Patent Application
20140212469
The present document relates to bioactive glass scaffolds to be used to regenerate large bone defects in mammals, and in particular to bioactive glass scaffolds having a modified surface area for enhancing bone repair and regeneration.
There is a clinical need for synthetic scaffolds that can be used to regenerate large bone defects in mammals which result from trauma, malignancy, and congenital diseases. Autologous bone grafts (taken from the patient) are the gold standard for treatment and bone allografts (taken from a cadaver) are alternatives to autologous bone grafts. However, both kinds of bone grafts suffer from limitations such as donor site morbidity, limited supply (autografts), and possible transmission of diseases and high cost (allografts). Synthetic scaffolds have been gaining interest as an alternative to bone grafts. However, the existing synthetic scaffolds are generally expensive, hard to manufacture, and suffer from slow bone regeneration. Furthermore, currently available synthetic bone graft substitutes have low strength and are limited to the repair of non-loaded bone only and cannot be used to replace structural bone loss.
Therefore, there is a need to provide new and improved synthetic scaffolds that include bioactive glass, which are inexpensive, easy to fabricate, and when treated to modify the surface, regenerate new bone tissue faster than the existing synthetic scaffolds. Additionally, there is a great need for porous biocompatible scaffolds that can replicate the structure and function of bone and have the requisite mechanical properties for reliable long-term cyclical loading during weight bearing.
In one embodiment, a bioactive glass scaffold is provided to enhance new bone formation including a bioactive glass material formed in a three-dimensional macroporous grid-like microstructure and having a modified surface layer on the surface of the bioactive glass scaffold. The modified surface layer increases the surface area of the bioactive glass, and at least part of the modified surface layer is converted to a calcium phosphate material.
In another embodiment, a method of manufacturing a bioactive glass scaffold is provided that includes: grinding a bioactive glass into fine particles; mixing the fine particles of bioactive glass with a processing aid and a liquid to form a slurry; fabricating a three-dimensional macroporous grid-like microstructure of the slurry to form a bioactive glass scaffold; and modifying the surface of the bioactive glass scaffold with a glass modifier. The modified surface layer increases the surface area of the bioactive glass scaffold to enhance new bone formation.
As described herein, embodiments of a bioactive glass scaffold having a bioactive glass and a modified surface layer for enhancing bone repair and regeneration. The bioactive glass scaffold may be a porous three-dimensional (3D) scaffold for implantation and promotion of bone growth. In addition, a method of manufacturing the bioactive glass scaffold is disclosed which includes forming a grid-like microstructure of bioactive glass and modifying the surface of the glass scaffold by a chemical treatment to enhance new bone formation in mammals. The bioactive glass scaffold can be implanted into a subject to promote bone growth. The bioactive glass scaffold may further include biomolecules such as growth factors which may be incorporated in the modified surface layer. In some embodiments, the modified surface layer of the bioactive glass scaffold may include osteoinductive growth factors such as bone morphogenetic protein (BMP) to stimulate bone formation.
Embodiments of the bioactive glass scaffold provide several advantages. First, the bioactive glass scaffolds may provide a low-cost alternative to bone autografts and allografts (the current treatment options); second, the bioactive glass scaffolds convert to hydroxyapatite or a hydroxyapatite-like material, the mineral constituent of bone, so there is no need to remove the bioactive glass scaffold from the body in a later surgery, as in the case for some synthetic bone graft substitutes; and third, the bioactive glass scaffolds exhibit superior strength to other synthetic bone graft substitutes, so such scaffolds can be applied to the regeneration of load-bearing or load-sharing bones, as well as non-loaded bone.
