AU2003266761A1

AU2003266761A1 - Biomimetic ceramic fibre

Info

Publication number: AU2003266761A1
Application number: AU2003266761A
Authority: AU
Inventors: Given Not
Original assignee: Kolos Elizabeth
Current assignee: Kolos Elizabeth
Priority date: 2003-10-10
Filing date: 2003-12-05
Publication date: 2005-05-12

Description

05/12 '03 FRI 21:37 FAX 61299255911 GRIFFITH HACK 444 IP AUSTRALIA 0004

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant: ELIZABETH KOLOS Invention Title: BIOMIMETIC CERAMIC FIBRE The following statement is a full description of this invention, including the best method of performing it known to me: 05/12 2003 FRI 21:37 [TX/RX NO 5034] ]004 05/12 '03 FRI 21:37 FAX 61299255911 GRIFFITH HACK 444 IP AUSTRALIA 2005 Biomimetic Ceramic Fibre Field of the Invention The present invention relates to the production of ceramic fibre. The invention will be hereinafter described with reference to the production of fibre, however, it is to be appreciated that the invention is not limited to this type of application.

Background of the Invention Synthetic hydroxyapatite (HA, Caio(PO 4 )s(OH)2) is generally accepted as an important biocompatible material. Owing to the inferior mechanical properties of hydroxyapatite, significant research activity has been associated with the development of hydroxyapatite coatings, composites and fibres. Fibrous materials are known to exhibit improved strength due to the interlocking of the fibres, crack deflection, and/or pullout.

Calcium phosphate contains only non-toxic species, such as Ca, P, (H20, OH-, CO 3 2 etc.). In contrast with fibrous materials such as glass, carbon, SiC, SisN 4 A1 2 0 3 ZrO 2 3 bioactive calcium phosphates exhibit excellent biocompatibility due to their chemical and crystallographic similarities with the mineral constituents of bones and teeth. Therefore, they have opportunity to be employed in biomedical applications, as it is believed that the essential requirement for artificial material to bond to living bone is the formation of a layer of biologically active apatite on the bone surface. This layer is carbonate-containing hydroxyapatite similar to bone apatite.

Some examples of applications of HA fibres include cotton-like cloth as a bone defect filler to encourage 1 05/12 2003 FRI 21:37 [TX/RX NO 5034] I005 05/12 '03 FRI 21:37 FAX 61299255911 GRIFFITH HACK 4-4 IP AUSTRALIA L006 bone growth in to the defect, or woven into current metal implants surface, yielding strong, three-dimensional interface for bone in-growth. They could also be employed to reinforce a ceramic or polymer matrix, resulting in a composite material. Fibrous calcium phosphate can also be used as thermal insulating agents and packing media for column chromatography. The main factors to be considered when addressing carcinogenicity are believed to be morphology and the chemical composition of the materials. Thus there is a need for preparation of safe fibrous materials that replace existing biohazardous whiskers and fibres in many applications.

There are various approaches to producing HA fibres in the prior art, including melt spinning and extrusion techniques, pyrolysis, homogeneous precipitation, electrophoretic deposition for coating.

Melt spinning and extrusion have been popular methods as fibrous apatite can be produced in a continuous fibre.

Mori et al. Mori, S. Fujii, M. Yoshizawa, K.

Miyasaka, J. Tabuchi, K. Egawa, M. Hirano, Y. Yoshida, US 4904257, February 27, 1990, Tao Nenryo Kogyo Kabushiki Kaisha, Tokyo, Japan] disclosed a method where a kneaded mixture of binder, hydroxyapatite particles and water is fed into spinning apparatus at room temperature. Sinterextrusion process is a method where microcrystalline hydroxyapatite powder is forged through an orifice at sintering temperature of 1200 0 C to create the filament.

