Vitreous composition, bioactive vitreous fibers and fabrics, and articles

Abstract

A vitreous composition according to Table (I) is described. Continuous vitreous fibers are obtained by downdrawing said molten composition, with a length ranging from millimeters to kilometers and diameters ranging from 2 μm to 3 mm. The fibers are covered with collagen and form vitreous fabrics. The fabrics form articles with a variety of medical uses.

Claims

1. A vitreous composition comprising the elements in wt % ranges according to Table I: TABLE-US-00004 TABLE I Element Quantity in wt % SiO.sub.2 43-52 Na.sub.2O   4-9.5 K.sub.2O 20.5-32   MgO 0.5-2.5 CaO 15-20 Au 0.1-3.5 Ag 0.1-3.5 B.sub.2O.sub.3 1.5-4   P.sub.2O.sub.5 1-6 ZnO 0.1-3.5 SrO  0.1-3.5.

2. The vitreous composition according to claim 1, wherein when the composition in a bulk form is subject to a simulated body fluid (SBF) in vitro a hydroxyapatite (HCA) layer is formed within 12 hours.

3. The vitreous composition according to claim 1, wherein the composition comprises a distribution powder with particle sizes between 1-10 μm.

4. The vitreous composition according to claim 1, wherein the composition comprises a distribution powder with particle sizes between 1-25 μm.

5. The vitreous composition according to claim 1, wherein the composition comprises antifungal and antimicrobial properties.

6. The vitreous composition according to claim 1, wherein the composition comprises a distribution particulate with particle sizes between 60-700 μm.

7. The vitreous composition according to claim 1, wherein the composition comprises a distribution particulate with particle sizes between 100 μm-1.5 mm.

8. A vitreous fiber prepared from the composition according to claim 1 prepared by a process comprising: heating the composition of claim 1 to between 1000-1250° C; decreasing the temperature to between 700-950° C; maintaining the temperature at least 20° C. higher than the liquidus temperature of the composition; obtaining a vitreous mass with viscosity from between 10.sup.4.0 to 10.sup.2.5 Poise; and pulling the vitreous mass while simultaneously applying a collagen I coating.

9. The vitreous fiber according to claim 8, wherein the collagen I coating has a thickness of at least 250 nanometers.

10. The vitreous fiber according to claim 8, wherein the fiber has length from between at least 1 millimeter to at least 1 kilometer.

11. The vitreous fiber according to claim 8, wherein the fiber has a diameter from between 2 μm to 3 mm.

12. The vitreous fiber according to claim 8, wherein heating the composition of claim 1 to between 1000-1250° C. comprises heating the composition of claim 1 to between 1000-1250° C. in downdrawing machine furnace.

13. The vitreous fiber according to claim 8 prepared by a process further comprising collecting the vitreous fiber through a controlled rotating drum.

14. A vitreous fabric and mesh prepared from the vitreous fiber according to claim 8 comprising a porosity of between 5 and 90%.

15. The vitreous fabric according to claim 14, wherein the fabric has a thickness of at least 2 μm (0.002 mm).

16. The vitreous fabric according to claim 15, wherein the fabric has a thickness of between 0.05 mm and 1 mm.

17. An article prepared from the vitreous composition according to claim 1 prepared by a process comprising: providing a particulate form of the vitreous composition; and optionally subjecting the particulate to a sintering processes.

18. The article according to claim 17 prepared by a process further comprising processing the particulate in a 3D printer.

19. The article according to claim 17, wherein the article is selected from the group consisting of 3D pieces and scaffolds.

20. The article according to claim 17, wherein the article comprises a distribution particulate with particle sizes between 60-700 μm.

21. The article according to claim 17, wherein the article comprises a distribution particulate with particle sizes between 100 μm-1.5 mm.

22. The article according to claim 17, wherein the article comprises a tangle of fibers either in a polymer or non-polymer matrix.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 attached is a picture that shows a) Titanium Mesh suitable for filling of orbital bone defect or dental application; and b) Titanium Mesh used in dental surgical procedure.

(2) FIG. 2 attached is a chart obtained through a DSC testing with monolithic samples (average 20 mg) for formulation of 45S5 Bioglass (curve 1, control), and BAGF (curve 2, invention).