The bioactive glass scaffold includes a bioactive glass as the foundation for the scaffold. In some embodiments, the bioactive glass is preferably a silicate bioactive glass. Alternatively, some embodiments of the bioactive glass can also include one or more glasses from other glass-forming systems, such as borate, phosphate, borosilicate, etc. In one embodiment, the bioactive glass may be a silicate bioactive glass with a composition designated as 13-93 (6Na2O, 12K2O, 5MgO, 20CaO, 4P2O5, 53SiO2, wt. %). In another embodiment, the bioactive glass scaffold may be B2O3-doped silicate bioactive glass scaffolds with a fibrous microstructure.
Embodiments of the bioactive glass scaffold are macroporous with a high-surface-area modified surface layer formed in a grid-like microstructure.
In some embodiments, the bioactive glass scaffold may be pretreated with a glass modifier to create a modified surface layer. The modified surface layer of the bioactive glass scaffold may create fine pores (nanometers to a few microns in size) to modify the surface roughness and increase the surface area of the bioactive glass scaffolds. The glass modifiers of the bioactive glass may include but are not limited to one or more elements from the alkali metals (e.g., Li, Na, K, etc), alkali-earth metals (Ca, Mg, etc), an aqueous phosphate solution, and other elements from the periodic table. The surface area of the modified surface layer may range from about 1 m2/g to about 100 m2/g.
Once treated with the glass modifier, the modified surface layer may be converted to include a calcium phosphate material. In an embodiment, the calcium phosphate material may be hydroxyapatite (HA) or a hydroxyapatite-like material. Once the bioactive glass scaffold is implanted, the calcium phosphate or HA-like material may further crystallize to HA. In an embodiment, the HA-like material on the modified surface layer may be formed by reaction of the bioactive glass scaffold in an aqueous phosphate solution. HA has been shown to improve the capacity of borate and silicate bioactive glass to support cell proliferation and differentiation in vitro. A rough modified surface layer of carbonated HA may improve the capacity of the bioactive glass scaffolds to support cell proliferation in vitro and to enhance bone formation in vivo.
In addition to material composition and microstructure, scaffold healing to bone in vivo can be markedly affected by other variables, such as the modified surface layer composition and structure, release of osteoinductive growth factors, and presence (or absence) of living cells. A bioactive glass scaffold with a modified surface layer may be implanted as prepared, or in combination with biomolecules, for the promotion of bone growth and regeneration.
In some embodiments, the surface modified bioactive glass scaffold may incorporate biomolecules, such as growth factors, drugs, antibodies, antibiotics or other molecules. The biomolecules may be adsorbed on the porous surface of the surface modified layer of the bioactive glass scaffold or the biomolecules can be chemically bonded onto the surface modified layer. In an embodiment, the modified surface layer of the bioactive glass scaffold may further be loaded with BMP-2. The bioactive glass scaffold may be loaded with about 1 μg/scaffold in one embodiment.
Bioactive glass scaffolds with silicate 13-93 may have promising mechanical properties for the repair of loaded bone. Pretreatment of the bioactive glass scaffolds in an aqueous phosphate solution to convert a modified surface layer to HA or HA-like material, or loading the pretreated bioactive glass scaffolds with BMP-2, may enhance the capacity of the bioactive glass scaffolds to regenerate bone in an osseous defect. The bioactive glass scaffolds pretreated with a glass modifier or BMP-loaded may support faster bone regeneration. Pretreatment of bioactive glass scaffolds to form an HA-like modified surface layer prior to implantation may provide a new and effective approach for regenerating bone in osseous defects. The modified surface layer can enhance the osteoconductivity of the bioactive glass scaffolds and provide a substrate for delivery of therapeutics such as growth factors.
Further provided herein is a method of fabricating the bioactive glass scaffold. The method may include the steps of 1) fabricating a porous bioactive glass scaffold in a desired 3D shape with bioactive glass, and 2) surface modifying the porous 3D bioactive glass scaffold. In some embodiments, the starting material includes a silicate bioactive glass, such as 13-93 glass. The bioactive glass may be a commercially available glass. The bioactive glass may be ground into fine particles (about 1-2 μm) using conventional materials processing methods which then may be combined with a liquid to form a slurry. In an embodiment, the liquid may be water. In some embodiments, the slurry may be composed of about 62.5 wt % glass particles, about 7.5 wt % Pluronic® F-127 (used as a processing aid), and 30 wt % distilled water.
