Comparison of Borate Bioactive Glass and Calcium Sulfate as Implants for the Local Delivery of Teicoplanin in the Treatment of Methicillin-Resistant Staphylococcus aureus-Induced Osteomyelitis in a Rabbit Model.

There is growing interest in biomaterials that can cure bone infection and also regenerate bone. In this study, two groups of implants composed of 10% (wt/wt) teicoplanin (TEC)-loaded borate bioactive glass (designated TBG) or calcium sulfate (TCS) were created and evaluated for their ability to release TEC in vitro and to cure methicillin-resistant Staphylococcus aureus (MRSA)-induced osteomyelitis in a rabbit model. When immersed in phosphate-buffered saline (PBS), both groups of implants provided a sustained release of TEC at a therapeutic level for up to 3 to 4 weeks while they were gradually degraded and converted to hydroxyapatite. The TBG implants showed a longer duration of TEC release and better retention of strength as a function of immersion time in PBS. Infected rabbit tibiae were treated by debridement, followed by implantation of TBG or TCS pellets or intravenous injection with TEC, or were left untreated. Evaluation at 6 weeks postimplantation showed that the animals implanted with TBG or TCS pellets had significantly lower radiological and histological scores, lower rates of MRSA-positive cultures, and lower bacterial loads than those preoperatively and those of animals treated intravenously. The level of bone regeneration was also higher in the defects treated with the TBG pellets. The results showed that local TEC delivery was more effective than intravenous administration for the treatment of MRSA-induced osteomyelitis. Borate glass has the advantages of better mechanical strength, more desirable kinetics of release of TEC, and a higher osteogenic capacity and thus could be an effective alternative to calcium sulfate for local delivery of TEC.

A novel injectable borate bioactive glass cement for local delivery of vancomycin to cure osteomyelitis and regenerate bone.

Osteomyelitis (bone infection) is often difficult to cure. The commonly-used treatment of surgical debridement to remove the infected bone combined with prolonged systemic and local antibiotic treatment has limitations. In the present study, an injectable borate bioactive glass cement was developed as a carrier for the antibiotic vancomycin, characterized in vitro, and evaluated for its capacity to cure osteomyelitis in a rabbit tibial model. The cement (initial setting time = 5.8 ± 0.6 min; compressive strength = 25.6 ± 0.3 MPa) released vancomycin over ~25 days in phosphate-buffered saline, during which time the borate glass converted to hydroxyapatite (HA). When implanted in rabbit tibial defects infected with methicillin-resistant Staphylococcus aureus (MRSA)-induced osteomyelitis, the vancomycin-loaded cement converted to HA and supported new bone formation in the defects within 8 weeks. Osteomyelitis was cured in 87 % of the defects implanted with the vancomycin-loaded borate glass cement, compared to 71 % for the defects implanted with vancomycin-loaded calcium sulfate cement. The injectable borate bioactive glass cement developed in this study is a promising treatment for curing osteomyelitis and for regenerating bone in the defects following cure of the infection.

Toward Strong and Tough Glass and Ceramic Scaffolds for Bone Repair.

The need for implants to repair large bone defects is driving the development of porous synthetic scaffolds with the requisite mechanical strength and toughness in vivo. Recent developments in the use of design principles and novel fabrication technologies are paving the way to create synthetic scaffolds with promising potential for reconstituting bone in load-bearing sites. This article reviews the state of the art in the design and fabrication of bioactive glass and ceramic scaffolds that have improved mechanical properties for structural bone repair. Scaffolds with anisotropic and periodic structures can be prepared with compressive strengths comparable to human cortical bone (100-150 MPa), while scaffolds with an isotropic structure typically have strengths in the range of trabecular bone (2-12 MPa). However, the mechanical response of bioactive glass and ceramic scaffolds in multiple loading modes such as flexure and torsion - as well as their mechanical reliability, fracture toughness, and fatigue resistance - has received little attention. Inspired by the designs of natural materials such as cortical bone and nacre, glass-ceramic and inorganic/polymer composite scaffolds created with extrinsic toughening mechanisms are showing potential for both high strength and mechanical reliability. Future research should include improved designs that provide strong scaffolds with microstructures conducive to bone ingrowth, and evaluation of these scaffolds in large animal models for eventual translation into clinical applications.