(3) FIG. 3 attached is a chart obtained through FITR with the infrared spectrums of the glass bioactivity tests of BAGF formulation on 4 to 16 hour periods (FIG. 3A) and 24 to 168 hour periods (FIG. 3B).

(4) FIG. 4 attached is a photographic chart of a bioactive glass fiber fabric obtained through downdrawing of the composition of the invention.

(5) FIG. 5 attached is an X-ray diffractogram of the powdered sample of BAGF formulation using the thermal treatment for crystal growth at 700° C. for 120 minutes.

(6) FIG. 6 attached are images obtained through the Heating Microscopy test for monolithic samples of the BAGF composition. Temperatures are: Sintering—708° C.; Softening—712° C.; Sphere—722° C.; Half-Sphere—820° C. and Meltdown—859° C.

(7) FIG. 7 attached are images obtained through the Heating Microscopy test for powdered samples of the BAGF and 45S5 Bioglass composition. Powder with particle size range between 25-75μ. a) BAGF with temperatures are: Sintering—575° C.; Softening—576° C.; Sphere—709° C.; Half-Sphere—802° C. and Meltdown—838° C. b) Bioglass 45S5 which does not feature these phases, since the powder cannot be sinterized due to crystallization.

(8) FIG. 8 attached is a viscosity chart expressed in Poise regarding the temperature for sampling of BAGF, 13-93 and 45S5.

(9) FIG. 9 attached is a bar chart showing a cell viability assay (MTT) of UMR 106 osteoblasts within periods of 1, 3 and 5 days (data with no statistic difference).

(10) FIG. 10 attached shows the Micrographs corresponding to the cell viability assay (MTT) of UMR 106 osteoblasts within periods of 1, 3 and 5 days.

(11) FIG. 11 attached is a photograph of the test with a Staphylococcus aureus strain, image related to the first dilution. FIG. 11A: Control (window glass only) and FIG. 11B: Bioactive fabric of the invention.

(12) FIG. 12 attached shows a bar chart of the cell proliferation of fibroblasts in different dilutions of the extract obtained through submersion of the bioactive tissue in a DMEM medium (100%, 50%, 25% and 12.5%), with (#)p<0.05 versus CG, (≠)p<0.05 versus CG, and (*)p<0.05 versus 50%, 25% and 2.5%.

(13) FIG. 13 attached shows a bar chart of osteoblast cell proliferation in different dilutions of the extract obtained through submersion of the bioactive fabric in a DMEM medium (100%, 50%, 25% and 12.5%), with (#)p<0.05 versus CG, (*)p<0.05 versus 12.5%, (a)p<0.05 versus CG, (≠)p<0.05 versus 25% and 12.5%, (b)p<0.05 versus CG, (†)p<0.05 versus 50%, 25% and 12.5%, and (**)p<0.05 versus 25% and 12.5%.

(14) FIG. 14 attached shows histological sections of the subcutaneous implant colored with Hematoxylin-Eosin for experimental periods of 15 (A-B), 30 (C-D) and 60 days (E-F). Scale bars represent 1 mm for a 10× magnification. Inflammatory Cells (IN), Granulation Tissue (G), Bioactive Glass Fibers (S), Fibrous Capsule (F) and Multinuclear Giant Cells. Scale bars represent 100 μm for a 100× magnification.

(15) FIG. 15 attached shows the histological analysis of a tibial lesion in periods of 15 (A), 30 (B) and 60 days (C). Scale bars represent 1 mm for a 10× magnification. Material: prepared scaffold from fibers obtained through downdrawing. The fibers were implanted on 3 mm defects of rat tibias.

(16) FIG. 16 attached shows histological sections of the tibial lesion colored with Hematoxylin-Eosin for groups GC (Control Group) and BG (group treated with the biomaterial of the invention) for the experimental periods of 15 (A-B), 30 (C-D), and 60 days (E-F). Inflammatory Cells (IN), Granulation Tissue (G), Bioactive Glass Fibers (S), Fibrous Capsule (F) and Multinuclear Giant Cells, Bone Neoformation (B) and Rim of the Lesion (D). Scale bars represent 100 μm for a 100× magnification.