In some embodiments, the bioactive glass scaffold may be formed from the slurry using a solid freeform fabrication method (also known as rapid prototyping), such as robocasting. In robocasting, the bioactive glass scaffold is formed layer-by-layer using a computer-aided design (CAD) file in the fabricating step. However, other methods used for forming glass and ceramics can be used, such as sintering of particles or fibers, sol-gel processing, polymer foam replication, and freezing of suspensions.
In the surface modifying step, the bioactive glass scaffold may be pretreated with a glass modifier. The glass modifier of the bioactive glass may include but is not limited to one or more elements from the alkali metals (e.g., Li, Na, K, etc), alkali-earth metals (Ca, Mg, etc), an aqueous phosphate solution, and other elements from the periodic table. In an embodiment, the glass modifier may be an aqueous phosphate solution treated under specified conditions. Alternatively, aqueous solutions without phosphate ions, or aqueous solutions containing ions or molecules in addition to phosphate ions can also be used.
In some embodiments, the bioactive glass scaffold may be pretreated with a glass modifier for about 1 to about 6 days. In one embodiment, the bioactive glass scaffold may be pretreated with a glass modifier for about 3 days. In various embodiments, the bioactive glass scaffold may be pretreated with a glass modifier at various times in an aqueous phosphate solution to create the modified surface layer on the bioactive glass scaffold. In an embodiment, the modified surface layer may be converted to calcium phosphate, hydroxyapatite (HA), or an HA-like material. The pretreatment may form a modified surface layer of HA on the surface of the bioactive glass scaffold.
In some embodiments, the thickness of the modified surface layer may be controlled by the time and temperature of the pretreatment of the bioactive glass scaffold with the glass modifier. The modified surface layer thickness may range from about 1 μm to about 20 μm. In various embodiments, the modified surface layer thickness may range from about 1 μm to about 10 μm, about 5 μm to about 15 μm, and about 10 μm to about 20 μm. In an embodiment, the modified surface layer may be about 5 μm thick. The thickness of the modified surface layer may be measured using Image J software. Table I shows the thickness of the modified surface layer on silicate (13-93) bioactive glass scaffolds with increasing reaction time in a 0.25M aqueous phosphate solution from 1-6 days at 60° C. and pH=12.0.
The surface modified bioactive glass scaffold can be used as prepared, or after incorporation of biomolecules or other species in the modified surface layer of the bioactive glass scaffold, such as growth factors, drugs, antibodies, antibiotics or other molecules. The biomolecules can be adsorbed on the porous surface of the surface modified layer of the bioactive glass scaffold or they can be chemically bonded onto the surface modified layer. In an embodiment, the modified surface layer of the bioactive glass scaffold may further be loaded with BMP-2. The bioactive glass scaffold may be loaded with about 1 μg/scaffold in one embodiment.
As-fabricated glass scaffolds prepared by sintering melt-derived glass particles may have a dense smooth surface, as seen in
Pretreatment of silicate 13-93 bioactive glass scaffolds prior to implantation with a glass modifier leads to the formation of a nanostructured calcium phosphate that is known to be favorable for protein adsorption and early bone formation. Conversion of bioactive glass in an aqueous phosphate solution forms a calcium phosphate modified surface layer which enhances adhesion, proliferation and differentiation of osteoblast cells in vitro.
