A high internal phase emulsion composition for use as a bone graft and the method for preparing the same

Abstract

The invention relates to an injectable (not requiring pre-molding), biodegradable, and porous high internal phase emulsion composition prepared using solvent-free, photo- crosslinkable, low molecular weight, three-arm polycaprolactone methacrylate-based high internal phase emulsions (3PCLMA HIPE) for use as a bone graft, and to the method for preparing the same. The composition according to the invention is produced using the emulsion templating technique.

Description

[0001] DESCRIPTION A HIGH INTERNAL PHASE EMULSION COMPOSITION FOR USE AS A BONE GRAFT AND THE METHOD FOR PREPARING THE SAME Technical Field of the Invention The invention relates to an injectable (requiring no pre-molding), biodegradable, and porous emulsion composition prepared using solvent-free, photo-crosslinkable, low molecular weight, three-arm polycaprolactone methacrylate-based high internal phase emulsions (3PCLMA (3-arm polycaprolactone methacrylate) HIPE) for use as a bone graft, and to the method for preparing the same. The composition according to the invention is produced by using the emulsion templating technique. State of the Art Bone graft transplantation is the second most commonly used tissue transplantation technique worldwide, following blood transfusion. In this application, autogenic and allogenic bone grafts are frequently used. Alternatively, in tissue engineering (TE), porous and biologically degradable matrices (TE scaffolds) derived from natural or synthetic materials, which aim to mimic the biochemical and structural characteristics of native tissues, are used for the regeneration of the damaged area. While these scaffolds provide temporary mechanical support, they fill the defect region to act as a three-dimensional substrate for cell adhesion and proliferation. These TE scaffolds must possess a highly interconnected porous architecture to allow for cell migration, nutrient flow, and integration of the material with the host tissue [1]. TE scaffolds are generally pre-formed using techniques such as particulate leaching, electrospinning, or 3D printing, and are transferred to the defect site in this form. Unlike other scaffold fabrication methods, injectable, in situ-shaped, and in situ-polymerizable grafts fill the irregularly shaped defect region without requiring pre-design, eliminate the need for costly molding techniques, maximize contact between the graft and surrounding tissue, and are applied in a minimally invasive manner, thereby reducing the risk of infection and postoperative pain. Additionally, bioactive molecules (such as drugs, growth factors, or cells depending on the material’s rheology) that support the bone healing process can be incorporated into the material composition, enabling their release at the defect site after in situ injection. Especially considering the size, morphology, and complex structure of dental and craniofacial tissues, injectable tissue scaffolds are considered advantageous over pre-formed scaffolds for the regeneration of these tissues. For example, during maxillary sinus lifting operation, it is necessary to create a window into the sinus cavity by cutting through healthy gingival and bone tissues to place the bone graft. In such cases, the use of injectable bone scaffolds minimizes surgical wound size and, consequently, the risk of infection. Both natural and synthetic polymers are used to produce biodegradable TE scaffolds. Natural polymers closely mimic the structure and composition of the native extracellular matrix; however, compared to synthetic polymers, they exhibit several disadvantages such as the risk of pathogen transmission, protein impurities that may potentially trigger immune responses, and a high degree of batch-to-batch variability [2]. Some advantages of synthetic polymers over naturally sourced polymers are that they are relatively inexpensive to produce and can be adjusted to create polymers with various mechanical and chemical properties. Polycaprolactone (PCL) is one of the most widely used synthetic, biocompatible and bioabsorbable polymers and is already used in the clinic for US Food and Drug Administration (FDA)-approved drug delivery systems and suture threads [3]. This makes PCL an attractive and promising biomaterial for use in other clinically relevant biomedical applications. PCL scaffolds are used for both hard and soft tissues such as bone, cartilage, vascular grafts, skin, and nerves [4, 5, 6]. In the state of the art, various fabrication techniques such as gas foaming, porogen leaching, and additive manufacturing are available to impart porosity to PCL-based scaffolds. However, gas foaming and porogen leaching often result in insufficient pore interconnectivity, while existing additive manufacturing techniques fall short in terms of precisely controlling (1– 50 μm) scaffold morphology due to resolution limitations [7]. Compared to these methods, emulsion templating offers an alternative approach to confer highly interconnected porosity to scaffold materials, and the process begins with the mixing of two immiscible liquids, wherein one liquid (the internal phase) is dispersed within a continuous phase (the polymer) in the presence of a surfactant that stabilizes the emulsion [8]. Emulsions are delicate systems that are directly affected by numerous parameters. When the internal phase volume fraction (φ) exceeds 74.05%, which is the maximum packing density of monodisperse spherical droplets, the emulsion is classified as a high internal phase emulsion (HIPE) [9]. When HIPEs are solidified by polymerization of the continuous phase, this structure is defined as polymerized HIPE (PolyHIPE) (Figure 1). The emulsion templating technique has advantages such as porosity up to 99%, the ability to offer high interconnectivity (open pore structure), the controllability of morphological structure and mechanical properties through control over multiple parameters, and the ability to design more complex scaffold architectures when used in conjunction with other scaffold fabrication methods. In the emulsion templating technique to achieve a high degree of control over both the stability of the resulting emulsion (HIPE) and the morphology of the resulting structure (PolyHIPE) after polymerization, the effects of each parameter in the system must be examined in detail. In the state of the art, there exist PolyHIPE-based porous materials made from biodegradable substances such as polypropylene fumarate (dimethacrylate), thiol-enes, and ethylene glycol dimethacrylate [10, 11, 12]. However, the production of PolyHIPE- based porous materials from PCL is challenging due to the high viscosity of the polymer, which limits the mixing of the two phases during emulsion formation. The formulation used in PCLMA PolyHIPE-based studies in the state of the art typically includes a prepolymer (4PCLMA, ~2000 g / mol or 20000 g / mol; 3PCLMA: ~1000 g / mol), a solvent used to reduce the viscosity of the prepolymer, a polymeric surfactant, a photoinitiator, and water (Figure 2). Here, the first four components that make up the oil phase are mixed, and then water is added as the internal phase. The resulting high internal phase emulsion is either transferred into a mold (a hydrophobic, mostly polydimethylsiloxane (PDMS) / silicone-based mold) and polymerized herein, or printed using a pneumatic 3D printer and polymerized with an LED at the nozzle tip. However, following polymerization, the scaffolds are subjected to extended (up to 1 week) sequential alcohol and water washes to eliminate the adverse effects of the diluting solvents used. However, since injectable bone grafts cannot be subjected to post- production washing processes, their composition must be free of cytotoxic solvents and materials as a basic prerequisite. In the state of art, in a study conducted by Moglia et al., a solvent-free, redox crosslinker-containing, biodegradable high internal phase emulsion composition is described for use as an injectable bone graft