Conversion of melt-derived microfibrous borate (13-93B3) and silicate (45S5) bioactive glass in a simulated body fluid.

Microfibrous bioactive glasses are showing a considerable capacity to heal soft tissue wounds, but little information is available on the mechanism of healing. In the present study, the conversion of microfibrous borate bioactive glass (diameter = 0.2-5 µm) with the composition designated 13-93B3 (5.5 Na2O, 11.1 K2O, 4.6 MgO, 18.5 CaO, 3.7 P2O5, 56.6 B2O3 wt%) was evaluated in vitro as a function of immersion time in a simulated body fluid (SBF) at 37 °C using structural and chemical techniques. Silicate 45S5glass microfibers (45 SiO2, 24.5 Na2O, 24.5 CaO, 6 P2O5 wt%) were also studied for comparison. Microfibrous 13-93B3 glass degraded almost completely and converted to a calcium phosphate material within 7-14 days in SBF, whereas >85 % of the silica remained in the 45S5 microfibers, forming a silica gel phase. An amorphous calcium phosphate (ACP) product that formed on the 13-93B3 microfibers crystallized at a slower rate to hydroxyapatite (HA) when compared to the ACP that formed on the 45S5 fibers. For immersion times >3 days, the 13-93B3 fibers released a higher concentration of Ca into the SBF than the 45S5 fibers. The fast and more complete degradation, slow crystallization of the ACP product, and higher concentration of dissolved Ca in SBF could contribute to the capacity of the microfibrous borate 13-93B3 glass to heal soft tissue wounds.

In vitro bioactivity, cytocompatibility, and antibiotic release profile of gentamicin sulfate-loaded borate bioactive glass/chitosan composites.

Borate bioactive glass-based composites have been attracting interest recently as an osteoconductive carrier material for local antibiotic delivery. In the present study, composites composed of borate bioactive glass particles bonded with a chitosan matrix were prepared and evaluated in vitro as a carrier for gentamicin sulfate. The bioactivity, degradation, drug release profile, and compressive strength of the composite carrier system were studied as a function of immersion time in phosphate-buffered saline at 37 °C. The cytocompatibility of the gentamicin sulfate-loaded composite carrier was evaluated using assays of cell proliferation and alkaline phosphatase activity of osteogenic MC3T3-E1 cells. Sustained release of gentamicin sulfate occurred over ~28 days in PBS, while the bioactive glass converted continuously to hydroxyapatite. The compressive strength of the composite loaded with gentamicin sulfate decreased from the as-fabricated value of 24 ± 3 MPa to ~8 MPa after immersion for 14 days in PBS. Extracts of the soluble ionic products of the borate glass/chitosan composites enhanced the proliferation and alkaline phosphatase activity of MC3T3-E1 cells. These results indicate that the gentamicin sulfate-loaded composite composed of chitosan-bonded borate bioactive glass particles could be useful clinically as an osteoconductive carrier material for treating bone infection.

Long-term conversion of 45S5 bioactive glass-ceramic microspheres in aqueous phosphate solution.

The conversion of 45S5 glass and glass-ceramics to a hydroxyapatite (HA)-like material in vitro has been studied extensively, but only for short reaction times (typically <3 months). In this paper, we report for the first time on the long-term conversion of 45S5 glass-ceramic microspheres (designated 45S5c) in an aqueous phosphate solution. Microspheres of 45S5c (75-150 µm) were immersed for 10 years at room temperature (~25 °C) in K(2)HPO(4) solution with a concentration of 0.01 M or 1.0 M, and with a starting pH of 7.0 or 9.5. The reacted 45S5c microspheres and solutions were analyzed using structural and analytical techniques. Only 25-45 vol% of the 45S5c microspheres were converted to an HA-like material after the 10 year reaction. In solutions with a starting pH of 9.5, an increase in the K(2)HPO(4) concentration from 0.01 to 1.0 M resulted in a doubling of the volume of the microspheres converted to an HA-like material but had little effect on the composition of the HA-like product. In comparison, reaction of the 45S5c microspheres in the solution with a starting pH of 7.0 resulted in an HA-like product in the 0.01 M K(2)HPO(4) solution but a calcium pyrophosphate product, Ca(10)K(4)(P(2)O(7))(6).9H(2)O, in the 1.0 M solution. The consequences of these results for the long-term use of 45S5 glass-ceramics in biomedical applications are discussed.