The bioactive glass scaffold may be implanted into or onto bone to stimulate bone growth. Without being limited to a particular theory, bone growth may be stimulated by the delivery of a plurality of biomolecules, the modified surface layer including HA, or ingrowth of cells into the pores of the bioactive glass scaffold. New bone formation in the bioactive glass scaffolds may be evaluated using histomorphometric techniques and scanning electron microscopy. The bioactive glass scaffold may remain implanted as it becomes incorporated into the bone. In addition, the bioactive glass scaffold with a modified surface layer may be converted to HA at a slower rate than a scaffold without pretreatment or modified surface layer. Without being limited to a particular theory, the slow conversion to HA may reduce local increases in pH and the concentration of ions released from the glass, which may help cell proliferation around the bioactive glass scaffold.
The greater capacity of the grid-like microstructure to support bone infiltration may result from the uniform microstructure of interconnected pores with a favorable size. As described earlier, interconnected pores of size about 100 μm are recognized as the minimum requirement for supporting tissue ingrowth, but pores of about 300 μm or larger may be required for enhanced bone ingrowth and capillary formation. In the grid-like microstructure, the pores are all interconnected, have the same size, and are not constricted at the necks between adjacent pores. In comparison, the pores in oriented scaffolds have a smaller pore diameter and limited interconnectivity between adjacent pores than the grid-like microstructure of the bioactive glass scaffold. In an embodiment, new bone formation may be osteoconductive and may infiltrate the pores of the bioactive glass scaffold along the edge of the bioactive glass scaffold.
The as-fabricated scaffolds were modified prior to implantation by reacting them in an aqueous phosphate solution to convert a surface layer of the bioactive glass to a hydroxyapatite (HA)-like modified surface layer. In the surface modification process, the bioactive glass scaffolds were immersed for 1, 3, and 6 days in 0.25 M K2HPO4 solution at 60° C. and a starting pH=12.0 (obtained by adding the requisite amount 2M NaOH solution). The mass of the bioactive glass scaffolds to the volume of the K2HPO4 solution was kept constant at 1 g per 200 ml, and the system was stirred gently each day. These reaction conditions were based on previous studies on the conversion of bioactive glasses to HA. After each reaction time, the bioactive glass scaffolds were removed from the solution, washed twice with deionized water, and twice with anhydrous ethanol to displace residual water from the bioactive glass scaffolds. The bioactive glass scaffolds were removed from the ethanol, dried for at least 24 h at room temperature, and stored in desiccator.
In this reaction process, a surface layer of the bioactive glass was converted to a modified surface layer composed of a calcium phosphate material. The thickness and the crystallinity of the modified surface layer were controlled by the time and temperature of the reaction. After the surface treatment, the bioactive glass scaffolds may be washed twice with distilled water and then twice with ethanol, and dried at room temperature for one day.
The morphology, crystallinity, specific surface area, and thickness of the converted layer of the bioactive glass scaffolds changed with an increase in the reaction time (1-6 days) in the K2HPO4 solution (
Table I shows the thickness and specific surface area of the modified surface layer formed by reacting silicate (13-93) bioactive glass scaffolds for the times shown in 0.25M K2HPO4 solution at 60° C. and starting pH=12.0.
The osteoconductivity and osteoinductivity of calcium phosphate and bioactive glass scaffolds for bone repair varied with the material characteristics, such as chemical composition, specific surface area, and micrometer size pores (0.5-10 μm) in the bioactive glass scaffold. While the scaffolds pretreated for 1, 3, and 6 days were all effective, the bioactive glass scaffold pretreated for 3 days showed a significantly better capacity to enhance new bone formation in vivo (
The modified surface layer of the bioactive glass scaffolds was sputter-coated with Au/Pd and examined in a scanning electron microscope, SEM (S-4700; Hitachi, Tokyo, Japan), using an accelerating voltage of 15 kV and a working distance of 8 mm. Some bioactive glass scaffolds were also mounted in epoxy resin, sectioned, polished to expose the cross-sections of the glass filaments, and examined in the SEM (S-4700; Hitachi). The thickness of the modified surface layer was determined from more than 15 measurements in the SEM images using the ImageJ software (National Institutes of Health, USA), and expressed as a mean value±standard deviation (sd).
