Photocrosslinkable poly(caprolactone fumarate)

Abstract

Fumaric acid or a salt thereof, such as a fumaryl halide (e.g. fumaryl chloride), which contains unsaturated carbon-carbon double bonds that can be used for in situ crosslinking, is copolymerized with poly(caprolactone) diol in the presence of an alkali metal salt. The resulting photocrosslinkable biocompatible and bioresorbable poly(caprolactone fumarate) biomaterial is useful in the fabrication of injectable an in-situ hardening scaffolds for application in skeletal reconstruction.

Claims

1. A photocrosslinkable, biodegradable composition comprising: poly(caprolactone fumarate); and a photoinitiator, wherein the poly(caprolactone fumarate) is colorless or light colored, and clear in the melt state.

2. The composition of claim 1, wherein the composition is injectable.

3. The composition of claim 1, wherein the composition is an injectable bone substitute.

4. The composition of claim 1, wherein, the composition is an injectable bone cement.

5. The composition of claim 1, further comprising a porogen.

6. The composition of claim 1, further comprising particulate or fiber reinforcement materials.

7. The composition of claim 1, wherein the composition does not include a crosslinker.

8. The composition of claim 1, wherein the photoinitiator is selected from the group consisting of benzoin and benzoin ether compounds, benzil ketal compounds, acetophenone compounds, aminoalkylphenone compounds, hydroxyalkylphenone compounds, acylphosphine oxides, acylphosphine sulfides, phenylglyoxylate compounds, benzophenone compounds, thioxanthone compounds, and mixtures thereof.

9. The composition of claim 1, wherein the poly(caprolactone fumarate) is prepared by reacting (i) a poly(-caprolactone) diol, and (ii) fumaric acid or a salt thereof.

10. The composition of claim 1, wherein the poly(caprolactone fumarate) is prepared by reacting (i) a poly(-caprolactone) diol, and (ii) fumaric acid or a salt thereof in the presence of an alkali metal salt.

11. The composition of claim 1, wherein the poly(caprolactone fumarate) is prepared by reacting (i) a poly(-caprolactone) diol, and (ii) fumaric acid or a salt thereof in the presence of an alkali metal carbonate.

12. The composition of claim 1, wherein the poly(caprolactone fumarate) is prepared by reacting (i) a poly(-caprolactone) diol, and (ii) fumaric acid or a salt thereof in the presence of potassium carbonate.

13. The composition of claim 1, wherein the poly(caprolactone fumarate) is prepared by reacting (i) a poly(-caprolactone) diol, and (ii) a fumaryl halide.

14. The composition of claim 1, wherein the poly(caprolactone fumarate) is prepared by reacting (i) a poly(-caprolactone) diol, and (ii) fumaryl chloride.

15. The composition of claim 1, further comprising a bioactive agent selected from enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, extracellular matrix and/or its individual components, pharmaceuticals, therapeutics and mixtures thereof.

16. The composition of claim 1, wherein the copolymer was prepared by reacting (i) a poly(-caprolactone)diol and (ii) fumaric acid or a salt thereof in the presence of potassium carbonate to form a repeating polymer of polycaprolactone fumarate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a scheme for the synthesis of PCLF and a self-crosslinking process for PCLF.

(2) FIG. 2 shows FTIR spectra of PCL530 and three PCLF macromers. FTIR means Fourier Transform Infrared Spectroscopy that is used to determine the chemical structure of the polymers. As marked in FIG. 2, the absorption peaks can be assigned to the chemical structure of the polymers herein.

(3) FIG. 3 shows .sup.1H NMR (400.1 MHz, CDCl.sub.3, reference TMS) spectra of PCL530 and PCLF530 where S=solvent and asterisks indicate signals due to methylene chloride. NMR means Nuclear Magnetic Resonance Spectroscopy which is used to determine the chemical structure of the polymers herein. All chemical shifts can be well assigned to different protons in the polymer backbone.

(4) FIG. 4 shows .sup.13C NMR (100.6 MHz, CDCl.sub.3, reference TMS) spectra of PCL530 and PCLF530 where S=solvent.

(5) FIG. 5 shows DSC curves of PCLF macromers and their PCL precursors. DSC means Differential Scanning Calorimetry which is used to determine melting temperature T.sub.m, glass transition temperature T.sub.g, and heat of fusion H.sub.m for polymers. The crystallinity .sub.c of PCL in the copolymers can be calculated by the H.sub.m values of copolymers and completely crystalline PCL (H.sub.m.sup.c=135 J/g) and the composition of PCL using the equation of .sub.c=[H.sub.m/(H.sub.m.sup.c)]100%. See Brandrup, J.; Immergut, E. H. Eds. Polymer Handbook, 3rd ed.; Wiley: New York, 1989.

