WO2009061908A2 - Ceramic/structural protein composites and method of preparation thereof - Google Patents
Ceramic/structural protein composites and method of preparation thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
- A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
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- Implantable medical devices such as orthopedic and dental prostheses, can be made more permanent if the interface between the existing bone and the device contains some natural bone growth to knit the two components together.
- Such ingrowth has advantages over the use of bone cement, both in terms of stability and permanency.
- Bioactive coatings on implantable medical devices allow for the ingrowth of natural bone into and around the device, forming chemical bonds between the device and natural bone.
- Bone is composed of substituted apatite crystals in an abundant collagen network.
- Type I collagen is the major protein of bone tissue, making up about thirty percent of the weight of bone. It has been shown that apatite crystals can grow and bond to collagen fibrils, and prepared apatite/collagen composites have been shown to promote direct bone apposition.
- apatite In addition to coatings, other materials made from apatite are used for bone repair and replacement.
- the cross-linked apatite/collagen porous scaffold materials have been studied for their excellent compatibility with human bone.
- Several approaches to preparing an apatite/collagen composite scaffold have been studied, but have exhibited drawbacks with respect to variable porosity of the composite.
- One known approach is to prepare a composite material containing protein osteoinductive factor, mineral (mixture of hydroxyapatite and tricalcium phosphate) and collagen in a water suspension by a mechanical mixing means.
- the apatite/collagen composite After gelation of collagen, the apatite/collagen composite is freeze-dried again to synthesis the apatite/collagen scaffold. Then the apatite/collagen scaffold is cross-linked and cleaned. This process requires two freeze-drying procedures and two cleaning procedures to form apatite/collagen composite scaffolds.
- a method of forming a composite scaffold comprises forming an aqueous scaffold system comprising a structural protein, a weak acid, water, Ca 2+ , HPO 4 2" , a buffer system, and optionally one or more of Mg 2+ , Na + , K + , Cl " , SO 4 2" ; or HCO 3 " ; wherein the aqueous scaffold system has an initial pH of about 6.5 to about 8.0; placing the aqueous system in container; sealing the container; isolating a gel; and freeze-drying the gel to form a composite scaffold.
- an implantable medical device comprises a composite scaffold prepared by the process comprising forming an aqueous scaffold system comprising a structural protein, a weak acid, water, Ca 2+ , HPO 4 2" , a buffer system, and optionally one or more of Mg 2+ , Na + , K + , Cl " , SO 4 2" ; or HCO 3 " ; wherein the aqueous scaffold system has an initial pH of about 6.5 to about 8.0; placing the aqueous scaffold system in container; sealing the container; allowing a gel to form; isolating the gel; and freeze-drying the gel to form a composite scaffold.
- composite scaffolds prepared by the processes, as well as uses for composite scaffolds.
- a controllable structural protein content apatite/structural protein scaffold can be formed.
- the resulting scaffold is a porous composite containing up to about ninety weight percent incorporated structural protein.
- the method involves preparing an aqueous scaffold system containing water, Ca + , HPO 4 " structural protein (e.g., collagen type I and the like), a weak acid (eg. acetic acid, and the like) and a buffer system; and optionally one or more of the following ions: Mg 2+ , Na + , K + , Cl " , SO 4 2" , HCO 3 " ; wherein the aqueous scaffold system has an initial pH of about 6.50 to about 8.00.
- the aqueous scaffold system is allowed to stand, for example at a temperature of about 20 0 C to about 45°C, to form a composite gel, the gel is optionally crosslinked, isolated, mixed with water and freeze-dried to form a porous ceramic/structural protein composite scaffold. Prior to freeze drying, the mixture can be placed in a mold.
- the structural protein used to prepare the scaffold can be any known structural protein such as collagens, elastin, and keratins, specifically collagen, and more specifically soluble collagen Types I, II, III, and V, and yet more specifically collagen Type I.
- soluble collagen means "collagen molecules or microfibrils which are soluble in an aqueous solution”.
- the structural protein may be obtained from commercial sources or extracted from natural sources using procedures well known in the art.
- the amount of structural protein (e.g., collagen) in the resulting scaffold can be about 1 to about 90 weight percent based on the total weight of the scaffold, specifically about 10 to about 80 weight percent, more specifically about 25 to about 65 weight percent, and yet more specifically about 40 to about 50 weight percent.
- the aqueous scaffold system generally comprises the following inorganic ions: Ca 2+ and HPO 4 2" ; and optionally one or more of the following ions: Mg 2+ , Na + , K + , Cl “ , SO 4 2" , HCO 3 " .
- the aqueous system can be prepared by dissolving in an aqueous solvent salts that when disassociated will result in the particular ions Ca 2+ , Mg 2+ , Na + , K + , Cl " , SO 4 2" , HPO 4 2" and HCO 3 " .
- the aqueous solvent can be deionized and purified water.