The modified surface layer was removed by vigorously shaking the bioactive glass scaffolds and used in determining its surface area and phase composition. Surface area measurements were made using nitrogen gas adsorption (Nova 2000e; Quantachrome, Boynton Beach, Fla., USA). The volume of nitrogen adsorbed and desorbed at different gas pressures was measured and used to construct adsorption-desorption isotherms. Eleven points of the adsorption isotherm, which initially followed a linear trend implying monolayer formation of adsorbate, were fitted by the Brunauer-Emmett-Teller equation to determine the surface area.
The presence of crystalline phases in the modified surface layer was determined using X-ray diffraction (XRD) (D/mas 2550 v; Rigaku, The Woodlands, Tex., USA). The material was ground into a powder and analyzed using Cu Kα radiation (A=0.15406 nm) at a scan rate of 1.8°/min in the 28 range 10-80°.
Referring to
The pretreated bioactive glass scaffolds showed a much slower conversion rate to HA in vivo than the as-fabricated scaffold (no pretreatment). After the six-week implantation, the thickness of the converted layer in the pretreated bioactive glass scaffolds showed little change, whereas the converted layer of the as-fabricated scaffolds had a thickness about 20 μm. When compared to the as-fabricated scaffolds, the slow conversion of the pretreated bioactive glass scaffolds in vivo had the effect of reducing local increases in pH and the concentration of ions released from the glass (e.g., Na+; K+), particularly soon after implantation when the conversion rate of the as-fabricated scaffolds is fast. Large increases in the local pH are detrimental to cell proliferation.
Some of the pretreated bioactive glass scaffolds described above were loaded with bone morphogenetic protein-2 (BMP-2) prior to implantation. In the process, a solution of BMP-2 (Shenandoah Biotechnology Inc., PA, USA) in citric acid was prepared by dissolving 10 μg of BMP-2 in 100 μl sterile citric acid (pH=3.0). Then 10 μl of the BMP-2 solution was pipetted on to each bioactive glass scaffold (4.6 mm in diameter×1.5 mm). The amount of BMP-2 loaded into the bioactive glass scaffolds was equivalent to 1 μg per bone defect (or per scaffold) in the animal model. The BMP-loaded bioactive glass scaffolds were kept for about 24 h in a refrigerator at 4° C. to dry them prior to implantation. For comparison, the as-fabricated scaffolds (no pretreatment in the phosphate solution) were also loaded with BMP-2 using the same procedure.
The release of BMP-2 from the bioactive glass scaffolds into a medium composed of equal volumes of fetal bovine serum (FBS) and phosphate-buffered saline (PBS) plus 1 vol % penicillin was measured as a function of time in vitro. Each bioactive glass scaffold was immersed in 500 μl of the solution in a 2.0 ml microtube and kept at 37° C. in an incubator. Three replicates were used for each group at each time point. At selected times (1 h, 8 h, 1 d, 3 d, 7 d, 14 d), the solution was completely removed and replaced with fresh solution. The amount of BMP-2 released into the solution was measured using by an enzyme-linked immunosorbent assay (ELISA) kit (Pepro Tech, Rocky Hill, N.J., USA) according to the manufacturer's instructions.
All animal experimental procedures were approved by the Animal Care and Use Committee, Missouri University of Science and Technology, in compliance with the NIH Guide for Care and House of Laboratory Animals (1985). Seven groups of bioactive glass scaffolds, described in Table II, were implanted in rat calvarial defects for 6 weeks. The bioactive glass scaffolds were assigned randomly, but bioactive glass scaffolds with and without BMP-2 were not mixed in the same animal. The implantation time was used because our previous studies had shown considerable bone regeneration in bioactive glass scaffolds composed of BMP-loaded hollow HA microspheres after implantation for the same time in rat calvarial defects.