Although these methods produce continuous fibres that enhance further processing for application, operating temperature of the sinter-extrusion method and porous nature of the calcined HA fibre from melt spinning 2 05/12 2003 FRI 21:37 [TX/RX NO 5034] Z 006 05/12 '03 FRI 21:37 FAX 61299255911 GRIFFITH HACK IP AUSTRALIA 2007 provide limitations for melt spinning and extrusion methods.

In the prior art, fibrous HA was prepared using a reaction of ion exchange of sodium alginate (Na-Alg.).

Essentially this pyrolysis method spins starting materials, Na 4

P

2

Q

7 Na-Alg, with mixed solution spinning solution, Ca(CH 3

COQ)

2 and CaCI 2 with pH of 6.0 to Gelatinous fibres were aged, dried and calcined. To remove pores appearing during the heating cycle, the dried fibres formed were heat-treated under reduced pressure. The resulting fibrous HA was polycrystalline material of about 30-40 gm in diameter.

Fibrous and whisker-like material can be obtained by homogeneous precipitation from Ca 2 and P0 4 3 solutions through an aqueous system. HA whiskers are obtained when the pH variation, temperature and reaction time are well controlled. Previously, a HA liquid reaction where HA was precipitated onto the surface of the pulp fibres using the same aqueous system for homogeneous precipitation Kawakatsu, Y. Okuda, S. Shirahata, Cytotechnology, 35 65-72 (2001)]. As the core of commercial softwood bleached is kraft pulp, sheets of hydroxyapatite pulp composite fibre can be produced using a paper-making sheet machine.

A biocompatibility testing technique developed by T.

Kokubo et al. Kokubo, S. Ito, Z.T. Huang. T. Hayashi, S. Sakka, T. Kitsugi and T. Yamamuro, J. Biomed. Mater.

Res. 24 331-343 (1990)] tests bioceramics for chemical stability in the physiological environment by placing the bioceramic in a simulated physiological environment.

The simulated physiological environment produced in the testing method attempts to mimic a process by which the biologically active, bone-like apatite layer is formed on 3- 05/12 2003 FRI 21:37 [TX/RX NO 5034] 1a007 05/12 '03 FRI 21:38 FAX 61299255911 GRIFFITH HACK IP AUSTRALIA z 008 the surfaces of the bioactive ceramic in the living body to determine their biocompatibility.

Summary of the Invention A first aspect of the invention relates to a method for producing ceramic fibre comprising the steps of: a. forming a ceramic coating on a cellulose fibre in a salt solution containing ions of Ca 2 and

PO

3 and b. heating the ceramic coating and cellulose fibre to volatilise the cellulose fibre.

The salt solution mimics the osteogenesis mechanism of the physiological environment and coats the cellulose fibre. The step of heating to volatilise the cellulose fibre enables the production of the bioactive fibre. In uses of the fibre in biological applications, the bioactive fibre allows bone on-growth and resorption of the fibre in the body.

The biocompatibility of the ceramic coating derives from the coating technique that utilises the salt solution in the deposition of the ceramic coating.

Preferably, the ion concentration of the salt solution is controlled as to form predominantly calcium phosphates in the ceramic coating.

These calcium phosphates formed in the coating and/or subsequent fibre are/is of a biocompatible nature. The formation of the ceramic phases can be controlled to produce fibres with a similar chemical composition to that of natural bone.

Biodegradability within the body is due to both the porosity of the fibre and the phases present in the 4 05/12 2003 FRI 21:37 [TX/RX NO 5034] [a008 05/12 '03 FRI 21:38 FAX 61299255911 GRIFFITH HACK -44 IP AUSTRALIA 1009 fibre. Increasing porosity facilitates the rate of bioresorption.

Preferably, phosphorylation of the cellulose fibre is performed prior to forming the ceramic coating.

Phosphorylation can be performed on various cellulose materials by treatments with urea/phosphorous acid, urea/phosphoric acid.

Preferably, the cellulose fibre is treated in a calcium containing solution prior to forming the ceramic coating.