(17) FIG. 17 attached shows an image obtained during the surgical procedure for implanting the material of the invention on a 6 mm defect on the cranial bone of Wistar rats.

(18) FIG. 18 attached shows the results obtained through histological sections colored with Hematoxylin-Eosin for control groups (a) and BAGF treated (b) for 2, 4, 8 and 16 weeks.

(19) FIG. 19 attached illustrates the analysis for verification of elimination of biofilm on the 4.sup.th and 5.sup.th dilutions, after 24 h. A) Window glass; B) Bioglass 45S5 and C) bioactive composition of the invention (BAGF).

(20) FIG. 20 attached is a chart that illustrates the analysis for verification of elimination of biofilm after 72 h. Results for window glass and for the bioactive composition of the invention (BAGF).

DETAILED DESCRIPTION OF THE INVENTION

(21) From a highly bioactive vitreous composition, continuous bioactive fibers are obtained with diameters controlled by the downdrawing process, as well as manufacturing of fibers in a highly bioactive, non-woven fabric, with oriented fibers and featuring certain porosity.

(22) The bioactive vitreous composition object of this application allows for obtaining not only fibers, but also distinct forms of presentation, such as 3D structures, scaffolds (highly porous hard structures), meshes, fabrics and similar.

(23) An aspect of the invention is the vitreous composition according to Table 1 below.

(24) TABLE-US-00003 TABLE 1 Element Quantity in mol % Quantity in wt % SiO.sub.2 46-52 43-52 Na.sub.2O  5-10   4-9.5 K.sub.2O 15-32 20.5-32   MgO 0.5-2.5 0.5-2.5 CaO 15-20 15-20 Au 0.1-3.5 0.1-3.5 Ag 0.1-3.5 0.1-3.5 B.sub.2O.sub.3 1.5-4   1.5-4   P.sub.2O.sub.5 1.5-3   1-6 ZnO 0.1-3.5 0.1-3.5 SrO 0.1-3.5 0.1-3.5

(25) Another aspect of the invention comprises the fibers obtained from said composition by downdrawing.

(26) Yet another aspect are the articles obtained from fibers, including fibrous fabrics and scaffolds, and articles obtained from particulates, such as 3D structures obtained through 3D printing, various scaffolds and articles for medical and dental applications.

(27) The use of vitreous formulations, which combine properties such as: rapid interaction with body fluids, that is, high bioactivity, and higher glass stability, with a broad working range, enables obtaining fibers through the downdrawing process.

(28) The proposed composition of this invention confers increased reactivity to the glass and low chemical durability, as expected for a highly bioactive glass. Its greater higher glass stability allows the glass to be manipulated and to withstand thermal and/or sintering treatments, without presenting an uncontrolled crystallization, thus enabling simpler processes, that requires low crystallization tendency, to be used.

(29) In order to maintain the vitreous fabric with its fibers oriented, a bioresorbable coating is applied. This coating may be composed of various bioresorbable polymers, but also, preferably, of a type I collagen thin layer that covers the surface of the fibers, providing support to the vitreous fabric and also protecting it against contact and reaction with air moisture.

(30) Type I collagen is the most abundant protein in the human body, present in connective tissue, skin, tendons, bones, fibrous cartilage, etc. Besides, it is a widely known compound for initial assistance in tissue regeneration processes.

(31) The formulation of the invention demonstrates outstanding results regarding stability, when tested via the Differential Scanning calorimetry technique (DSC) and does not show evident crystallization peaks when compared to the 45S5 formulation, as demonstrated in FIG. 2.

(32) It also features outstanding bioactive properties when tested and analyzed in vitro using the SBF-K9 solution, forming the hydroxycarbonate apatite (HCA) layer in only 12 hours. FIGS. 3A and 3B show the curves obtained by the FTIR testing (Fourier Transform Infrared Spectroscopy), evidencing the formation of HCA after only 12 hours of testing in a solution for BAGF bioglass discs. The test was conducted from 4 to 168 hours of exposure of the BAGF glass to the solution.