- Exemplary salts include those that result in an aqueous solution of the desired ions, for example, alkali metal halides, alkaline earth metal halides, alkali metal hydrogen carbonates, alkali metal phosphates, and alkali metal sulfates.
- Specific salts include, NaCl, KCl, K 2 HPO 4 , MgCl 2 , Na 2 SO 4 , CaCl 2 and NaHCO 3 .
- Mg 2+ at about 0 to about 5.0 mM, specifically about 0.05 to about 1.0 mM, and more specifically about 0.2 to about 0.4 mM;
- SO 4 2" at about 0 to about 2.0 mM, specifically about 0.1 to about 1.5 mM, and more specifically about 0.4 to about 0.6 mM;
- HPO 4 2" at about 0.05 to about 10.0 mM, specifically about 0.1 to about 3.0 mM, and more specifically about 0.5 to about 1.0 mM;
- HCO 3 at about 0 to about 30.0 mM, specifically about 0.5 to about 10.0 mM, and more specifically about 2.0 to about 5.0 mM.
- An additional component present in the aqueous scaffold system is a buffer system.
- the buffer system can contain HEPES (4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; Molecular formula: C 8 Hi 7 N 2 SO 3 ; CAS No: 7365-45-9) and an alkali metal hydrogen carbonate (e.g. NaHCO 3 , KHCO3, etc.) which are added to the aqueous scaffold system in amounts to substantially stabilize the aqueous system.
- HEPES 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
- an alkali metal hydrogen carbonate e.g. NaHCO 3 , KHCO3, etc.
- the concentration of HEPES present in the aqueous scaffold system can be at about 5.0 grams per liter (g/L) to about 80.0 g/L, specifically about 10.0 g/L to about 60.0 g/L, and more specifically about 12.0 g/L to about 48.0 g/L.
- Additional buffer systems may include tris-hydroxymethyl aminomethan (TRIS), HEPES salts, piperazine-l,4-bis(2-ethanesulfonic acid) (PIPES), PIPES salts, combinations of the foregoing with an alkali metal carbonate, and combinations thereof.
- TMS tris-hydroxymethyl aminomethan
- PEPES piperazine-l,4-bis(2-ethanesulfonic acid)
- PIPES salts combinations of the foregoing with an alkali metal carbonate, and combinations thereof.
- the aqueous scaffold system may optionally contain additional ionic components such as silicate, strontium, zinc, silver, fluoride, combinations thereof, and the like.
- the weak acid present in the aqueous scaffold system can be any acid with a pKa of about 3.5 to about 5.5.
- exemplary acids include organic acids, specifically alkyl carboxylic acids such as acetic acid, propionic acid, and the like.
- the aqueous scaffold system can have an initial pH of about 6.5 to about 8.0, specifically about 7.0 to about 7.5.
- the temperature of during the process to prepare the scaffold can be about 15 to about 5O 0 C, specifically about 20 to about 45 0 C, and yet more specifically about 25 to about 4O 0 C.
- the incubation time for preparing the composite gel can be about 0.5 to about 10 hours, specifically about 1.0 to about 9 hours, and yet more specifically about 2.0 to about 8.0 hours.
- crosslinking agents such as a carbodiimide
- exemplary crosslinking agents include glutaraldehyde, l-ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride optionally in combination with N- hydroxysuccinimide or N-hydroxysulfosuccinimide; dimethyl suberimidate, bis(sulfosuccinimidyl)suberate (BS 3 ), 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate (Sulfo-SMCC), dithiobis(succinimidyl)propionate (DSP), sulfosuccinimidyl 6-(3'-[2-pyridyldithi
- a method of forming a composite scaffold comprises forming an aqueous scaffold system comprising a structural protein, a weak acid, water, Ca 2+ , Mg 2+ , Na + , K + , Cl " , SO 4 2" , HPO 4 2" , HCO 3 " and a buffer system, wherein the aqueous scaffold system has an initial pH of about 6.5 to about 8.0; placing the aqueous system in container; sealing the container; allowing a gel to form; isolating the gel; and freeze-drying the gel to form a composite scaffold, hi another embodiment, the method of forming a composite scaffold further comprises molding the gel prior to freeze-drying.
- the resulting ceramic is generally a bone-like apatite, but can also be other types of calcium phosphate.
- Exemplary calcium phosphate minerals include Cas(PO 4 ) 3- x (OH)i_ y (CO 3 ) x+y , Ca 5 (PO 4 ) 3 (OH), Ca 3 (PO 4 ) 2 , CaHPO 4 , Ca(H 2 PO 4 ),, and the like.
- the scaffolds can be used to prepare medical, surgical, reconstructive, orthopedic, orthodontic, prosthodontic, endodontic or dental devices, implants, appliances, or a component thereof.
- a soluble collagen solution was prepared from extraction of three rat tails in IL solution ( ⁇ 1.5 g/L) according to the following procedure.
