic inducer of bone regeneration. Hollow hydroxyapatite microspheres (100±25 µm) with a core (60±18 µm) and a mesoporous shell (180±42 m(2)/g surface area) were prepared by a glass conversion technique and loaded with recombinant human BMP2 (1 µg/mg). There was a gentle burst release of BMP2 from microspheres into the surrounding phosphate-buffered saline in vitro within the initial 48 hours, and continued at a low rate for over 40 days. In comparison with hollow hydroxyapatite microspheres without BMP2 or soluble BMP2 without a carrier, BMP2-loaded hollow hydroxyapatite microspheres had a significantly enhanced capacity to reconstitute radial bone defects in rabbit, as shown by increased serum alkaline phosphatase; quick and complete new bone formation within 12 weeks; and great biomechanical flexural strength. These results indicate that BMP2-loaded hollow hydroxyapatite microspheres could be a potential new option for bone graft substitutes in bone regeneration.