[0014] . The composition includes ethylene glycol dimethacrylate (EGDMA), butanediol dimethacrylate (BDMA), propylene fumarate dimethacrylate (PFDMA), water, redox initiators such as trimethylaniline (TMA) and benzoyl peroxide (BPO), and polyglycerol polyricinoleate as the polymeric surfactant. By increasing the concentration of redox initiators in the double-barreled syringe system described in the aforementioned document, the polymerization time decreases. However, increasing the concentration not only reduces the polymerization time, but also limits the physician's manipulation time until polymerization. Once the HIPEs are injected from the syringe, the redox initiators immediately begin to interact, initiating polymerization. The operator is therefore required to shape the graft and apply it to the defect site within a short period. Additionally, according to the document, the crosslinking reaction initiated by the redox initiators is exothermic, and the temperature can rise up to 42^°C during the reaction. This temperature level may damage surrounding tissue cells during clinical applications. Due to the limitations and insufficiencies of the solutions in the state of the art, the inability of PolyHIPE compositions to be applied to bone defects as injectables because they contain cytotoxic solvents and components, and the toxicity of the photoinitiators used and their inability to be incorporated into injectable systems, a development in high internal phase emulsion compositions has become necessary. Brief Description and Objects of the Invention The invention describes an injectable (not requiring pre-molding), biodegradable, and porous emulsion composition prepared using solvent-free, photo-crosslinkable, low molecular weight, three-arm polycaprolactone methacrylate-based high internal phase emulsions (3PCLMA HIPE) for use as a bone graft, as well as a method for preparing the same. The object of the invention is to provide an emulsion composition for use as a bone graft. The composition according to the invention is capable of integrating into the defect and merging with the surrounding tissue, resulting in complete integration with the bone after polymerization. An object of the invention is to reduce the viscosity of the oil phase in emulsions. In the invention, the oil phase components of the emulsion, prepolymer and surfactant, are replaced with lower-viscosity equivalents compared to those described in the state of the art, thereby reducing the viscosity of the oil phase. As a result of this viscosity reduction, a solvent-free PCLMA (PCL-methacrylate) HIPE is obtained. An object of the invention is to provide a high internal phase emulsion that is applicable to bone defects in an injectable form, without requiring pre-molding. Since the compositions in the state of the art contain toxic components, a post-polymerization washing is required to remove these toxic components. Therefore, the HIPE must be polymerized in a mold prior to implantation in the patient, and implantation should take place after all toxic components have been removed by washing. However, in the composition of the present invention, since there are no toxic components, the HIPE is directly injected into the site without the need for pre-molding and subsequently polymerized. In this way, a graft that precisely matches the shape of the defect site is obtained, ensuring maximum contact between the tissue and the graft. Another object of the invention is to obtain a photo-crosslinkable high internal phase emulsion. In the invention, 3-arm polycaprolactone (~300 g / mol) is commercially available and methacrylated to obtain a photocrosslinkable polymer, resulting 3PCLMA pre-polymer. Description of the Figures Figure 1. Stages of PolyHIPE production. ((A, B) Addition of the aqueous phase into the oil phase during mixing, (C) polymerization of the HIPE using a light source, (D) formation of pores in the PolyHIPE after removal of the aqueous phase which acts as a template (E: oil phase before polymerization, F: aqueous phase, G: oil phase after polymerization, H: pores formed after removal of water, J: light source)). Figure 2. (A) Scanning electron microscope (SEM) image showing an open porous morphology obtained using 4PCLMA (~2000 g / mol) and a solvent; (B) SEM image of the structure obtained using the same composition without the inclusion of diluting solvents; (C) Viscosity of oil phase components when no diluting solvent is used with the same polymer (a: polymer + surfactant, b: polymer, c: polymer + surfactant + photoinitiator); (D) Viscosity of the oil phase when different volumes of chloroform are used as a diluting solvent with the same polymer. Figure 3. H-NMR spectrum of 3-arm polycaprolactone methacrylate (3PCLMA). Figure 4. (A) Viscosity measurement graph of 3PCLMA; (B) Electron microscope image of solvent-free PCLMA PolyHIPE obtained from preliminary studies. Figure 5. SEM image of the solvent-free, injectable, photo-crosslinkable bone graft produced with the composition according to the invention (composition: internal phase containing 5% (w / w) PGPR and 0.1% (w / v) LAP). Detailed Description of the Invention The invention relates to an injectable (not requiring pre-molding), biodegradable, and porous emulsion composition prepared using solvent-free, photo-crosslinkable, low molecular weight, three-arm polycaprolactone methacrylate-based high internal phase emulsions (3PCLMA HIPE) for use as a bone graft, and to the method for preparing the same. The emulsion composition according to the invention comprises a prepolymer, surfactant, crosslinker, and water. In one embodiment of the invention, the emulsion composition according to the invention comprises a prepolymer, surfactant, photoinitiator, and water. In another embodiment, the emulsion composition according to the invention comprises a prepolymer, a surfactant in an amount of 1–30% by weight of the prepolymer (optimally 5%), a photoinitiator in an amount of 0.01–10% up to 75% of total volume (optimally 0.1%), and 1–99% water (optimally 75%). The prepolymer in question is 3-arm polycaprolactone methacrylate, the surfactant is polyglycerol polyricinoleate (PGPR), and the photoinitiator is lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP). The composition has an internal phase volume of 1–99%. Here, if the internal phase volume is 30-74%, polymerized MIPE (medium internal phase emulsion) is obtained; if the internal phase volume is below 30%, polymerized LIPE (low internal phase emulsion) is obtained; and if the internal phase volume is at least 74.048%, PolyHIPE is obtained. In general, an increase in internal phase volume leads to higher porosity, which is advantageous for cell penetration and the diffusion of nutrients and waste. However, increasing porosity results in reduced mechanical strength of the material. Therefore, if materials with higher mechanical strength are desired, PolyMIPE or PolyLIPE may be preferred. The method for preparing the emulsion composition according to the invention comprises the following process steps: i. dissolving 3-arm polycaprolactone (polycaprolactone triol; 3PCL) in dichloromethane (DCM) and forming a mixture; ii. adding triethylamine (TEA) and DCM to the mixture; iii. stirring the mixture with a magnetic stirrer and placing the resulting mixture in an ice bath; iv. dissolving methacrylic anhydride (MAAn) in DCM and adding it dropwise to the system in the ice bath using a dropping funnel; v. once MAAn addition is complete, bringing the mixture to room temperature and allow it to polymerize with continuous stirring; vi. after the polymerization is complete, performing washing processes to remove residual TEA, MAAn, and salts from the resulting 3PCLMA pre-polymer; vii. removing DCM from the system using a rotary evaporator after washing processes; viii. after removing DCM, transferring the polymer into a bottle for methanol washing, filling the bottle with methanol, and shaking and storing it until a precipitate forms; ix. once the polymer precipitate forms, removing methanol in the upper phase, liquidizing the frozen polymer precipitate at room temperature, and repeating the process twice by adding fresh methanol to the bottle; x. following methanol washing, removing the remaining methanol completely using a rotary evaporator, and storing the methacrylated 3-arm polycaprolactone (3PCLMA); xi. for HIPE preparation, mixing the oil phase components, pre-polymer (3PCLMA) and surfactant (PGPR), in a glass bottle with a magnetic stirrer; xii. while stirring continues, adding the internal phase containing LAP dropwise at a rate of 1 drop per second, and stirring the mixture to obtain a stable emulsion. In one embodiment of the invention, the method for preparing the emulsion composition according to the invention comprises the following process steps: i. dissolving 3-arm polycaprolactone (~300 g / mol) in 0.133 mol polycaprolactone triol and 50–500 mL dichloromethane (DCM) and forming a mixture; ii. adding 0.1-1 mol triethylamine (TEA) and 