(33) From this composition, it was also possible to obtain fibers through downdrawing and also a vitreous fabric, as illustrated in FIG. 4.

(34) The fiber obtained through the method of the invention is continuous and not fragmented, with a length ranging from millimeters to kilometers, obtained in a single step, thus, eliminating the need for additional techniques to obtain the fabric (such as spraying, as described in the aforementioned patent), since it is possible, to obtain non-woven fabrics with random or oriented fibers and controlled porosity with the downdrawing machine itself.

(35) The articles manufactured from the fibers obtained through downdrawing may have the porosity ranging from 5 to 90%, depending on the arrangement of the fibers and whether bioresorbable polymers are used or not.

(36) Furthermore, it is possible to previously determine the diameter of the fibers and choose if only a single fixed diameter is used for the entire fabric or different diameters, alternatively.

(37) Via the downdrawing process, it is possible to obtain fibers with various diameters depending on rotation speed of the collecting drum. The diameters obtained range from 2 μm to 3 mm.

(38) The downdrawing technique is consolidated and known for enabling the creation of glass fibers in industrial scale in a simple and inexpensive manner. In this technique, glasses in high temperature and adjusted viscosity may pass through orifices located at the bottom of crucibles or platinum devices (nozzles), thus forming filaments, which are quickly cooled, yielding the shape of fibers.

(39) With the help of an X-ray Diffraction technique, information is collected about the crystals eventually formed when the machine is used, the average diameter of fibers and also definition of processing parameters such as heating rate, glass viscosity and drawing speed were defined throughout tests performed with the composition informed in Table 1, suitable for the fiber obtainment.

(40) FIG. 5 shows the X-ray diffraction test, with a crystallized sample of the bioactive glass of the invention. A BAGF glass sample from the downdrawing machine (after 7 hours of processing) was treated in muffle at 700° C. for 120 minutes for growth and identification of crystals, which eventually are formed during the process. However, it should be remembered that the process is done in order for such event to be avoided, since it would affect obtaining the fibers via the downdrawing process. In spite of the great amount of amorphous material, the crystal KCaPO.sub.4 was detected.

(41) Another test performed, which evidences the vitreous stability of the composition of the invention, is the heating microscopy technique (FIGS. 6 and 7), in which it was possible to observe that through the entire heating/cooling cycle, there was no crystal formation and the material was kept vitreous, which did not occur with the Bioglass 45S5, since it did not sintered due to the crystallization process.

(42) FIG. 8 is a chart of the viscosity curve for the bioactive glass of the invention and for 13-93 and 45S5 bioactive glasses. In this chart, it may be once again observed that obtaining viscosity information for the Bioglass 45S5 is not possible on temperatures lower than 1200° C. due to the crystallization process that quickly takes place. Glass 13-93, on the other hand, was found to be more viscous than BAGF and this is due to the higher amount of silica in its formulation, but obtaining points from 1100° C. was also proven impossible due to the beginning of the crystallization process. Through this chart, it is possible to determine the working range temperature for the BAGF glass of the invention.

(43) Various methods of application of a bioresorbable polymeric coating as well as different polymers were researched and proven to be applicable. The type I collagen is one of the biopolymers that demonstrated to have the necessary properties for coating this vitreous fiber fabric and it also takes part in various reactions of tissue regeneration and healing.

(44) In order to obtain bioactive fibers by the downdrawing process, a certain amount of bioactive glass, in a monolithic form that results from the melting process of the mixture of raw materials in an furnace with 1000-1350° C. temperature, is heated within the downdrawing machine furnace until its melts completely, in a temperature ranging from 1000-1250° C.

(45) Subsequently, the temperature of the furnace is decreased to 700-950° C., for adjustment of the viscosity of the cast material, obtaining a vitreous mass with viscosity between 10.sup.4.0 and 10.sup.2.5 Poise. This viscosity range is considered ideal for obtaining fibers in industrial scale, while keeping the furnace temperature, at least, 20 Celsius degrees above the liquidus temperature.

(46) During the drawing process, the selected coating may be applied simultaneously, with the help of an applier device. Thus, fibers are coated with the polymer and, subsequently, collected by a controlled rotation drum, enabling a precise control of their obtained diameters.