- Type I collagen was extracted from rat tail tendon as previously described W. Zhang, S. S. Liao, F. Z. Cui, Chem. Mater. 2003, 15, 3221.
- the rat tail tendon was soaked in 0.5 M acetic acid for 3-4 days at 4 0 C.
- the solution was centrifuged at 10,000 rpm at 4°C for 15 minutes and filtered with No.l filter paper to remove the insoluble components. NaCl (5% wt%) was added to induce precipitation of collagen, and the precipitates were collected by centrifuging at 10,000 rpm for 15 minutes at 4 0 C.
- Collagen was then dissolved in 0.5 M acetic acid to form a collagen solution.
- the collagen solution was added to an aqueous system containing Ca 2+ and HPO 4 2" , Na + , K + , Mg 2+ , Cl " , HCO 3 " , SO 4 2" and acetic acid; prepared from NaCl, NaHCO 3 , Na 2 CO 3 , KCl, K 2 HPO 4 -3H 2 O, MgCl 2 -H 2 O, HEPES, CaCl 2 , Na 2 SO 4 , and glacial acetic acid.
- the amount of inorganic salts used in the aqueous system was varied to explore the apatite/collagen ratio in the final composite (Table 3).
- the initial pH of the collagen containing aqueous system was adjusted to 7.5 using 5M NaOH.
- apatite/collagen composite Fifty milliliters of collagen containing aqueous system was placed in a sealed 100 ml bottle and allowed to form an apatite/collagen composite. The composite formation process is carried out at 4O 0 C. After 4 hours, the collagen started to form hydro gel-like material. Five ml of glutaraldehyde is then added to further cross-link the collagen. After one-hour for the crosslinking, the apatite/collagen hydrogel is collected and rinsed with 50 ml deionized water four times using centrifuge (7000-12000 rpm). The apatite/collagen composite is then transferred into a cylinder mold and mixed with water. The porosity of the scaffold can be controlled by the amount of water added at this point in the process. The more water is present, the more porous the scaffold becomes. After that, the mixture was freeze-dried to obtain a porous apatite/collagen scaffold.
- An apatite/collagen composite scaffold was prepared by extracting Type I collagen from rat tails and dissolved in 0.5 M acetic acid.
- the components used to make the aqueous system including NaCl, CaCl 2 , K 2 HPO 4 , MgCl 2 , NaHCO 3 and HEPES, were added to the collagen solution (approximately 1.5 g/L) to prepare collagen containing aqueous system.
- the concentrations of these components are listed in Table 5.
- the initial pH of the solution was adjusted to 7.0 at 4O 0 C using dilute HCl or NaOH.
- the solution was aged for 4 hours to allow co-precipitation of apatite nanoparticles and collagen fibers in the solution.
- the scaffold is crosslmked using 2 w/v% l-ethyl-3-(3-dimethylarninopropyi) carbodiimide hydrochloride (EDC) at 4 0 C for 24 hours, then rinsed and freeze-dried to attain the final scaffold.
- EDC carbodiimide hydrochloride
- the scaffold was cut into 5 millimeter (mm) discs, and seeded with MC3T3 cells.
- the cell seeding density was 1.6x10 5 cells/scaffold.
- DMEM Dulbecco's Modified Eagle's Medium
- FCS fetal calf serum
- penicillin 100 units/ml
- streptomycin 100 ⁇ g/ml
- non-essential amino acids 100 ⁇ M
- a mouse calvaria model was used with two 3.5 mm defects created at each side of the suture line at the calvaria site.
- One positive control and one apatite/collagen scaffold were implanted. The implantation period was 28 days. After harvest, the implants were embedded and frozen-sectioned. Adjacent images were obtained from a Zeiss Axiovert and AxioObserver work station and tiled together to reproduce a full-size image of the bone section. It was found that the apatite/collagen composite scaffold supports bone formation. The new bone formation was mainly contributed by donor cells.
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Abstract
Ceramic/structural protein composite scaffolds and their preparation in a simple one-step process are shown.
Description
CERAMIC/STRUCTURAL PROTEIN COMPOSITES AND METHOD OF
PREPARATION THEREOF
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0001] The U.S. Government has certain rights in this invention pursuant to Grant No. DMI 0500269 awarded by the National Science Foundation.
BACKGROUND OF INVENTION
[0002] Implantable medical devices, such as orthopedic and dental prostheses, can be made more permanent if the interface between the existing bone and the device contains some natural bone growth to knit the two components together. Such ingrowth has advantages over the use of bone cement, both in terms of stability and permanency.
[0003] "Bioactive" coatings on implantable medical devices allow for the ingrowth of natural bone into and around the device, forming chemical bonds between the device and natural bone. Bone is composed of substituted apatite crystals in an abundant collagen network. Type I collagen is the major protein of bone tissue, making up about thirty percent of the weight of bone. It has been shown that apatite crystals can grow and bond to collagen fibrils, and prepared apatite/collagen composites have been shown to promote direct bone apposition.