50-500 mL DCM to the resulting mixture; iii. stirring the mixture with a magnetic stirrer and placing it in an ice bath; iv. dissolving 0.1-1 mol methacrylic anhydride (MAAn) in 10-150 mL DCM and adding it to the system in an ice bath dropwise using a dropping funnel; v. after MAAn addition is completed, bringing the mixture to room temperature and allowing it to polymerize by stirring for 24-150 hours at 100-1000 rpm; vi. after the polymerization is complete, performing washing processes to remove residual TEA, MAAn, and salts from the 3PCLMA pre-polymer; vii. removing DCM from the system using a rotary evaporator after washing operations; viii. after removing DCM, transferring the polymer into a bottle for methanol washing, then filling the bottle with methanol, and shaking and storing it at -80°C until a precipitate forms; ix. once the polymer precipitate forms, removing methanol in the upper phase, liquidizing the frozen polymer precipitate at room temperature, and repeating the process twice more with adding fresh methanol to the bottle; x. following methanol washing, removing the remaining methanol completely using a rotary evaporator, and storing the methacrylated 3-arm polycaprolactone (3PCLMA) at -20^°C; xi. for HIPE preparation, mixing the oil phase components, pre-polymer (3PCLMA) and surfactant 1-30% by weight of the pre-polymer (PGPR), in a glass bottle using a magnetic stirrer at 100-1000 rpm for 1-10 minutes; xii. while stirring continues, adding the internal phase containing 0.01-10% (w / v) LAP dropwise at a rate of 1 drop per second, and stirring the mixture for an additional 1-10 minutes to obtain a stable emulsion. In another embodiment, the method for preparing the emulsion composition according to the invention comprises the following process steps (Figure 5): i. dissolving 3-arm polycaprolactone (~300 g / mol) in 0.133 mol 3PCL and 100 mL of dichloromethane (DCM) and forming a mixture, ii. adding 0.8 mol of triethylamine (TEA) and 150 mL of DCM to the resulting mixture, iii. stirring the mixture with a magnetic stirrer and placing the resulting mixture in an ice bath, iv. dissolving 0.8 mol of methacrylic anhydride (MAAn) in 150 mL of DCM, then adding it to the system in the ice bath dropwise using a dropping funnel, v. once the addition of MAAn is completed, bringing the mixture to room temperature and allowing it to polymerize by stirring continously at 380 rpm for 68 hours, vi. after the polymerization is completed, performing washing processes to remove residual TEA, MAAn, and salts from the 3PCLMA pre-polymer, vii. removing DCM from the system using a rotary evaporator after washing process, viii. after removing DCM, transferring the polymer into a bottle for methanol washing, then filling the bottle with methanol, and shaking and storing it at -80°C until a precipitate forms, ix. once the polymer precipitate forms, removing methanol in the upper phase, liquidizing the frozen polymer precipitate at room temperature, and repeating the process twice by adding fresh methanol to the bottle, x. following methanol washing, removing the remaining methanol completely using a rotary evaporator, and storing the methacrylated 3-arm polycaprolactone (3PCLMA) at −20°C, xi. for HIPE preparation, mixing the oil phase components, 0.4 g of the pre-polymer (3PCLMA) and surfactant 5% by weight of the pre-polymer (0.02 g) (PGPR), in a glass bottle using a magnetic stirrer at 380 rpm for 2 minutes, xii. while stirring continues, adding 1.1 mL of internal phase containing 0.1% (w / v) LAP dropwise at a rate of 1 drop per second and stirring the mixture for an additional 2 minutes to obtain a stable emulsion. In the invention, the first step involves the preparation of a photo-crosslinkable polymer. In this step, 3-arm polycaprolactone (~300 g / mol) is commercially available and methacrylated to obtain a photocrosslinkable polymer. For methacrylation, 0.133 mol 3PCL is dissolved in 50-500 mL dichloromethane (DCM), and 0.1-1 mol triethylamine (TEA) is added to the mixture. After adding another 150-500 mL of DCM to the mixture, a homogeneous mixture was obtained by stirring with a magnetic stirrer and the resulting mixture was placed in an ice bath.0.1-1 mol of methacrylic anhydride (MAAn) was dissolved in 10-150 mL DCM and added to the system placed in an ice bath with a dropping funnel (∼1 drop per second). Upon complete addition of MAAn, the mixture is brought to the room temperature and allowed to polymerize by stirring continuously at 100-1000 rpm for 24-150 