Hollow hydroxyapatite microspheres as a device for controlled delivery of proteins.

Hollow hydroxyapatite (HA) microspheres were prepared by reacting solid microspheres of Li(2)O-CaO-B(2)O(3) glass (106-150 µm) in K(2)HPO(4) solution, and evaluated as a controlled delivery device for a model protein, bovine serum albumin (BSA). Reaction of the glass microspheres for 2 days in 0.02 M K(2)HPO(4) solution (pH = 9) at 37°C resulted in the formation of biocompatible HA microspheres with a hollow core diameter equal to 0.6 the external diameter, high surface area (~100 m(2)/g), and a mesoporous shell wall (pore size ≈ 13 nm). After loading with a solution of BSA in phosphate-buffered saline (PBS) (5 mg BSA/ml), the release kinetics of BSA from the HA microspheres into a PBS medium were measured using a micro bicinchoninic acid (BCA) protein assay. Release of BSA initially increased linearly with time, but almost ceased after 24-48 h. Modification of the BSA release kinetics was achieved by modifying the microstructure of the as-prepared HA microspheres using a controlled heat treatment (1-24 h at 600-900°C). Sustained release of BSA was achieved over 7-14 days from HA microspheres heated for 5 h at 600°C. The amount of BSA released at a given time was dependent on the concentration of BSA initially loaded into the HA microspheres. These hollow HA microspheres could provide a novel inorganic device for controlled local delivery of proteins and drugs.

Freeze extrusion fabrication of 13-93 bioactive glass scaffolds for bone repair.

A solid freeform fabrication technique, freeze extrusion fabrication (FEF), was investigated for the creation of three-dimensional bioactive glass (13-93) scaffolds with pre-designed porosity and pore architecture. An aqueous mixture of bioactive glass particles and polymeric additives with a paste-like consistency was extruded through a narrow nozzle, and deposited layer-by-layer in a cold environment according to a computer-aided design (CAD) file. Following sublimation of the ice in a freeze dryer, the construct was heated according to a controlled schedule to burn out the polymeric additives (below ~500°C), and to densify the glass phase at higher temperature (1 h at 700°C). The sintered scaffolds had a grid-like microstructure of interconnected pores, with a porosity of ~50%, pore width of ~300 µm, and dense glass filaments (struts) with a diameter or width of ~300 µm. The scaffolds showed an elastic response during mechanical testing in compression, with an average compressive strength of 140 MPa and an elastic modulus of 5-6 GPa, comparable to the values for human cortical bone. These bioactive glass scaffolds created by the FEF method could have potential application in the repair of load-bearing bones.

Corrigendum to "Strontium modulates osteogenic activity of bone cement composed of bioactive borosilicate glass particles by activating Wnt/ß-catenin signaling pathway" [Bioact. Mater. 5 (2020) 334-347].

[This corrects the article DOI: 10.1016/j.bioactmat.2020.02.016.].

Effect of pyrophosphate ions on the conversion of calcium-lithium-borate glass to hydroxyapatite in aqueous phosphate solution.

The conversion of glass to a hydroxyapatite (HA) material in an aqueous phosphate solution is used as an indication of the bioactive potential of the glass, as well as a low temperature route for preparing biologically useful materials. In this work, the effect of varying concentrations of pyrophosphate ions in the phosphate solution on the conversion of a calcium-lithium-borate glass to HA was investigated. Particles of the glass (150-355 µm) were immersed for up to 28 days in 0.25 M K(2)HPO(4) solution containing 0-0.1 M K(4)P(2)O(7). The kinetics of degradation of the glass particles and their conversion to HA were monitored by measuring the weight loss of the particles and the ionic concentration of the solution. The structure and composition of the conversion products were analyzed using X-ray diffraction, scanning electron microscopy, and Fourier transform infrared spectroscopy. For K(4)P(2)O(7) concentrations of up to 0.01 M, the glass particles converted to HA, but the time for complete conversion increased from 2 days (no K(4)P(2)O(7)) to 10 days (0.01 M K(4)P(2)O(7)). When the K(4)P(2)O(7) concentration was increased to 0.1 M, the product consisted of an amorphous calcium phosphate material, which eventually crystallized to a pyrophosphate product (predominantly K(2)CaP(2)O(7) and Ca(2)P(2)O(7)). The consequences of the results for the formation of HA materials and devices by the glass conversion route are discussed.