The formation of amorphous calcium phosphate materials by treatment in the calcium containing solution stimulates the growth of calcium phosphates on the fibres during subsequent steps. The calcium containing solution for example can be Ca(OH) 2 However, alternatives to the calcium containing solution include solutions of calcium chloride.

More preferably, the calcium containing solution treatment is performed after the phosphorylation treatment.

Preferably, heating of the ceramic coating is performed at temperatures sufficient to achieve at least partial sintering of the ceramic coating.

Preferably, the salt solution includes Ca 2 P0 3 Na K Mg 2 So42-, C0 3 2- and Cl' ions. These ions are ions present in body plasma.

More preferably, the ratio of the ions is similar to the ratio of ions in body plasma.

More preferably, the concentration of the one or more of the ions is in the range of 1 to 5 times the concentration of ions in body plasma.

5 05/12 2003 FRI 21:37 [TX/RX NO 5034] I009 Y~ 05/12 '03 FRI 21:38 FAX 61299255911 GRIFFITH HACK IP AUSTRALIA J0O10 Preferably, sintering is performed at a temperature between 800-1250°C.

Preferably, sintering below a temperature of 950°C produces predominately tubular ceramic fibre.

Preferably, sintering above a temperature of 1150°C produces predominately tape-like ceramic fibre- Preferably, the cellulose fibre is cotton.

A second aspect of the invention relates to a ceramic fibre wherein the ceramic fibre is tape-like or tube-like in morphology.

Preferably, the ceramic fibre predominantly comprises a combination of one or more of the phases of calcium phosphate, hydroxyapatite and whitlockite.

Preferably, the surface morphology of a proportion of the ceramic fibre has a cauliflower-like appearance.

Preferably, the surface morphology of a proportion of the ceramic fibre has a dendrite-like appearance.

6 05/12 2003 FRI 21:37 [TX/RX NO 5034] I010 05/12 '03 FRI 21:38 FAX 61299255911 GRIFFITH HACK IP AUSTRALIA loi0 Brief Description of the Drawings Notwithstanding any other forms which may fall within the scope of the invention, preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings and/or the accompanying examples, in which: Figure 1 shows a flow sheet for a method for the producing predominately calcium phosphate fibres; Figure 2 shows a scanning electron microscope image of a calcium phosphate fibre prepared in 1.5 times concentration of SBF; Figure 3 shows a scanning electron microscope calcium phosphate fibre prepared in 5.0 times concentration of SBF; Figure 4 shows a scanning electron microscope calcium phosphate fibre prepared in 1.5 times concentration of SBF and sintered at 950C; Figure 5 shows a scanning electron microscope calcium phosphate fibre prepared in 1.5 times concentration of SBF and sintered at 1150 0

C;

Figure 6 shows a scanning electron microscope calcium phosphate fibre prepared in 1.5 times concentration of SBF and sintered at 1250 0

C;

Figure 7 shows a scanning electron microscope calcium phosphate fibre prepared in 5.0 times concentration of SBF and sintered at 950°C; Figure 8 shows a scanning electron microscope calcium phosphate fibre prepared in 5.0 times concentration of SBF and sintered at 1150 0

C;

image of a image of a image of a image of a image of a image of a 7- 05/12 2003 FRI 21:37 [TX/RX NO 5034] I011 mm III 05/12 '03 FRI 21:39 FAX 61299255911 GRIFFITH HACK IP AUSTRALIA 1012 Figure 9 shows a scanning electron microscope image of a calcium phosphate fibre prepared in 5.0 times concentration of SBF and sintered at 1250 0

C;

Figure 10 shows and x-ray diffraction scan of calcium phosphate fibres prepared in 1.5 times concentration of SBF and sintered at 950 0 C, 1150 0 C,1250 0

C;

Figure 11 shows and x-ray diffraction scan of calcium phosphate fibres prepared in 5.0 times concentration of SBF and sintered at 950 0 C, 1150 0 C,1250 0

C;