Thirty male Sprague Dawley rats (3 months old; weight=350-400 g, Harlan Laboratories Inc., USA) were maintained in the animal facility for 2 weeks to become acclimated to diet, water and housing. The rats were anesthetized with a combination of ketamine (72 mg/kg) and xylazine (6 mg/kg) and maintained under anesthesia with ether gas in oxygen. The surgical site was shaved, scrubbed with iodine and draped. Using sterile instruments and aseptic technique, a full-thickness defect (4.6 mm in diameter) was created in the central area of each parietal bone using a saline-cooled trephine drill. The dura mater was not disturbed. The bilateral defects were randomly implanted with 5 or 10 bioactive glass scaffolds per group. The periosteum and skin were repositioned and closed using wound clips. All animals were given a dose of ketoprofen (3 mg/kg) intramuscularly and about 200 μl penicillin subcutaneously post-surgery. The animals were monitored daily for condition of the surgical wound, food intake, activity and clinical signs of infection. After 6 weeks, the animals were sacrificed by CO2 inhalation, and the calvarial defect sites with surrounding bone and soft tissue were harvested for subsequent evaluation.
The amount of new bone formed in the as-fabricated 13-93 bioactive glass scaffolds, determined as a fraction of the available pore area, was 32±13% after the six-week implantation. In comparison, the amount of new bone formed in 13-93 bioactive glass scaffolds with an oriented microstructure (porosity=50%; pore diameter=50-100 μm) was 37±8% after implantation for 12 weeks in the same osseous defect model (rat calvarial model) (Table III). Table III shows the comparison of new bone formed in bioactive glass scaffolds composed of silicate 13-93 glass with different microstructures after implantation in rat calvarial defects (4.0-4.6 mm in diameter □ 1.5 mm). The amount of new bone is shown as a percent of the available pore space in the bioactive glass scaffolds.
For 13-93 bioactive glass scaffolds with a trabecular microstructure (similar to dry human trabecular bone) (porosity=80%; pore size=100-500 μm), only 25±12% of the available porosity was infiltrated with new bone after an implantation time of 12 weeks in the same osseous defect model. Bioactive glass scaffolds of 13-93 glass with a fibrous microstructure of thermally bonded short fibers (porosity=50%; pore size=50-500 μm), showed new bone formation equal to 8.5% of the total defect size when implanted in rat calvarial defects for 12 weeks. Since the porosity of the bioactive glass scaffolds was 50%, the amount of new bone estimated as a fraction of the pore area was about 17%. The results indicate that bioactive glass scaffolds with the grid-like microstructure had a microstructure more conductive to supporting bone ingrowth when compared to the oriented, trabecular, and fibrous microstructures. The amount of new bone formed after 6 weeks in vivo was approximately the same or greater than that in the oriented, trabecular, and fibrous microstructures implanted for 12 weeks.
The calvarial samples, including the surgical sites with surrounding bone and tissue, were fixed in 10% buffered formaldehyde for 3 days, then transferred into 70% ethyl alcohol, and cut in half. Half of each sample was for paraffin embedding and the other half for methyl methacrylate embedding. The samples for paraffin embedding were de-siliconized by immersion for 2 h in 10% hydrofluoric acid, decalcified in 14% ethylenediaminetetraacetic acid (EDTA) solution for 4 weeks, dehydrated in a series of graded ethanol, and embedded in paraffin using routine histological techniques. Then the specimens were sectioned to 5 μm and stained with hematoxylin and eosin (H&E). The undecalcified samples were dehydrated in ethanol and embedded in PMMA. Sections were affixed to acrylic slides and ground down to 40 μm using a surface grinder (EXAKT 400CS, Norderstedt, Germany), and stained using the von Kossa technique. Transmitted light images of the stained sections were taken with an Olympus BX 50 microscope connected with a CCD camera (DP70, Olympus, Japan).