Bioactive borate glass scaffolds: in vitro and in vivo evaluation for use as a drug delivery system in the treatment of bone infection.

The objective of this work was to evaluate borate bioactive glass scaffolds (with a composition in the system Na(2)O-K(2)O-MgO-CaO-B(2)O(3)-P(2)O(5)) as devices for the release of the drug Vancomycin in the treatment of bone infection. A solution of ammonium phosphate, with or without dissolved Vancomycin, was used to bond borate glass particles into the shape of pellets. The in vitro degradation of the pellets and their conversion to a hydroxyapatite-type material in a simulated body fluid (SBF) were investigated using weight loss measurements, chemical analysis, X-ray diffraction, and scanning electron microscopy. The results showed that greater than 90% of the glass in the scaffolds degraded within 1 week, to form poorly crystallized hydroxyapatite (HA). Pellets loaded with Vancomycin provided controlled release of the drug over 4 days. Vancomycin-loaded scaffolds were implanted into the right tibiae of rabbits infected with osteomyelitis. The efficacy of the treatment was assessed using microbiological examination and histology. The HA formed in the scaffolds in vivo, resulting from the conversion of the glass, served as structure to support the growth of new bone and blood vessels. The results in this work indicate that bioactive borate glass could provide a promising biodegradable and bioactive material for use as both a drug delivery system and a scaffold for bone repair.

Effect of Process Variables on the Microstructure of Hollow Hydroxyapatite Microspheres Prepared by a Glass Conversion Method.

Solid microspheres (diameter = 106-150 µm) of a Li(2)O-CaO-B(2)O(3) glass were reacted in a K(2)HPO(4) solution to form hollow hydroxyapatite (HA) microspheres. The effect of the temperature (25°-60°C), K(2)HPO(4) concentration (0.01-0.25M), and pH (9-12) of the solution on the diameter (d) of the hollow core normalized to the diameter (D) of the HA microspheres, the surface area, and the pore size of the microsphere wall was studied. The statistically significant process variables that influenced these microstructural characteristics were evaluated using a factorial design approach. While the pH had little effect, the concentration of the solution had a marked effect on d/D, surface area, and pore size, whereas temperature markedly influenced d/D and pore size, but not the surface area. The largest hollow core size (d/D value ≈ 0.6) was obtained at the lowest temperature (25°C) or the lowest K(2)HPO(4) concentration (0.02M), while microspheres with the highest surface area (140 m(2)/g), with pores of size 10-12 nm were obtained at the highest concentration (0.25M). The consequences of these results for potential application of these hollow HA microspheres as devices for local delivery of proteins, such as drugs or growth factors, are discussed.

Relaxin enhances bone regeneration with BMP-2-loaded hydroxyapatite microspheres.

Our aims were to 1) evaluate the capacity of hollow hydroxyapatite (HA) microspheres (212-250 µm) to serve as a delivery system for controlled release of BMP-2 in vitro and 2) examine relaxin as an enhancer of BMP-2 for bone regeneration. Hollow HA microspheres were converted from borate glass microspheres and characterized using X-ray diffraction, Fourier-transform infrared spectroscopy, scanning electron microscopy, and the Brunauer-Emmett-Teller method. The microspheres loaded with BMP-2 and relaxin were implanted for 6 weeks in Sprague Dawley rats with calvarial defects. BMP-2 alone in the range up to 1 µg per defect exhibited dose-dependent bone regeneration while relaxin alone in the range up to 0.25 µg per defect did not promote bone regeneration. When compared with BMP-2 alone (1 µg per defect), a 50% reduction in the BMP-2 dose was achieved with the addition of 0.05, 0.1, or 0.25 µg of relaxin per defect. These results show that loading HA microspheres with a combination of relaxin and BMP-2 can significantly reduce the BMP-2 dose required to regenerate an equivalent amount of bone.