hours. After completion of polymerization, washing steps are performed to remove residual TEA, MAAn, and salts from 3PCLMA prepolymer. The polymer is transferred to a separation funnel, and 50-250 mL hydrochloric acid (HCl) solution (1 M) is added and shaken vigorously. The system is allowed to stand at room temperature until polymer precipitates and two phases form. The precipitated polymer is collected, and the process is repeated twice. After acid washing, deionized water washing is started and the polymer is washed with deionized water 3 times by applying the same steps. DCM is then removed from the system using a rotary evaporator after the washing processes. After DCM is removed, the methanol washing step is started, the polymer is transferred to a bottle, the bottle is filled with methanol and shaken. The bottle is kept at -80°C until a precipitate forms at the bottom. When the polymer precipitate is formed, the methanol in the upper phase is removed, the frozen polymer precipitate is liquidized at room temperature and fresh methanol is added to the bottle and the process is repeated 2 more times. After methanol washing, the residual methanol is completely removed using a rotary evaporator, and the resulting 3PCLMA (~600 g / mol) is stored at -20°C. Rı Rıı1 Reaction 1 mentioned above pertains to the synthesis of 3-arm polycaprolactone methacrylate. When Rı in Formula 1, Formula 1 is reacted in the presence of DCM, TEA and MAA to synthesize Formula 2 (polycaprolactone methacrylate). In Formula 2, Rıı. For HIPE preparation, the oil phase components, prepolymer (3PCLMA) and a surfactant (PGPR) in an amount of 1–30% by weight of the prepolymer (preferably 5%), are mixed in a glass bottle using a magnetic stirrer at 100–1000 rpm (preferably 380 rpm) for 1–10 minutes (preferably 2 minutes). While stirring continues, the internal phase containing 0.01–10% (w / v) LAP (preferably 0.1% (w / v)) is added dropwise at a rate of 1 drop per second, and to obtain a stable emulsion, the mixture is stirred for an additional 1–10 minutes (preferably 2 minutes). The resulting high internal phase emulsion is injected into the defect site using a syringe and needle. LAP is a photoinitiator with a maximum absorption peak at 375 nm and demonstrated activity at 405 nm. It allows polymerization within the visible light spectrum (380-700 nm). The injected emulsion is polymerized within 0.5-15 minutes (preferably 1-4 minutes) depending on the sample size with a 50-1500 w (preferably 150 w) light source in the wavelength range of 375-425 nm. In the invention, instead of 3PCL obtained by methacrylation of 1000 g / mol PCL triol used in PolyHIPE studies in the state of the art, a pre-polymer (Formula 2) with a molecular weight of about 600 g / mol to be obtained by methacrylation of 300 g / mol PCL is used. The H-NMR spectrum of 3PCLMA is provided in Figure 3. Using 300 g / mol PCL triol, methacrylated 3PCLMA is synthesized (3PCLMA) and its viscosity is measured. It is observed that the viscosity of the obtained pre-polymer has a viscosity value of approximately 0.1 Pa.s, which is the viscosity value that can only be obtained when solvent is used (Figure 4(A)). Due to the reduced molecular weight, the viscosity of PCLMA pre-polymer shows a viscosity decrease of about 20 times without the use of solvent. As a preliminary study, to test the HIPE-forming potential of the synthesized prepolymer, an emulsion is prepared as the oil phase comprising 3PCLMA, PGPR (1- 30% by weight of the polymer), and a photoinitiator (diphenyl(2,4,6-trimethylbenzoyl) phosphine oxide / 2-hydroxy-2-methylpropiophenone (TPOHM) (1-20% by weight of the polymer), and 80% internal phase can be successfully added to the emulsion without visible phase separation and the emulsion is polymerized. The morphological image of the resulting open-porous scaffold is shown in Figure 4(B). Industrial Applicability of the Invention The invention relates to an injectable (not requiring pre-molding), biodegradable, and porous emulsion composition prepared using solvent-free, photo-crosslinkable, low molecular weight, three-arm polycaprolactone methacrylate-based high internal phase emulsions (3PCLMA HIPE) for use as a bone graft, and to the method for preparing the same, and it is industrially applicable. The invention is not limited to the above descriptions, and a person skilled in the art may easily propose various embodiments of the invention. These shall be considered within the scope of protection claimed by the patent claims.