(6) FIG. 6 shows polarized optical microscopic graphs of PCLF macromers (a, b, c: PCLF530, 1250, and 2000) and the corresponding PCL diols (d, e, f: PCL530, 1250, and 2000). (Magnification: 200, scale bar in (a): 100 m is applicable for all these six graphs).

(7) FIG. 7 shows TGA thermograms of PCLF macromers and their PCL precursors. TGA is thermogravimetric analysis which is used to determine the weight loss and the thermal degradation temperature T.sub.d when the temperature increases. It can be seen in FIG. 7 that the thermal stability of the PCLF530 is lower than those of the other two PCLF macromers; nevertheless, all the PCLF macromers show higher thermostability than their PCL precursors because of higher molecular weights.

(8) FIG. 8 shows typical force-deformation curves of a crosslinked PCLF530 multi-channel tube and a cylinder determined using DMA (Dynamic Mechanical Analyzer.

(9) FIG. 9 shows a porous structure of one PCLF scaffold made using a 3D printing method.

DETAILED DESCRIPTION OF THE INVENTION

(10) The invention provides a copolymer including caprolactone units and fumarate units wherein the copolymer is colorless or light-colored poly(caprolactone fumarate) so that the copolymer is self-crosslinkable by both redox initiation and photoinitiation. Preferably, the copolymer is clear when it is in solution or melt state. In one form, the copolymer has a number average molecular weight of 4000 daltons or greater.

(11) A copolymer according to the invention can be prepared by reacting a poly(caprolactone) diol, and fumaric acid or a salt (preferably, fumaryl chloride) thereof in the presence of an alkali metal salt. Preferably, the alkali metal salt is an alkali metal carbonate, and most preferably, the alkali metal salt is potassium carbonate.

(12) The invention also provides a photocrosslinkable, biodegradable material including a copolymer including caprolactone units and fumarate units according to the invention, and a photoinitiator. Example photoinitiators include benzoin and benzoin ether compounds, benzil ketal compounds, acetophenone compounds, aminoalkylphenone compounds, hydroxyalkylphenone compounds, acylphosphine oxides, acylphosphine sulfides, phenylglyoxylate compounds, benzophenone compounds, thioxanthone compounds, and mixtures thereof. In one example material, the photoinitiator is bisacylphosphinoxide.

(13) The material may be an injectable bone substitute or an injectable bone cement. The injectable nature of the material allows for the filling of defects of limited accessibility or irregular shape. For example, minimally invasive endoscopic techniques now in clinical use may allow the injectable form of the material to be inserted for posterolateral intertransverse process spinal fusion. The injectable material could be placed into cancellous voids from periarticular fractures, osteoporotic spinal fractures, or bone cysts without creating a large access hole in the surrounding cortical bone.

(14) With respect to the injectable nature of a copolymer according to the invention, the temperature range of injection can be broad, between the melting point of the mixture and the boiling point of the solvent used in the mixture. Normally the polymer mixture is injected at room temperature for convenience. For PPF, one component in the copolymer, the highest temperature during the crosslinking would be around 48 C., while polymethylmethacrylate, the currently used bone cement, may cause as high as 100 C. during crosslinking. Thus, PPF has advantages over polymethylmethacrylate. For the copolymers according to the invention, the temperature would be even lower than 48 C. because the content of fumarate group the only crosslinkable segment in copolymers, is lower than 10%.

(15) Because the biodegradable material according to the invention is self-crosslinking, the material does not need to include a crosslinker. A crosslinker is typically used to help bridge the neighboring double bonds in crosslinking. Because the self-crosslinkable and/or photocrosslinkable, biodegradable material according to the invention does not need any crosslinkers, toxicity concerns in biomedical applications are minimized; however, a crosslinker can be used.

(16) The material according to the invention is suitable for forming a scaffold for tissue regeneration. In one form, the material includes a porogen to allow for the formation of a porous scaffold. Suitable porogens include salt crystals (e.g., sodium chloride) that may be used in a salt leaching technique that forms a porous scaffold. Examples of this type of particle leaching technique can be found in U.S. Pat. Nos. 6,436,426, 6,379,962 and 5,514,378. The porogen may also be a hydrogel porogen as described in PCT International Publication No. WO 2005/020849. The choice of porogen may be dictated by the crosslinking process. Porogens can be used in making a photo-crosslinked film; however, it depends the physical properties and color of the porogen. Also, some porogens may block the UV light thereby make the photocrosslinking procedure inefficient. Thus, the photocrosslinkable, biodegradable material according to the invention may or may not include a porogen depending on the final product desired.