Mechanical and degradation properties of poly(methyl methacrylate) cement/borate bioactive glass composites.

Bone cement is used extensively in orthopedics to anchor prostheses to bone and fill voids. Incorporating bioactive glass into poly(methyl methacrylate) (PMMA)-based bone cement could potentially improve its effectiveness for these tasks. This study characterizes the mechanical and degradation properties of composites containing PMMA-based bone cement and particles of borate bioactive glass designated as 13-93B3. Glass particles of size 5, 33, and 100 µm were mixed with PMMA bone cement to create composites containing 20, 30, and 40 wt % glass. Composites and a bone cement control were soaked in phosphate-buffered saline. Compressive strength, Young's modulus, weight loss, water uptake, solution pH, and ionic concentrations were measured over 21 days. The compressive strengths of composites decreased over 21 days. Average Young's moduli of the composites remained below 3 GPa. Weight loss and water uptake of specimens did not exceed 2 and 6%, respectively. Boron concentrations and pH of all solutions increased over time, with higher glass weight fractions leading to higher pH values. Results demonstrated that the composite can sustain glass degradation and ionic release without compromising short-term mechanical strength.

Characterization of the conversion of bone cement and borate bioactive glass composites.

Borate bioactive glass 13-93B3 converts into an osteoconductive hydroxyapatite-like material in a liquid medium. In this study, 13-93B3 was incorporated into a commercial PMMA (poly(methyl methacrylate)) bone cement, and the conversion of the glass into a precipitate in solution was investigated with scanning electron microscopy, energy dispersive X-ray spectroscopy, Fourier transform infrared (spectroscopy)-attenuated total reflection, and micro-Raman spectroscopy. Glass particles of 5, 33, and 100 µm diameter were each mixed with the PMMA cement to create 20, 30, and 40% glass-loaded composites. Precipitate formation was found to be a calcium-deficient apatite partially substituted with magnesium ions that resembles native bone material and would ideally encourage bone growth better than stoichiometric hydroxyapatite. Composites of bone cement and 13-93B3 show promise as a means of encouraging bone attachment to the surface of the bone cement.

Strontium modulates osteogenic activity of bone cement composed of bioactive borosilicate glass particles by activating Wnt/ß-catenin signaling pathway.

There is a need for synthetic grafts to reconstruct large bone defects using minimal invasive surgery. Our previous study showed that incorporation of Sr into bioactive borate glass cement enhanced the osteogenic capacity in vivo. However, the amount of Sr in the cement to provide an optimal combination of physicochemical properties and capacity to stimulate bone regeneration and the underlying molecular mechanism of this stimulation is yet to be determined. In this study, bone cements composed of bioactive borosilicate glass particles substituted with varying amounts of Sr (0 mol% to 12 mol% SrO) were created and evaluated in vitro and in vivo. The setting time of the cement increased with Sr substitution of the glass. Upon immersion in PBS, the cement degraded and converted more slowly to HA (hydroxyapatite) with increasing Sr substitution. The released Sr2+ modulated the proliferation, differentiation, and mineralization of hBMSCs (human bone marrow mesenchymal stem cells) in vitro. Osteogenic characteristics were optimally enhanced with cement (designated BG6Sr) composed of particles substituted with 6mol% SrO. When implanted in rabbit femoral condyle defects, BG6Sr cement supported better peri-implant bone formation and bone-implant contact, comparing to cements substituted with 0mol% or 9mol% SrO. The underlying mechanism is involved in the activation of Wnt/ß-catenin signaling pathway in osteogenic differentiation of hBMSCs. These results indicate that BG6Sr cement has a promising combination of physicochemical properties and biological performance for minimally invasive healing of bone defects.

Proliferation and function of MC3T3-E1 cells on freeze-cast hydroxyapatite scaffolds with oriented pore architectures.