Histomorphometric analysis was carried out using optical images of the stained sections and Image J software. The percent new bone formed in the defects was evaluated from the H&E stained sections. The entire defect area was determined as the area between the two defect margins, including the entire bioactive glass scaffold and the tissue within. The available pore area within the bioactive glass scaffold was determined by subtracting the area of the bioactive glass scaffold from the total defect area. The newly formed bone, fibrous tissue, and bone marrow-like tissue within the defect area were then outlined and measured. The area of each tissue was expressed as a percentage of the total defect area as well as a percentage of the available pore area within the bioactive glass scaffold.
Unstained sections of the bioactive glass scaffolds in PMMA were coated with carbon and examined using a field-emission scanning electron microscope (SEM) (S-4700; Hitachi, Tokyo, Japan) operating in the backscattered electron (BSE) mode. The specimens were examined at an accelerating voltage of 15 kV and a working distance of 12 mm.
As fabricated, the bioactive glass scaffolds implanted in the rat calvarial defects had a grid-like microstructure (
After reaction of the bioactive glass scaffolds in the K2HPO4 solution, the smooth dense surface of the glass filaments (
The XRD pattern of the converted layer formed after the one-day reaction also showed a broad bump at about 22° 28 in the vicinity of the major peak for the cristobalite phase of silica. The height of the bump gradually weakened with increasing reaction time. A similar bump has been observed in the XRD pattern of silicate 45S5 and 13-93 bioactive glass, and it has been attributed to the polymerization of silanol groups during the early stage of the conversion process, leading to the formation of a silica gel phase.
The results of this example show that the characteristics of the modified surface layer can have a marked effect on the capacity of the pretreated 13-93 bioactive glass scaffolds to enhance bone formation. H&E and von Kossa stained sections of bioactive glass scaffolds composed of the as-fabricated scaffolds and the bioactive glass scaffolds pretreated in the K2HPO4 solution for 1 day, 3 days, and 6 days which were implanted for 6 weeks in rat calvarial defects are shown in
All bioactive glass scaffolds showed the formation of new bone into the edges (periphery) of the bioactive glass scaffolds (adjacent to the old bone), indicating good integration of the bioactive glass scaffolds with the surrounding bone. New bone formation was observed mainly within the pores of the bioactive glass scaffolds, and the amount of new bone formed was dependent on the pretreatment in the aqueous phosphate solution. Scaffolds composed of the as-fabricated scaffolds showed a limited amount of new bone within the pores of the scaffolds, predominantly in the form of “islands” (FIGS. 8A1-A3). In comparison, the pretreated bioactive glass scaffolds showed a greater capacity to support new bone formation (FIGS. 8B1-D3). In particular, the bioactive glass scaffolds pretreated for 3 days in the K2HPO4 solution showed the greatest capacity to support new bone formation; the bioactive glass scaffold was well integrated with the old bone and the pores of the scaffold was almost completely infiltrated with new bone (FIG. 8C2). Blood vessels were observed within all of the implanted bioactive glass scaffolds in the defects (FIGS. 8A3-D3).
Infiltration of new bone into the grid-like bioactive glass scaffolds was also different in nature when compared to bioactive glass scaffolds of silicate 13-93 and borate-based bioactive glasses with the oriented and fibrous microstructures implanted in the same animal model. New bone infiltrated the grid-like bioactive glass scaffolds mainly from the edge (adjacent to old bone), indicating that new bone formation was mainly osteoconductive in nature, but some “islands” of new bone were also observed within the interior pores of the bioactive glass scaffold (FIG. 8A2). In comparison, while bone formation in the oriented and fibrous bioactive glass scaffolds was mainly osteoconductive, new bone formed mainly on the dural side of the bioactive glass scaffold with little infiltration into the edge for implantation times of up to 12 weeks. In the case of 13-93 bioactive glass scaffolds with the trabecular microstructure, new bone formation was found predominantly at the periphery of the defect. Differences in the nature of the new bone infiltration (from the edge vs. along the dural side of the bioactive glass scaffold) appear to be dependent on the size and interconnectivity of the pores. Larger pores with better pore interconnectivity appear to support greater bone infiltration from the edge of the bioactive glass scaffold.