(17) The material may further include particulate or fiber reinforcement materials. Hydroxyapatite is especially advantageous to serve as a reinforcement material because of its similarity in composition to bone mineral, bioactivity and promotion of cellular function, and osteoconductivity. The reinforcement materials may also include single-wall carbon nanotube.

(18) The material may further include one or more bioactive agents. A bioactive agent as used herein includes, without limitation, physiologically or pharmacologically active substances that act locally or systemically in the body. A bioactive agent is a substance used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness, or a substance which affects the structure or function of the body or which becomes biologically active or more active after it has been placed in a predetermined physiological environment. Bioactive agents include, without limitation, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, extracellular matrix and/or its individual components, pharmaceuticals, and therapeutics.

(19) The self-crosslinkable and/or photocrosslinkable, biodegradable material may also include an accelerator. Non-limiting example accelerators include toluidines (such as N,N-diethyl-p-toluidine (DET) and N,N-dimethyl-o-toluidine (DMT)), acetyl phenylhydrazine, maleic acid, quinines (such as napthaquinone and anthraquinone), and alkyl mercaptans. Often, in a photocrosslinking process, an accelerator is not needed because the whole procedure is rather short (e.g., less than 30 minutes).

(20) In another aspect, the invention provides a biocompatible scaffold for tissue regeneration. The scaffold includes a biodegradable matrix including a copolymer including caprolactone units and fumarate units wherein the copolymer is prepared by reacting a poly(caprolactone) diol and fumaric acid or a salt (preferably, fumaryl chloride) thereof in the presence of an alkali metal salt. The copolymer may have a number average molecular weight of 4000 daltons or greater. Preferably, the copolymer is clear in solution or melt state. The matrix may include particulate or fiber reinforcement materials such as hydroxyapatite. The matrix may include one or more bioactive agents. The scaffold may be porous.

(21) In an example embodiment of this invention, we have made colorless or light-colored poly(caprolactone fumarate) using potassium carbonate as the proton scavenger. Furthermore, we have modified prior PCLF synthesis processes to make the time consumption much shorter and the molecular weights of the final products higher. The newly synthesized PCLF has been tested to show it is self-crosslinkable by both redox initiation and photoinitiation. It is potentially useful as an injectable material in various research and clinical applications. In addition, the dark color in the previously developed PCLF interferes with various cell and tissue staining techniques. We expect the new colorless PCLF will allow much greater visibility of cells and tissues for in vitro and in vivo assays.

(22) The scaffold may be formed from a copolymer according to the invention using various techniques. For example, a block copolymer of poly(propylene fumarate) and poly(-caprolactone) according to the invention may be extruded, injection molded or compression molded into a scaffold. Alternatively, solid free-form fabrication methods may also be used to form the scaffold from a copolymer according to the invention. Non-limiting examples of solid free-form fabrication methods include stereo-lithography, selective laser sintering, ballistic particle manufacturing, fusion deposition modeling; and three dimensional printing. The macrostructure and porosity of the scaffold can be manipulated by controlling printing parameters, and these features can be designed and tailored using computer assisted design (CAD) for individual patients. U.S. Pat. Nos. 6,530,958, 5,869,170, 5,518,680 and 5,490,962 provide examples of solid free-form fabrication methods. See also, Hutmacher et al., Scaffold-based tissue engineering: rationale for computer-aided design and solid free-form fabrication systems, Trends in Biotech. 2004, 22(7):354. These patents and publications and all other patents and publications cited herein are incorporated herein by reference.

(23) The applications of the material extend beyond scaffolds and bone cement. The self-crosslinkable and/or photocrosslinkable, biodegradable material including a copolymer according to the invention is suitable as a crosslinkable polymer in many biomedical applications. Since it is crosslinkable, a micropatterned surface can be made using this material. The material can also form a polymer network with controlled swelling ratios in a variety of solvents which make the material a sorbent for organic solvents or a carrier for catalysts.

(24) As used herein, a biocompatible material is one which stimulates only a mild, often transient, implantation response, as opposed to a severe or escalating response. As used herein, a biodegradable material is one which decomposes under normal in vivo physiological conditions into components which can be metabolized or excreted. As used herein, a bioresorbable material is one that breaks down over a finite period of time due to the chemical/biological action of the body. By injectable, we mean the copolymer may be delivered to a site by way of a medical syringe. By self-crosslinkable, we mean the functional groups of a polymer according to the invention may crosslink with the functional groups of the same polymer or another polymer according to the invention without a crosslinker that forms crosslinks between the functional groups of a polymer according to the invention and the functional groups of the same or another polymer according to the invention. By photocrosslinkable, we mean the functional groups of a copolymer according to the invention may crosslink with the functional groups of the same polymer or another copolymer according to the invention by application of photons (e.g., UV light) in the presence of a photoinitiator.