Figure 12 shows a scanning electron microscope image of a calcium phosphate fibre prepared in 1.5 times concentration of SBF, sintered at 950 0 C and with human derived MG63 osteoplast cells cultured; Figure 13 shows a scanning electron microscope image of a calcium phosphate fibre prepared in 1.5 times concentration of SBF and sintered at 1150 0 C and with human derived MG63 osteoplast cells cultured; Figure 14 shows a scanning electron microscope image of a calcium phosphate fibre prepared in 1.5 times concentration of SBF and sintered at 1250°C and with human derived MG63 osteoplast cells cultured; Figure 15 shows a scanning electron microscope image of a calcium phosphate fibre prepared in 5.0 times concentration of SBF and sintered at 950 0 C and with human derived MG63 osteoplast cells cultured; Figure 16 shows a scanning electron microscope image of a calcium phosphate fibre prepared in 5.0 times concentration of SBF and sintered at 11500C and with human derived MG63 osteoplast cells cultured; and Figure 17 shows a scanning electron microscope image of a calcium phosphate fibre prepared in 5.0 times 8 05/12 2003 FRI 21:37 [TX/RX NO 5034] @012 05/12 '03 FRI 21:39 FAX 61299255911 GRIFFITH HACK 444 IP AUSTRALIA Q 013 concentration of SBF and sintered at 1250°C and with human derived MG63 osteoplast cells cultured; Figure 18 shows a table of ion concentration of 1.OSBF and 1.5SBF solution in comparison with those of blood plasma; Detailed Description Referring to Figure 1, a first embodiment of the invention involves the production of a ceramic fibre using the steps of: a. phosphorylation of a plurality of cotton fibres; b. treating the cotton fibres in a solution of Ca(OH) 2 c. growth of a ceramic coating on the cotton fibres in a simulated body fluid (SBF); and d. heating of cellulose fibres and the ceramic coating.

Each steps is described in more detail below together with analysis of products at stages and A cell attachment study was performed on the resultant hydroxyapatite fibres to assess biocompatibility.

Phosphorylation Treated Cotton Phosphorylation of cotton samples were carried out.

Cotton pieces were placed in a round bottom flask equipped with a thermometer, mechanical stirrer, condenser and N 2 gas inlet tube. Urea dissolved in dimethyl formamide (DMF) was added to the flask and heated to 130°C, upon which phosphorous acid (H30P3) was added and heated to 1450C. The reaction was allowed to reflux for thirty minutes. Cotton fibres were then washed repeatedly in distilled water and dried in an oven at 50 0

C.

9 05/12 2003 FRI 21:37 [TX/RX NO 5034] d013 05/12 '03 FRI 21:39 FAX 81299255911 GRIFFITH HACK 4-4 IP AUSTRALIA J014 Ca(OH) 2 Treatment The phosphorylated cotton was soaked (without stirring) in a saturated solution of Ca(OH) 2 (pH 11-12) in closed screw-top glass bottle for periods of up to 8 days. The Ca(OH)z solution was renewed every 4 days. Upon completion of the soaking period the samples were subsequently filtered, rinsed thoroughly with distilled water and dried in an oven at 500C.

Growth of Calcium Phosphate times the concentration of Simulated Body Fluid was prepared. SBF simulates the ionic concentration of blood plasma and prepared according to Figure 18. The pH was measured and adjusted to pH 7.4 with tris(hydroxymethyl)aminomethane ((CHaOH) 3

CNH

2 and dilute hydrochloric acid (HC1). Samples of pre-treated cotton were placed in 1.5SBF in closed screw-top glass jars and re-buffered to pH of 7.4. Immersion of the glass jars in a shaking water bath at 36.5 0 C was for two weeks. The 1.5SBF solution was renewed every two days to maintain pH 7.4 and ion concentration. Upon completion of soaking period, samples were washed with distilled water and dried in air before further examination. A solution was prepared in a similar way.

Heat Treatment Cotton Burnout To determine both the effect of sintering behaviour at various temperatures on the calcium phosphate apatite, and to volatilise (burn out) the cotton substrate, coated cotton fibres were fired in air to 950, 1150 and 1250°C at a rate of 100°C per hour, with a soaking time of 1 hour.