[0004] In addition to coatings, other materials made from apatite are used for bone repair and replacement. The cross-linked apatite/collagen porous scaffold materials have been studied for their excellent compatibility with human bone. Several approaches to preparing an apatite/collagen composite scaffold have been studied, but have exhibited drawbacks with respect to variable porosity of the composite.
[0005] One known approach is to prepare a composite material containing protein osteoinductive factor, mineral (mixture of hydroxyapatite and tricalcium phosphate) and collagen in a water suspension by a mechanical mixing means.
[0006] Another approach is by mixing insoluble collagen with calcium chloride and tribasic sodium phosphate at pH around 11.0. However, insoluble collagen was used to directly mix with apatite to form into composites, which may render an inhomogeneous apatite/collagen composite.
[0007] Finally, it is known to prepare an apatite/collagen composite using soluble collagen, phosphoric acid and calcium salt. Instead of forming apatite/collagen composite in one step, once the soluble collagen, phosphoric acid and calcium salt mix, the slurry-like mixture is freeze-dried. The gelation of collagen is carried out after freeze-drying apatite/collagen composite at a pH around 11.0. After gelation of collagen, the apatite/collagen composite is freeze-dried again to synthesis the apatite/collagen scaffold. Then the apatite/collagen scaffold is cross-linked and cleaned. This process requires two freeze-drying procedures and two cleaning procedures to form apatite/collagen composite scaffolds.
[0008] None of the above processes addresses the variable porosity of apatite/collagen scaffold, a factor that is important to control the regeneration of new bone tissue.
[0009] There remains a need in the art for improved apatite composite scaffolds, as well as improved processes to prepare porosity controllable apatite composite scaffolds.
BRIEF DESCRIPTION OF THE INVENTION
[0010] In one embodiment, a method of forming a composite scaffold comprises forming an aqueous scaffold system comprising a structural protein, a weak acid, water, Ca2+, HPO4 2", a buffer system, and optionally one or more of Mg2+, Na+, K+, Cl", SO4 2"; or HCO3 "; wherein the aqueous scaffold system has an initial pH of about 6.5 to about 8.0; placing the aqueous system in container; sealing the container; isolating a gel; and freeze-drying the gel to form a composite scaffold.
[0011] In another embodiment, an implantable medical device comprises a composite scaffold prepared by the process comprising forming an aqueous scaffold system comprising a structural protein, a weak acid, water, Ca2+, HPO4 2", a buffer system, and optionally one or more of Mg2+, Na+, K+, Cl", SO4 2"; or HCO3 "; wherein the aqueous scaffold system has an initial pH of about 6.5 to about 8.0; placing the aqueous scaffold system in container; sealing the container; allowing a gel to form; isolating the gel; and freeze-drying the gel to form a composite scaffold.
[0012] Also disclosed herein are composite scaffolds prepared by the processes, as well as uses for composite scaffolds.
DETAILED DESCRIPTION
[0013] Disclosed herein are methods of forming ceramic/structural protein composite scaffolds in a simple one-step process; composite scaffolds prepared therefrom; and articles prepared therefrom. With the disclosed method, a controllable structural protein content apatite/structural protein scaffold can be formed.
[0014] Disclosed herein is a method to prepare ceramic/structural protein composite scaffolds in a convenient, one-step process. The resulting scaffold is a porous composite containing up to about ninety weight percent incorporated structural protein. The method involves preparing an aqueous scaffold system containing water, Ca +, HPO4 " structural protein (e.g., collagen type I and the like), a weak acid (eg. acetic acid, and the like) and a buffer system; and optionally one or more of the following ions: Mg2+, Na+, K+, Cl", SO4 2", HCO3 "; wherein the aqueous scaffold system has an initial pH of about 6.50 to about 8.00. The aqueous scaffold system is allowed to stand, for example at a temperature of about 200C to about 45°C, to form a composite gel, the gel is optionally crosslinked, isolated, mixed with water and freeze-dried to form a porous ceramic/structural protein composite scaffold. Prior to freeze drying, the mixture can be placed in a mold.
[0015] The structural protein used to prepare the scaffold can be any known structural protein such as collagens, elastin, and keratins, specifically collagen, and more specifically soluble collagen Types I, II, III, and V, and yet more specifically collagen Type I. As used herein, soluble collagen means "collagen molecules or microfibrils which are soluble in an aqueous solution".
[0016] There is no particular limitation as to the source of the structural protein. The structural protein may be obtained from commercial sources or extracted from natural sources using procedures well known in the art.
[0017] When collagen Type I is used as the structural protein, the collagen gelation and apatite precipitation happen simultaneously after incubation for about 2 to about 8 hours. The gel-like composite is then cross-linked, cleaned with pure water and freeze-dried to form apatite/collagen scaffold with varying porosity depending upon the amount of water contained in the initial scaffold. The scaffold's collagen to apatite ratio is controlled by the initial collagen concentration of the aqueous scaffold system.