(25) The term molecular weight in this specification refers to weight average molecular weight (M.sub.w=.sub.i N.sub.iM.sub.i.sup.2/.sub.iN.sub.iM.sub.i). Although weight average molecular weight (M.sub.w) can be determined in a variety of ways, with some differences in result depending upon the method employed, it is convenient to employ gel permeation chromatography. As used herein, the term number average molecular weight (M.sub.n) refers to the total weight of all the molecules in a polymer sample divided by the total number of moles present (M.sub.n=.sub.iN.sub.iM.sub.i/.sub.iN.sub.i). Although number average molecular weight can be determined in a variety of ways, with some differences in result depending upon the method employed, it is convenient to employ gel permeation chromatography. As used herein, the term polydispersity (DPI in Table 1) refers to the ratio of a materials' weight average molecular weight divided by its number average molecular weight (M.sub.w/M.sub.n).

EXAMPLES

(26) The following examples have been presented in order to further illustrate the invention and are not intended to limit the invention in any way.

Example 1

Synthesis of Poly(Caprolactone Fumarate) Macromer

(27) A general synthesis scheme for poly(caprolactone fumarate) is shown in FIG. 1. PCL diols [,-dihydroxy poly(-caprolactone)] with nominal molecular weights of 530, 1250, and 2000 g.Math.mol.sup.1 were purchased from Aldrich Co. (Milwaukee, Wis.) and had the chemical structure as H[O(CH.sub.2).sub.5CO].sub.mOCH.sub.2CH.sub.2OCH.sub.2CH.sub.2O[OC(CH.sub.2).sub.5O].sub.nH. Prior to copolymerization, a certain amount of PCL diol was dried overnight in a vacuum oven at 50 C. All the other chemicals in the invention were also purchased from Aldrich Co. Methylene chloride was dried and distilled over calcium hydride before the reaction. Fumaryl chloride was purified by distillation at 161 C. Ground potassium carbonate was dried at 100 C. for 2 days and then cooled down at vacuum condition.

(28) Fumaryl chloride, poly(-caprolactone) diol, and potassium carbonate were measured out in a 0.95:1:1.2 molar ratio. The PCL diol was dissolved in methylene chloride (1:2 by volume) and placed in a 1 L three-neck flask along with the powdered potassium carbonate. This mixture was stirred with an overhead mechanical stirrer to form a slurry. Fumaryl chloride dissolved methylene chloride (1:1 volume ratio) was added dropwise to the slurry. The reaction mixture was maintained at 50 C. under reflux using a condenser. After reacting for 12 hours, the mixture was cooled down and transferred to centrifuge tubes and spun down for 15 minutes at 4000 rpm until the potassium carbonate was completely removed. The supernatant was then added dropwise to petroleum ether to force the polymer out of solution, and the precipitate was rotary-evaporated to yield a wax-like product.

Characterizations

(29) Gel Permeation Chromatography (GPC) was used to determine the molecular weight and polydispersity of the polymers herein. The GPC was carried out with a Waters 717 Plus autosampler GPC system (Waters, Milford, Mass., USA) connected to a model 515 HPLC pump and model 2410 refractive index detector. Fourier Transform Infrared Spectroscopy (FTIR) spectra were obtained on a Nicolet 550 spectrometer. All the polymers were analyzed using a zinc selenide ATR crystal. The resolution of the instrument was specified as 4 cm.sup.1 at a wavenumber of 1000 cm.sup.1. Proton and carbon Nuclear Magnetic Resonance (NMR) spectra were acquired on Varian Mercury Plus NMR spectrometer (.sup.1H=400.1 MHz, .sup.13C=100.6 MHz) using CDCl.sub.3 solutions containing TMS. Differential Scanning Calorimetry (DSC) was measured on a TA Instruments DSC Q1000 differential scanning calorimeter at a heating rate of 10 C./min in a nitrogen atmosphere. To keep the same thermal history, each sample was preheated from room temperature to 100 C. and cooled to 90 C. at a cooling rate of 5 C./min. Then the DSC scan was recorded via heating from 90 C. to 100 C. In order to support the DSC results, the amorphous structure of the copolymers and the crystalline form of three PCL samples were observed using a Zeiss Axioskop Polarizing Optical Microscope (POM). Thermogravimetric Analysis (TGA) was done using a TA model Q500 thermal analyst. The TGA data were obtained in flowing nitrogen at a heating rate of 20 C./min. Intrinsic viscosities [] of the polymers were measured in toluene at 30.00.05 C. with a calibrated Cannon Ubbehlobe capillary viscometer (Model OC, Cannon Instrument Co.) in water bath equipped with a Lauda ECO-Line Immersion Circulator (Brinkmann Co.). Toluene was distilled from CaH.sub.2 before being used as the solvent.