Since all the bioactive glass scaffolds had the same overall microstructure, the capacity of the bioactive glass scaffolds to regenerate bone was compared on the basis of new bone formed as a percent of the pore area of the bioactive glass scaffolds (
The amount of new bone formed in bioactive glass scaffolds composed of the as-fabricated scaffolds after the six-week implantation was 32±13%. In comparison, the percent new bone formed in the bioactive glass scaffolds pretreated in K2HPO4 solution for 1 day, 3 days, and 6 days was 46±10%, 57±14%, and 45±11%, respectively. The bioactive glass scaffold pretreated for 3 days had a significantly higher percent of new bone growth than the as-fabricated scaffold (p<0.05). The amount of new bone formed in the bioactive glass scaffolds pretreated for 1 day, 3 days, and 6 days and loaded with BMP-2 was 65±7%, 61±8%, and 64±11%, respectively; these values were significantly higher than the percent new bone formed in the as-fabricated scaffolds and the bioactive glass scaffolds pretreated for 1 and 6 days.
The in vivo results show that when loaded with BMP-2 (1 μg/defect), all three bioactive glass scaffolds, pretreated for 1 day, 3 days, and 6 days in K2HPO4 solution, significantly enhanced bone regeneration when compared to the as-fabricated scaffold. The capacity of the BMP-loaded bioactive glass scaffolds to enhance bone regeneration after the six-week implantation was independent of the pretreatment time (
When compared to the bioactive glass scaffolds subjected to the pretreatment alone (no BMP-2), the amount of new bone formed in the BMP-loaded bioactive glass scaffolds (61-65%) was not significantly greater than the value (57%) for the bioactive glass scaffolds pretreated for 3 days. In comparison, for pretreatment times of 1 day and 6 days, the BMP-loaded bioactive glass scaffolds showed a significantly greater amount of new bone formation when compared to the pretreated bioactive glass scaffolds (no BMP-2). Presumably because the three-day pretreatment alone was very effective, there was little opportunity for the beneficial effect of the BMP-2 to be felt.
While the capacity of the BMP-loaded bioactive glass scaffolds to regenerate bone and integrate with old bone was similar to that of the bioactive glass scaffolds pretreated for 3 days (no BMP-2), there were differences in the quality of the new bone. Apart from the new bone that infiltrated the pores of the bioactive glass scaffolds, the remaining pore space in the pretreated bioactive glass scaffolds was filled with soft tissue whereas, in the BMP-loaded bioactive glass scaffolds, the remaining pore space was filled with bone marrow-like tissue (
The glass filaments of the bioactive glass scaffolds consisted of three regions after implantation: an unconverted glass core (light-gray), a silica-rich layer (dark-gray) on the unconverted glass, and an HA-like modified surface layer (light-gray). (The cracks in the bioactive glass scaffolds and delamination of the HA layer presumably resulted from capillary drying stresses during the sample preparation for SEM examination.)
The bioactive glass scaffolds are composed of the as-fabricated scaffolds (
New bone formed during the six-week implantation did not appear to bond to the as-fabricated scaffolds (
For the bioactive glass scaffolds pretreated for 3 days in the K2HPO4 solution prior to implantation (without or with BMP-2) (
While the invention has been described in connection with specific embodiments thereof, it will be understood that the inventive device is capable of further modifications. This patent application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein before set forth.
This application is a non-provisional that claims benefit to U.S. Provisional Patent Application No. 61/849,512, filed on Jan. 28, 2013, which is herein incorporated by reference in its entirety.
This invention was made with Government support under Grant No. R15AR056119-01 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61849512 | Jan 2013 | US |