10 05/12 2003 FRI 21:37 [TX/RX NO 5034] 1014 05/12 '03 FRI 21:39 FAX 61299255911 GRIFFITH HACK 444 IP AUSTRALIA 1 015 Experimental Procedure of Cell Cultures of Sintered Fibres The sintered calcium phosphate fibres and sintered calcium phosphate fibre compacts using 1.5SBF and the were weighed to 3tg and autoclaved for 45 minutes at 135°C. Both fibres and the compacts of the 1.5SBF and the 5.OSBF groups were seeded with human derived MG63 cells at a density of 50000 cells per ml of cell media.

The media of the cells was replaced after 4 days. After one week, the media was pipetted out of each well and the specimens were rinsed twice with Phosphate Buffer Solution (PBS). The specimens were then fixed with 2% glutaraldehyde, rinsed with PBS, dehydrated in a series through a graded series of ethanol and critical point dried. Finally they were mounted on aluminium plates and coated with a thin layer of gold.

Analysis of Products Figures 2 to 3 respectively show SEM images of coated cotton with a 1.5SBF and a 5.OSBF. These images show fairly uniform coverage of calcium phosphate on cotton fibres at each of the SBF concentrations with no heat treatment. Figures 4 to 6 show the heat treatment of fibres coated using a 1.5SBF at 950, 1150 and 1250°C respectively. Heat treatment at 950 0 C shows the cotton substrate has been burnt out, however very little sintering of the calcium phosphate phase has occurred.

With heat treatment at 1150°C there seems to be necking within the coat, the earlier stage of sintering. While with heat treatment at 1250 0 C there is a considerable amount of sintering, resulting in sintered porosity.

There is also a reduction in surface area at 1250°C compared with heat treatment at 950 0 C. It is also evident from these SEM pictures that the fibres maintain a tubular morphology. At 950°C, the cotton has burnt out 11 05/12 2003 FRI 21:37 [TX/RX NO 5034] a015 05/12 '03 FRI 21:40 FAX 61299255911 GRIFFITH HACK IP AUSTRALIA z 016 leaving a fairly thick walled (approximately lpm) hollow fibre. Increasing the sintering temperature to 1150 0

C,

the tubular morphology is maintained but at 1250°C sintering occurs such that the hollow fibres start to open to take the form of tapes.

Figures 7 to 9 show the heat treatment of fibres coated using a S.OSBF at 950, 1150 and 1250°C respectively and show similar characteristics to the fibres treated using a 1.5 SBF.

XRD analysis was performed to confirm the calcium phosphate phase was low crystalline apatite. XRD plots in Figures 10 and 11 show the crystallinity of the apatite phases generally increasing with increasing heat treatment temperature for 5.0SBF, but 950 0 C treatment of samples has higher crystallinity than the 1150 0

C.

This may be an experimental error and requires further investigation. The main calcium phosphate phase present at the increasing heat treatment temperature is whitlockite, a decomposing phase of hydroxyapatite.

Figures 12 to 14 show a scanning electron microscope images of a calcium phosphate fibre prepared in 1.-5 times concentration of SBF, sintered at 950, 1150 and 1250 0

C

respectively and with human derived MG63 osteoplast cells cultured. It was observed that all samples were favourable to cell attachment and the sintering temperature did not directly influence the proliferation and attachment of the cells. The purpose for cell cultures was to test the general biocompatibility of the fibres, but also to see if any residue of cotton from the burnout stage could deleteriously affect the biocompatibility. The osteoblasts grew along length of the fibres and did not attempt to enter the hollow area.

They were well attached and spread over the surface with 12 05/12 2003 FRI 21:37 [TX/RX NO 5034] a016 05/12 '03 FRI 21:40 FAX 61299255911 GRIFFITH HACK -44 IP AUSTRALIA 2 017 contact to other cells. Thus the results show the biomimetically produced hollow fibres are biocompatible with no cytotoxic effects.