[0002] REFERENCES [1] FJ;, M. C. M. (n.d.). The effect of mean pore size on cell attachment, proliferation and migration in collagen-glycosaminoglycan scaffolds for bone tissue engineering. Biomaterials. [2] Bhatia, S. (1970, January 1). Natural polymers vs synthetic polymer. SpringerLink. [3] Woodruff, M.A. and Hutmacher, D.W. (2010) The return of a forgotten polymer polycaprolactone in the 21st century. progress in polymer science, 35, 1217-1256. - references - scientific research publishing. (n.d.). [4] Ghasemi-Mobarakeh, L., Prabhakaran, M. P., Morshed, M., Nasr-Esfahani, M. H., & Ramakrishna, S. (1970b, January 1). Bio-functionalized PCL nanofibrous scaffolds for nerve tissue engineering. Materials Science and Engineering. C, Biomimetic Materials, Sensors and Systems. [5] Nottelet B., Pektok E., Mandracchia D., Tille J.-C., Walpoth B., Gurny R. and Moeller M. 2009, “Factorial design optimization and in vivo feasibility of poly(ε-caprolactone)- micro- and nanofiber-based small diameter vascular grafts”, Journal of Biomedical Materials Research Part A, 89A(4), pp: 865-875. [6] E;, B. H. S. A. (n.d.). Human unrestricted somatic stem cells loaded in nanofibrous PCL scaffold and their healing effect on skin defects. Artificial cells, nanomedicine, and biotechnology. [7] Subia, B., Kundu, J., & Kundu, S. C. (2010, March 1). Biomaterial scaffold fabrication techniques for potential tissue engineering applications. IntechOpen. [8] Cameron, N. R., Krajnc, P., & Silverstein, M. S. (2011, January 25). Colloidal Templating. [9] Park JY;Park SH;Kim MG;Park SH;Yoo TH;Kim MS; (n.d.). Biomimetic scaffolds for Bone Tissue Engineering. Advances in experimental medicine and biology.