Crosslinking Process and Scaffold Fabrication

(30) A general crosslinking diagram is shown in FIG. 1.

(31) Thermal-crosslinking process: Benzoyl peroxide (BPO) and N-dimethyl toluidine (DMT) were used as the free radical initiator and accelerator, respectively. A typical procedure for fabrication of scaffolds was as follows. One hundred microliters of initiator solution (50 mg of BPO in 250 microliters of 1-vinyl-2-pyrrolidinone (NVP)) and 40 microliters of accelerator solution (20 microliters of DMT in 980 microliters of methylene chloride) were added in 1.5 g PCLF solution in 500 microliters of methylene chloride and mixed thoroughly. The polymerizing scaffold was transferred into various Teflon molds, such as multi-channel tube mode. The mold was placed in a convection oven for overnight to facilitate crosslinking. After crosslinking, cylinders or tubes were removed from the mold before the mold was cooled to ambient temperature.

(32) Photocrosslinking process: Photocrosslinking was initiated with ultraviolet (UV) (=380315 nm) using a photoinitiator bisacylphosphinoxide (BAPO, Ciba Geigy). 75 L of BAPO solution (30 mg BAPO in 150 L methylene chloride) was added into 1.5 g PCLF solution in 500 microliters of methylene chloride and mixed thoroughly. The mixture was poured to a Petri dish and the Petri dish was placed directly under UV light for 30 minutes to facilitate crosslinking.

(33) Scaffold fabrication: Similar crosslinking processes can be done to the mixture of PCLF and porogen (salt with various size distributions) to make scaffolds with different porosity, which can be controlled by the content of porogen. After crosslinking, salt was leached out by place the scaffolds in distilled water for 3 days, during which time water changes frequently. The scaffolds were dried in vacuum for at least 12 hours.

(34) Characterizations of materials used and copolymers produced in the Examples are shown in FIGS. 2-9.

(35) Table 1 below shows molecular characteristics and physical properties of PCL precursors, PCLF macromers, and their crosslinked products.

(36) TABLE-US-00001 TABLE 1 % PCL (Wt. %) Thermal Properties ( C.) M.sub.w M.sub.n Feed [] T.sub.g T.sub.m H.sub.m X.sub.c T.sub.d Polymer (Dalton) (Dalton) DPI Ratio NMR (dL .Math. g.sup.1) ( C.) ( C.) (J/g) (%) ( C.) PCL530 1270 770 1.65 100 100 0.04 80.6 26.2 52.2 38.6 354 PCL1250 3030 1710 1.77 100 100 0.07 73.5 43.4 61.1 45.3 386 PCL2000 5320 3970 1.34 100 100 0.11 68.5 48.7 76.7 56.8 392 PCLF530a 6050 3520 1.72 91.4 89.5 59.1 29.2 46.2 37.4 387 PCLF530b 6120 3450 1.77 91.0 89.3 61.0 29.2 46.7 38.0 392 PCLF530c 6100 3420 1.78 91.0 89.0 0.10 57.6 29.5 45.4 37.0 390 PCLF1250 15800 9000 1.76 95.7 94.3 0.19 62.7 43.9 61.0 47.2 399 PCLF2000 12900 7300 1.76 98.1 96.7 0.16 62.7 45.7 67.0 50.6 399 Crosslinked 91.0 54.5 27.5 1.27 0.01 PCLF530 Crosslinked 95.7 58.5 35.7 26.8 20.7 PCLF1250 Crosslinked 98.1 58.7 43.1 47.1 35.6 PCLF2000

(37) Thus, the invention provides photocrosslinkable, biodegradable poly(caprolactone fumarate) that in one application can be injected and then hardened in situ to form scaffolds for tissue and/or skeletal reconstruction.

INDUSTRIAL APPLICABILITY

(38) The present invention relates to photocrosslinkable, biodegradable polymeric materials for forming scaffolds for tissue and/or skeletal reconstruction.

(39) Although the present invention has been described in considerable detail with reference to certain embodiments, one skilled in the art will appreciate that the present invention can be practiced by other than the described embodiments, which have been presented for purposes of illustration and not of limitation. Therefore, the scope of the appended claims should not be limited to the description of the embodiments contained herein.