[0018] The amount of structural protein (e.g., collagen) in the resulting scaffold can be about 1 to about 90 weight percent based on the total weight of the scaffold, specifically about 10 to about 80 weight percent, more specifically about 25 to about 65 weight percent, and yet more specifically about 40 to about 50 weight percent.
[0019] The aqueous scaffold system generally comprises the following inorganic ions: Ca2+ and HPO4 2"; and optionally one or more of the following ions: Mg2+, Na+, K+, Cl", SO4 2", HCO3 ". The aqueous system can be prepared by dissolving in an aqueous solvent salts that when disassociated will result in the particular ions Ca2+, Mg2+, Na+, K+, Cl", SO4 2", HPO4 2" and HCO3 ". The aqueous solvent can be deionized and purified water. Exemplary salts include those that result in an aqueous solution of the desired ions, for example, alkali metal halides, alkaline earth metal halides, alkali metal hydrogen carbonates, alkali metal phosphates, and alkali metal sulfates. Specific salts include, NaCl, KCl, K2HPO4, MgCl2, Na2SO4, CaCl2 and NaHCO3.
[0020] The particular concentrations of each of the above-described ions initially present in the aqueous system can be as follows:
[0021] Ca2+ at about 0.1 to about 15.0 mM, specifically about 0.5 to about 10.0 mM, and more specifically about 1.0 to about 2.0 mM;
[0022] Mg2+ at about 0 to about 5.0 mM, specifically about 0.05 to about 1.0 mM, and more specifically about 0.2 to about 0.4 mM;
[0023] Na+ at about 0 to about 300.0 mM, specifically about 5.0 to about 100.0 mM, and more specifically about 20.0 to about 50.0 mM;
[0024] K+ at about 0 to about 10.0 mM, specifically about 0.1 to about 5.0 mM, and more specifically about 1.0 to about 2.0 mM;
[0025] Cl" at about 0 to about 300.0 mM, specifically about 5.0 to about 100.0 ]mM, and more specifically about 20.0 to about 50.0 mM;
[0026] SO4 2" at about 0 to about 2.0 mM, specifically about 0.1 to about 1.5 mM, and more specifically about 0.4 to about 0.6 mM;
[0027] HPO4 2" at about 0.05 to about 10.0 mM, specifically about 0.1 to about 3.0 mM, and more specifically about 0.5 to about 1.0 mM; and
[0028] HCO3 " at about 0 to about 30.0 mM, specifically about 0.5 to about 10.0 mM, and more specifically about 2.0 to about 5.0 mM.
[0029] An additional component present in the aqueous scaffold system is a buffer system. The buffer system can contain HEPES (4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; Molecular formula: C8Hi7N2SO3; CAS No: 7365-45-9) and an alkali metal hydrogen carbonate (e.g. NaHCO3, KHCO3, etc.) which are added to the aqueous scaffold system in amounts to substantially stabilize the aqueous system. The concentration of HEPES present in the aqueous scaffold system can be at about 5.0 grams per liter (g/L) to about 80.0 g/L, specifically about 10.0 g/L to about 60.0 g/L, and more specifically about 12.0 g/L to about 48.0 g/L.
[0030] Additional buffer systems may include tris-hydroxymethyl aminomethan (TRIS), HEPES salts, piperazine-l,4-bis(2-ethanesulfonic acid) (PIPES), PIPES salts, combinations of the foregoing with an alkali metal carbonate, and combinations thereof.
[0031] The aqueous scaffold system may optionally contain additional ionic components such as silicate, strontium, zinc, silver, fluoride, combinations thereof, and the like.
[0032] The weak acid present in the aqueous scaffold system can be any acid with a pKa of about 3.5 to about 5.5. Exemplary acids include organic acids, specifically alkyl carboxylic acids such as acetic acid, propionic acid, and the like.
[0033] The aqueous scaffold system can have an initial pH of about 6.5 to about 8.0, specifically about 7.0 to about 7.5.
[0034] The temperature of during the process to prepare the scaffold can be about 15 to about 5O0C, specifically about 20 to about 450C, and yet more specifically about 25 to about 4O0C.
[0035] The incubation time for preparing the composite gel can be about 0.5 to about 10 hours, specifically about 1.0 to about 9 hours, and yet more specifically about 2.0 to about 8.0 hours.
[0035] Various crosslinking agents, such as a carbodiimide, can be used to crosslink the collagen. Exemplary crosslinking agents include glutaraldehyde, l-ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride optionally in combination with N- hydroxysuccinimide or N-hydroxysulfosuccinimide; dimethyl suberimidate, bis(sulfosuccinimidyl)suberate (BS3), 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate (Sulfo-SMCC), dithiobis(succinimidyl)propionate (DSP), sulfosuccinimidyl 6-(3'-[2-pyridyldithio]- propionamido)hexanoate, and the like. The amount of crosslinking agent used can be about 0.1 to about 0.4 M, specifically about 0.2 to about 0.3 M.