Figures 15 to 17 show a scanning electron microscope images of a calcium phosphate fibre prepared in 5.0 times concentration of SBF, sintered at 950, 1150 and 1250 0

C

respectively and with human derived MG63 osteoplast cells cultured.

It would be advantageous if embodiments of the invention provide a biodegradable fibre material with good thermal insulating properties and biocompatibility. Applications for such fibre includes tissue scaffolds, orthopaedic surgical bone filler, and as a drug carrier. Other nonbiomedical applications include fire protection, thermal insulation and furnace linings.

It is to be understood that a reference herein to a prior art publication does not constitute an admission that the publication forms a part of the common general knowledge in the art in Australia, or any other country.

13 05/12 2003 FRI 21:37 [TX/RX NO 5034] 01017

Claims

1. A method for producing ceramic fibre comprising the steps of: a. forming a ceramic coating on a cellulose fibre in a salt solution containing ions of Ca2 and PO43-; and b. heating the ceramic coating and cellulose fibre to volatilise the cellulose fibre

2. A method as claimed in claim 1 wherein the ion concentration of the salt solution is controlled as to form predominantly calcium phosphates in the ceramic coating.

3. A method as claimed in either claim 1 or 2 wherein phosphorylation of the cellulose fibre is performed prior to forming the ceramic coating.

4. A method as claimed in any one of claims 1 to 3 wherein the cellulose fibre is treated in a calcium containing solution prior to forming the ceramic coating. A method as claimed in claim 4 wherein the calcium containing solution treatment is performed after the phosphorylation treatment.

6. A method as claimed in any one of claims 1 to wherein heating of the ceramic coating is performed at temperatures sufficient to achieve at least partial sintering of the ceramic coating.

7. A method as claimed in any one of claims 1 to 6 wherein 14 05/12 2003 FRI 21:37 [TX/RX NO 5034] a018 05/12 '03 FRI 21:40 FAX 61299255911 GRIFFITH HACK 4+44 IF AUSTRALIA a 0O19 1 the salt solution inzcludes Ca 2 pot3, NSa+, Mg 2 SO 4 CO3 2 and Cl- ions.

8- A method as claimed in any one of claims 1 to 7 where in the ratios of the ions are similar to the ratio of ions in body plasma.

9. A method as claimed in any one of claims I. to 8 wherein the concentration of the one or more of the ions is in the range of I to 5 times the concentration of ions in body plasma. A method as claimed in any one of claims 6 to 9 wherein sintering is performed at a temperature between 800- 12500C.

11- A method as claimed in any one of claims 6 to wherein sintering below a temperature of 950 0 C produces predominately tubular ceramic fibre.

12. A method as claimed in any one of claims 6 to 11 wherein sintering above a temperature of 1150 0 C produces predominately tape-like ceramic fibre.

13. A method as claimed in any one of claims 1 to 12 wherein the cellulose fibre is cotton.

14. A ceramic fibre, the ceramic fibre contains predominantly phases of calcium phosphates wherein the ceramic fibre is tape-like or tube-like in morphology. is1 05/12 2003 FRI 21:37 [TX/RX NO 5034) Q~019 05/12 '03 FRI 21:41 FAX 61299255911 GRIFFITH HACK P IP AUSTRALIA 1020 A ceramic fibre as claimed in claim 14 wherein the ceramic fibre predominantly comprises a combination of one or more of the phases of calcium phosphate, hydroxyapatite and whitlockite.

16. A ceramic fibre as claimed in either claim 14 or wherein the surface morphology of a proportion of the ceramic fibre has a cauliflower-like appearance.

17. A ceramic fibre as claimed in any one of claims 14 to 16 wherein the surface morphology of a proportion of the ceramic fibre has a dendrite-like appearance. Dated 5 December 2003 16 05/12 2003 FRI 21:37 [TX/RX NO 5034] a020