(47) The thickness of the coating may also be controlled, with a scale ranging from nanometers to millimeters (thicknesses equal or higher than 250 nanometers more precisely).

(48) The vitreous fabric is obtained simultaneously to the collection of bioactive glass fibers. The fabric is manufactured in a device coupled to the downdrawing machine.

(49) The thickness of the vitreous fabric, as well as the interlacing between fibers may be controlled with the help of devices present in the machine and through variation of the colleting times of fiber.

(50) Vitreous fabrics can be obtained with a single fiber layer, therefore from 2 μm, until multiple layers, reaching up to centimeters in thickness. The optimal working range for vitreous fabrics with extremely high flexibility and rapid degradation rate is from 0.05 mm to 1 mm thickness.

(51) In summary, the thickness of the vitreous fabric, fiber diameter, thickness of the coating may vary and be adjusted depending on the desired application for the final product. These, among other characteristics of the product, shall grant control of the degradation rate both of the polymer and the bioactive glass for in vitro and in vivo procedures.

(52) These vitreous fabrics were tested in vitro using osteoblasts. Cell viability assays were performed and the developed glass presented very favorable and similar results to the standard market Bioglass45S5. That is, the bioactive glass of the invention enables adhesion and viability of bone tissue cells as well as the golden standard Bioglass 45S5.

(53) Results may be observed on FIGS. 9 and 10.

Antibacterial Properties

(54) Essays for verification of antibacterial capacity of the new vitreous composition and fabrics were conducted following the guidelines of JIS 2801:2010 and ISO 22196:2011 standards. The tests used E. coli and Staphylococcus aureus strains.

(55) The material was capable of interacting and eliminating viable colony forming units in both strains, in all dilutions and, therefore, it has antibacterial (bactericide) properties as demonstrated by FIG. 11. FIG. 11A is the control sample (window glass), with countless colony forming units (CFU) and FIG. 11B is the vitreous composition sample of the invention (BAGF) with no CFU.

Cytotoxicity Assays

(56) Cytotoxicity assays (MTT) were performed with L929 fibroblasts, as well as OSTEO-1 osteoblasts with experimental times of 24, 72 and 144 hours for dilutions obtained from the submerged material in DMEM (Dulbeco's Modified Eagle Medium) culture medium, being 100% the solution directly extracted from the submerged material and percentages presented, dilutions arising from this solution. The results are shown in FIG. 12 and FIG. 13.

(57) The tests shown in FIGS. 12 and 13 present the higher level of cell proliferation either for fibroblasts or osteoblasts, particularly in the 100% group. It indicates cell metabolism acceleration with respect to the control group and evidences the non-cytotoxicity of the new bioactive glass material.

In Vivo Results

(58) In vivo tests were performed in order to assess the genotoxicity, cytotoxicity and verification of the aid towards the tissue regenerative capacity of the invention biomaterial.

Genotoxicity Assay

(59) Comet Assay II tests were performed for fibroblasts and osteoblasts from samples implemented subcutaneously in the dorsal of Wistar rats, in the periods of 15, 30 and 60 days.

(60) From this test and histological analysis, it was possible to verify the absence of any damage to the cells genetic code and the material provided the growth of an organized tissue without the presence of a fibrous capsule. The material showed to be reabsorbable, as appears in FIG. 14.

(61) In bone regeneration applications, the material presented a similar reabsorbing rate to the new bone formation in 3 mm diameter tibial defects in 60 Wistar rats. The experimental times chosen were 15, 30 and 60 days. Results also indicate that the material stimulate new bone formation as presented in FIGS. 15 and 16.

(62) Essays identifying the BAGF capacity of bone regeneration in rat calvarial defects were also carried out. Bone defects with 6 mm diameter were made on the cranial bone of 48 animals (Wistar rats). FIG. 17 shows the surgical procedure with the implementation of the bioactive glass fibers obtained by downdrawing.

(63) FIG. 18 shows the results obtained from this study. Animals treated with the invention biomaterial had the formation of a more organized hard tissue, with quite accelerated growth when compared to the control group (without the invention material) and the material's degradation rate accompanying the tissue regeneration process may be once again verified, indicating that the material degrades at an approximate rate as the tissue regeneration.