[0036] In one embodiment, a method of forming a composite scaffold comprises forming an aqueous scaffold system comprising a structural protein, a weak acid, water, Ca2+, Mg2+, Na+, K+, Cl", SO4 2", HPO4 2", HCO3 " and a buffer system, wherein the aqueous scaffold system has an initial pH of about 6.5 to about 8.0; placing the aqueous system in container; sealing the container; allowing a gel to form; isolating the gel; and freeze-drying the gel to form a composite scaffold, hi another embodiment, the method of forming a composite scaffold further comprises molding the gel prior to freeze-drying.
[0037] The resulting ceramic is generally a bone-like apatite, but can also be other types of calcium phosphate. Exemplary calcium phosphate minerals include Cas(PO4)3- x(OH)i_y(CO3)x+y, Ca5(PO4)3(OH), Ca3(PO4)2, CaHPO4, Ca(H2PO4),, and the like.
[0038] The scaffolds can be used to prepare medical, surgical, reconstructive, orthopedic, orthodontic, prosthodontic, endodontic or dental devices, implants, appliances, or a component thereof.
EXAMPLES
Example 1. Apatite/collagen composite: Scaffold
[0039] A soluble collagen solution was prepared from extraction of three rat tails in IL solution (~ 1.5 g/L) according to the following procedure. Type I collagen was extracted
from rat tail tendon as previously described W. Zhang, S. S. Liao, F. Z. Cui, Chem. Mater. 2003, 15, 3221. The rat tail tendon was soaked in 0.5 M acetic acid for 3-4 days at 40C. The solution was centrifuged at 10,000 rpm at 4°C for 15 minutes and filtered with No.l filter paper to remove the insoluble components. NaCl (5% wt%) was added to induce precipitation of collagen, and the precipitates were collected by centrifuging at 10,000 rpm for 15 minutes at 40C. Collagen was then dissolved in 0.5 M acetic acid to form a collagen solution. The collagen solution was added to an aqueous system containing Ca2+ and HPO4 2", Na+, K+, Mg2+, Cl", HCO3 ", SO4 2" and acetic acid; prepared from NaCl, NaHCO3, Na2CO3, KCl, K2HPO4-3H2O, MgCl2-H2O, HEPES, CaCl2, Na2SO4, and glacial acetic acid. The amount of inorganic salts used in the aqueous system was varied to explore the apatite/collagen ratio in the final composite (Table 3). The initial pH of the collagen containing aqueous system was adjusted to 7.5 using 5M NaOH.
Table 3 The ion concentrations of collagen containing aquequs^system
Ions Sample 1 Sample 2 Sample 3 Sample 4
Na+ 109.5 mM 43.6 mM 21.8 mM 10.9 mM K+ 6.O mM I 2.4 mM 1.2 mM 0.6 mM
Mg2+ 1.5 mM 0.6 mM 0.3 mM 0.15 mM Ca2+ 1 7 5 mM 3 mM 1.5 mM 0.75 mM
Cl 110.O mM 44 mM 22 mM H mM HC(V i 17.5 mM 7 mM 3 5 mM 1.75 mM HPO4 2 I 3 O mM 1.2 mM 0.6 mM 0.3 mM
Collagen ~1.5g/L ~1.5g/L ~1.5g/L ~1 5g/L
[0040] Fifty milliliters of collagen containing aqueous system was placed in a sealed 100 ml bottle and allowed to form an apatite/collagen composite. The composite formation process is carried out at 4O0C. After 4 hours, the collagen started to form hydro gel-like material. Five ml of glutaraldehyde is then added to further cross-link the collagen. After one-hour for the crosslinking, the apatite/collagen hydrogel is collected and rinsed with 50 ml deionized water four times using centrifuge (7000-12000 rpm). The apatite/collagen composite is then transferred into a cylinder mold and mixed with water. The porosity of the scaffold can be controlled by the amount of water added at this point in the process. The more water is present, the more porous the scaffold becomes. After that, the mixture was freeze-dried to obtain a porous apatite/collagen scaffold.
[0041] The resulting composite was then characterized by thermo gravimetric analysis (TGA). The analysis revealed that the collagen content of the resulting composite was prepared in a controlled manner (20%-90wt%) (Table 4).