Previous work by the authors showed that hydroxyapatite (HA) scaffolds with different types of oriented microstructures and a unique 'elastic-plastic' mechanical response could be prepared by unidirectional freezing of suspensions. The objective of the present work was to evaluate the in vitro cellular response to these freeze-cast HA scaffolds. Unidirectional scaffolds with approximately the same porosity (65-70%) but different pore architectures, described as 'lamellar' (pore width = 25 +/- 5 microm) and 'cellular' (pore diameter = 100 +/- 10 microm), were evaluated. Whereas both groups of scaffolds showed excellent ability to support the proliferation of MC3T3-E1 pre-osteoblastic cells on their surfaces, scaffolds with the cellular-type microstructure showed far better ability to support cell proliferation into the pores and cell function. These results indicate that freeze-cast HA scaffolds with the cellular-type microstructure have better potential for bone repair applications.

Fabrication and testing of silicon nitride bearings in total hip arthroplasty: winner of the 2007 "HAP" PAUL Award.

Total hip arthroplasty (THA) bearings were fabricated from silicon nitride (Si(3)N(4)) powder. Mechanical testing showed that Si(3)N(4) had improved fracture toughness and fracture strength over modern alumina (Al(2)O(3)) ceramic. When tested with Si(3)N(4) cups in a hip simulator, both cobalt-chromium (CoCr) and Si(3)N(4) femoral heads produced low wear rates that were comparable to Al(2)O(3)-Al(2)O(3) bearings in THA. This study offers experimental support for a novel metal-ceramic THA bearing couple that combines the reliability of CoCr femoral heads with the wear advantages of ceramic surfaces.

Growth and differentiation of osteoblastic cells on 13-93 bioactive glass fibers and scaffolds.

This in vitro study was conducted to evaluate the ability of two types of constructs of bioactive, silica-based 13-93 glass fibers to support the growth and differentiation of MC3T3-E1 osteoblastic cells. The two types of constructs tested included single-layer 13-93 glass fiber rafts and three-dimensional porous scaffolds formed from sintered 13-93 fibers. Scanning electron micrographs showed a closely adhering, well-spread morphology of MC3T3-E1 cells seeded on both types of constructs. The scanning electron microscopy images also showed a continuous increase in cell densities during a 6 day incubation on 13-93 glass fiber rafts and scaffolds. Quantitative fluorescence measurements of DNA also revealed a linear increase in cell density during a 6 day incubation on both types of 13-93 constructs. Examination of scaffolds incubated in MTT containing medium showed the presence of metabolically active viable cells within the interior of the scaffold. The addition of ascorbic acid to MC3T3-E1 cells cultured on the 13-93 glass fibers triggered a threefold increase in alkaline phosphatase, a key indicator of osteoblast differentiation. The sintered scaffolds were found to have open, interconnected pores favorable for tissue ingrowth with a compressive strength similar to cancellous bone. Collectively, the results indicate that 13-93 glass fiber scaffolds are a favorable substrate for the growth and differentiation of osteoblasts and a promising material for bone tissue engineering and repair of bone defects.

In vitro study on different cell response to spherical hydroxyapatite nanoparticles.

Hydroxyapatite (HA) is widely used in filling of bone defects and coating on metal parts of prosthetic implants due to its excellent biocompatibility, bioactivity, and bone-bonding properties. It has been demonstrated that micro-sized HA particles cause inflammatory reaction, especially for the needle shaped particles. However, little effort has been concentrated on the cell responses of the spherical HA nanoparticles. The aim of the present work is to chemically and physically characterize the synthesized HA nanoparticles and to investigate the in vitro cell responses. X-ray diffraction, electron microscopy, nitrogen adsorption, and Fourier transform infrared spectroscopy revealed that the particles consisted of nearly spherical crystallites of carbonate-substituted HA with size of 20-40 nm and specific surface area of 75 m(2)/g. L929 cell proliferation experiments demonstrate that the spherical HA nanoparticles is more biocompatible than commercially available HA. On the other hand, U2-OS cell test results show that the inhibition rate of the spherical HA nanoparticles increases with time and concentration. The half effective inhibitory concentration (IC50) of the nanoparticles was determined to be 50.8 mug/mL at 72 h. All these data indicated that the synthesized spherical nanocrystalline HA particles can function as an effective biomaterial for bone tumorectomy repair, while having little adverse effect.