(64) In addition to FIG. 11 concerning the antimicrobial activity of the invention bioactive material, FIGS. 19 and 20 as follows also show that the vitreous bioactive composition of the invention, as well as the products from its dissolution (ions dissolved in the medium) show antibacterial and antifungal properties.

(65) The bioactive materials of the invention having antimicrobial properties are useful at the cosmetics-pharmaceutical, agricultural or food areas, civil construction industry, paper, textile and environment areas, without limitation thereto.

(66) At first, in order to reach such properties, the material may be used under different presentation formats, such as powder, granules or porous or non-porous 3D pieces.

(67) The elimination of biofilm capability, a feature that does not usually occur in materials, such as glass and other, is fully achieved with the invention material, as shown in experiments from FIG. 9. FIGS. 19A and 19B show control materials, in 4.sup.th and 5.sup.th dilutions, which formed biofilm when tested under the conditions provided at the test. The test may be inquired in Brazilian Dental Journal (2014) 25, available at http://dx.doi.Org/10.1590/0 03-6440201302398.

(68) However, the glass prepared from the invention material, FIG. 19C, does not show any formation of biofilm, since it has antibacterial/antifungal properties.

(69) FIG. 20 also shows the biofilm elimination capacity after 72 hours, very higher than that from any control material under the same testing conditions.

(70) Therefore, the fabric developed enables a great advance in the tissue engineering field, since this biomaterial may be applied as a graft in order to aid healing and in situ regeneration processes for different human body tissues, as well as in in vitro-tissue engineering processes.

(71) Its application and manipulation also show an advantage with respect to other alloplastic materials, since it has flexibility and can adjust itself to different fracture contours, bone cavity or injury from different tissues.

(72) Other clear advantage is that in grafting procedures this fibers or fabrics can decrease surgical steps, since this material is reabsorbable and, therefore, it does not require any further removal, diminishing patient's discomfort, contamination risk and the total procedure cost.

(73) The vitreous fabrics developed are classified as third generation biomaterials and show a broad applicability. They can be applied in hard tissue regeneration, skin wound healing and in soft tissue regeneration in medical and dental procedures.

(74) In other presentation forms, this vitreous composition may also be used as scaffolds and granules for bone regeneration and as powder of different granulometric ranges may be used for remineralization of dental tissues after chemical erosion or teeth whitening.

(75) In the following, several methods of usage for this new biomaterial are presented. As powder with granulometric range from 1-10 μm for tooth enamel remineralization, after chemical or mechanical erosive procedures and teeth whitening as well. As powder with granulometric range from 1-25 μm for obliteration of dentinal tubules for dentin hypersensitivity treatments. As particulates with granulometric range from 60-700 μm for bone regeneration on dental grafting procedures for periodontal disease recovery. As particulates with granulometric range from 100 μm-1.5 mm for grafting procedures, on dental implant pre-procedure, for grafting on oral-maxillofacial procedures and treatment of grafting under traumatic conditions. As vitreous fabric on orthopedic procedures, traumatic post-conditions, such as bone fractures. As vitreous fabric on medical and dental procedures requiring mesh, tissues or membranes for rehabilitation or containment of other alloplastic, xenogenous or autogenous material. As vitreous fabric or mesh operating as a membrane for hard and/or soft tissue regeneration guide. As vitreous fabric to replace the titanium mesh, since it requires to be removed after the osseous consolidation, as this tissue is reabsorbable, not requiring the second surgical step. As vitreous fabric in using wound pads, which may be applied for skin wound regeneration, either in perfectly healthy patients or patients having poor skin regeneration due to any illness, such as type I or II diabetes, osteoporosis, etc. As vitreous fabric for skin burn regeneration and protection. As vitreous fabric for the regeneration of chondrocytes, that is, cartilaginous tissue. As scaffolds manufactured from a tangle of fibers either dipped or not in a highly porous polymer matrix or also synthesized from powder, for bone regeneration in procedures that do not require any load support, that is, do not require high mechanical strength of the material. As fabric in first-aid or surgical procedures requiring grafts and/or plasters with antimicrobial properties.