Table 4 Collagen content in apatite/collagen composite materials (calculated using TGA)
Specimen Sample 1 Sample 2 Sample 3 Sample 4
Collagen content
22 36 70 87 (wt%)
Example 2. In vitro cell culture test
[0042] An apatite/collagen composite scaffold was prepared by extracting Type I collagen from rat tails and dissolved in 0.5 M acetic acid. The components used to make the aqueous system, including NaCl, CaCl2, K2HPO4, MgCl2, NaHCO3 and HEPES, were added to the collagen solution (approximately 1.5 g/L) to prepare collagen containing aqueous system. The concentrations of these components are listed in Table 5. The initial pH of the solution was adjusted to 7.0 at 4O0C using dilute HCl or NaOH. The solution was aged for 4 hours to allow co-precipitation of apatite nanoparticles and collagen fibers in the solution. After aging, the precipitates were collected and freeze-dried to form a scaffold. The scaffold is crosslmked using 2 w/v% l-ethyl-3-(3-dimethylarninopropyi) carbodiimide hydrochloride (EDC) at 40C for 24 hours, then rinsed and freeze-dried to attain the final scaffold.
Table 5
[0043] The scaffold was cut into 5 millimeter (mm) discs, and seeded with MC3T3 cells. The cell seeding density was 1.6x105 cells/scaffold. After five days of incubation in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal calf serum (FCS), penicillin (100 units/ml), streptomycin (100 μg/ml), and non-essential amino acids (100 μM), the cell seeded scaffolds were harvested, embedded, stained with hematoxylin, and frozen- sectioned at a thickness of 10 μm. The stained samples were then observed under light microscope. Microscopic examination revealed many cells have penetrated into the scaffold, suggesting that the scaffold supports cell attachment.
Example 3. In vivo test
[0044] Five day old mouse calvaria digest cells, a rich source of osteogenic progenitor cells, were harvested from a litter derived from a homozygous Col3.6GFP father and a non-transgenic mother. All the off spring carries one copy of the Col3.6GFP transgene, which is inactive at the time the cells are harvested. The cells were loaded onto the surface of an apatite/collagen composite scaffold at a density of 1.0 x 10 cells/scaffold. The apatite/collagen composite scaffold was prepared similarly to Example 2, except the collagen concentration was approximately 1.0 g/L. The scaffold was punched into 3.5 mm diameter discs with a thickness of approximately 1 mm. A mouse calvaria model was used with two 3.5 mm defects created at each side of the suture line at the calvaria site. One positive control and one apatite/collagen scaffold were implanted. The implantation period was 28 days. After harvest, the implants were embedded and frozen-sectioned. Adjacent images were obtained from a Zeiss Axiovert and AxioObserver work station and tiled together to reproduce a full-size image of the bone section. It was found that the apatite/collagen composite scaffold supports bone formation. The new bone formation was mainly contributed by donor cells.
[0045] The terms "a" and "an" herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. The suffix "(s)" as used herein is intended to include both the singular and the plural of the term that it modifies, thereby including one or more of that term (e.g., the metal(s) includes one or more metals). Ranges disclosed herein are inclusive and independently combinable (e.g., ranges of "up to about 25 wt%, or, more specifically, about 5 wt% to about 20 wt %", is inclusive of the endpoints and all intermediate values of the ranges of "about 5 wt% to about 25 wt%," etc).
[0046] While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims
1. A method of forming a composite scaffold, comprising:
forming an aqueous scaffold system comprising a structural protein, a weak acid, water, Ca2+, HPO4 2" , a buffer system, and optionally one or more OfMg2+, Na+, K+, CY, SO4 2"; or HCO3 "; wherein the aqueous scaffold system has an initial pH of about 6.5 to about 8.0;
placing the aqueous scaffold system in container;
sealing the container;
allowing a gel to form;
isolating the gel; and
freeze-drying the gel to form a composite scaffold.
2. The method of claim 1, wherein the structural protein is collagen Type I, II, III, or V.
3. The method of claim 2, wherein the collagen content in the composite scaffold is about 10 to about 90 weight percent based on the total weight of the composite scaffold.
4. The method of claim 1, further comprising molding the gel prior to freeze- drying.
5. The method of claim 1 , wherein
the structural protein is collagen Type I present in an amount of about 0.1 g/L to about 5.0g/L of the aqueous scaffold system;
Ca2+ is present in an amount of about 0.1 to about 15.0 mM;
Mg2+ is present in an amount of about 0.05 to about 5.0 mM;
Na+ is present in an amount of about 5.0 to about 300.0 mM;
K+ is present in an amount of about 0.1 to about 10.0 mM;
Cl" is present in an amount of about 5.0 to about 300.0 mM; SO4 2" is present in an amount of about 0 to about 2.0 mM;
HPO4 2" is present in an amount of about 0.05 to about 10.0 mM; and
HCO3 " is present in an amount of about 5 about 0.5 to about 30.0 mM.
6. The method of claim 1, wherein the weak acid has a pKa of about 3.5 to about 5.5.
7. The method of claim 1, further comprising adding a crosslinking agent to crosslink the gel prior to isolating.
8. A composite scaffold prepared by the process of claim 1.
9. An implantable medical device, comprising:
a composite scaffold prepared by the process comprising
forming an aqueous scaffold system comprising a structural protein, a weak acid, water, Ca2+, HPO4 2" , a buffer system, and optionally one or more OfMg2+, Na+, K+, Cl", SO4 2"; or HCO3 "; wherein the aqueous scaffold system has an initial pH of about 6.5 to about 8.0;
placing the aqueous scaffold system in container;
sealing the container;
allowing a gel to form;
isolating the gel; and
freeze-drying the gel to form a composite scaffold.
10. The implantable medical device of claim 9, wherein the structural protein is collagen Type I, II, III, or V.
11. The implantable medical device of claim 10, wherein the collagen content in the composite scaffold is about 10 to about 90 weight percent based on the total weight of the composite scaffold.
12. The implantable medical device of claim 10, wherein the collagen content in the composite scaffold is about 25 to about 80 weight percent based on the total weight of the composite scaffold.
13. The implantable medical device of claim 10, wherein the collagen content in the composite scaffold is about 40 to about 65 weight percent based on the total weight of the composite scaffold.
14. The implantable medical device of claim 9, further comprising molding the gel prior to freeze-drying.
15. The implantable medical device of claim 9, wherein
the structural protein is collagen Type I present in an amount of about 0.1 g/L to about 5.0g/L of the aqueous scaffold system;
Ca2+ is present in an amount of about 0.1 to about 15.0 mM;
Mg2+ is present in an amount of about 0.05 to about 5.0 mM;
Na+ is present in an amount of about 5.0 to about 300.0 mM;
K+ is present in an amount of about 0.1 to about 10.0 mM;
CF is present in an amount of about 5.0 to about 300.0 mM;
SO4 2" is present in an amount of about 0 to about 2.0 mM;
HPO4 2" is present in an amount of about 0.05 to about 10.0 niM; and
HCO3 " is present in an amount of about 5 about 0.5 to about 30.0 mM.
16. The implantable medical device of claim 9, wherein the weak acid has a pKa of about 3.5 to about 5.5.
17. The implantable medical device of claim 9, further comprising adding a crosslinking agent to crosslink the gel prior to isolating.
18. The implantable medical device of claim 17, wherein the crosslinking agent is a carbodiimide, glutaraldehyde, or a combination thereof.
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WO2013178852A1 (en) * | 2012-06-01 | 2013-12-05 | Universitat Politècnica De València | Polymer/ceramic hybrid material |
US9078832B2 (en) | 2012-03-22 | 2015-07-14 | The University Of Connecticut | Biomimetic scaffold for bone regeneration |
CN108159499A (en) * | 2017-12-06 | 2018-06-15 | 广西医科大学 | A kind of plural gel and its preparation method and application |
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US20020018797A1 (en) * | 2000-05-19 | 2002-02-14 | Fuzhai Cui | Nano-calcium phosphates/collagen based bone substitute materials |
EP1566186A1 (en) * | 2002-11-06 | 2005-08-24 | National Institute for Materials Science | Apatite/collagen crosslinked porous material containing self-organized apatite/collagen composite and process for producing the same |
US20060204491A1 (en) * | 2004-03-29 | 2006-09-14 | Tadashi Kokubo | Titanium oxide-organic polymer conjuction suitable for artificial bone |
-
2008
- 2008-11-06 CA CA2704673A patent/CA2704673A1/en not_active Abandoned
- 2008-11-06 EP EP08846420A patent/EP2211923A2/en not_active Withdrawn
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US20020018797A1 (en) * | 2000-05-19 | 2002-02-14 | Fuzhai Cui | Nano-calcium phosphates/collagen based bone substitute materials |
EP1566186A1 (en) * | 2002-11-06 | 2005-08-24 | National Institute for Materials Science | Apatite/collagen crosslinked porous material containing self-organized apatite/collagen composite and process for producing the same |
US20060204491A1 (en) * | 2004-03-29 | 2006-09-14 | Tadashi Kokubo | Titanium oxide-organic polymer conjuction suitable for artificial bone |
Non-Patent Citations (1)
Title |
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HAIBO QU, MEI WEI: "the effect of initial pH on morphology of biomimetic apatite coating" KEY ENGINEERING MATERIALS, vol. 330-332, 1 February 2007 (2007-02-01) , pages 757-760, XP008123095 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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US9078832B2 (en) | 2012-03-22 | 2015-07-14 | The University Of Connecticut | Biomimetic scaffold for bone regeneration |
WO2013178852A1 (en) * | 2012-06-01 | 2013-12-05 | Universitat Politècnica De València | Polymer/ceramic hybrid material |
ES2437183A1 (en) * | 2012-06-01 | 2014-01-09 | Universidad Politécnica De Valencia | Polymer/ceramic hybrid material |
CN108159499A (en) * | 2017-12-06 | 2018-06-15 | 广西医科大学 | A kind of plural gel and its preparation method and application |
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CA2704673A1 (en) | 2009-05-14 |
WO2009061908A3 (en) | 2010-08-05 |
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