[0010] R.S. Moglia, J.L. Holm, N.A. Sears, C.J. Wilson, D.M. Harrison, E. Cosgriff- Hernandez, Injectable polyHIPEs as high-porosity bone grafts Biomacromolecules, 12 (10) (2011), pp.3621-3628

[0011] E. Lovelady, et al. Preparation of emulsion-templated porous polymers using thiol– ene and thiol–yne chemistry Polym. Chem., 2 (3) (2011), pp.559-562

[0012] R. Moglia, M. Whitely, M. Brooks, J. Robinson, M. Pishko, E. Cosgriff-Hernandez Solvent-free fabrication of polyHIPE microspheres for controlled release of growth factors,” Macromol Rapid Commun., 35 (14) (2014), pp.1301-1305

[0013] Aldemir Dikici, B.; Sherborne, C.; Reilly, G.C.; Claeyssens, F., (2019), "Emulsion templated scaffolds manufactured from photocurable polycaprolactone", Polymer (Guildf) (Q1), 175, 243–254. https: / / doi.org / 10.1016 / j.polymer.2019.05.023.

[0014] Moglia RS, Whitely M, Dhavalikar P, Robinson J, Pearce H, Brooks M, Stuebben M, Cordner N, Cosgriff-Hernandez E. Injectable polymerized high internal phase emulsions with rapid in situ curing. Biomacromolecules. 2014 Aug 11;15(8):2870-8. doi: 10.1021 / bm500754r. Epub 2014 Jul 22. PMID: 25006990; PMCID: PMC4130241.

Claims

CLAIMS 1. An emulsion composition, characterized in that it has 1–99% internal phase volume and comprises a prepolymer, a surfactant, a crosslinker, and water.

2. The composition according to claim 1, characterized in that it comprises a prepolymer, a surfactant in an amount of 1–30% by weight of the prepolymer, 0.01-10% photoinitiator up to 75% of the total volume and 1–99% water.

3. The composition according to claim 1 or 2, characterized in that said prepolymer is a 3-arm polycaprolactone methacrylate whenin the compound shown with formula .

4. The composition according to claim 1that said surfactant is polyglycerol polyricinoleate (PGPR).

5. The composition according to claim 1 or 2, characterized in that said photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

6. The composition according to claim 1 or 2, characterized in that the mode of application is injection.

7. The composition according to claim 1, characterized in that when the internal phase volume is 30–74%, the emulsion is a polymerized MIPE (medium internal phase emulsion).

8. The composition according to claim 1, characterized in that when the internal phase volume is below 30%, the emulsion is a polymerized LIPE (low internal phase emulsion).

9. The composition according to claim 1, characterized in that when the internal phase volume is at least 74.048%, the emulsion is a polymerized HIPE (high internal phase emulsion).

10. Use of the composition according to any of the preceding claims as a bone graft.

11. A method for preparing an emulsion composition, characterized in that it comprises the process steps of: i. dissolving 3-arm polycaprolactone (polycaprolactone triol; 3PCL) in dichloromethane (DCM) and forming a mixture; ii. adding triethylamine (TEA) and DCM to the mixture; iii. stirring the mixture with a magnetic stirrer and placing the resulting mixture in an ice bath; iv. dissolving methacrylic anhydride (MAAn) in DCM and adding it dropwise to the system in the ice bath using a dropping funnel; v. once MAAn addition is complete, bringing the mixture to room temperature and allow it to polymerize with continuous stirring; vi. after the polymerization is complete, performing washing processes to remove residual TEA, MAAn, and salts from the resulting 3PCLMA pre- polymer; vii. removing DCM from the system using a rotary evaporator after washing processes; viii. after removing DCM, transferring the polymer into a bottle for methanol washing, filling the bottle with methanol, and shaking and storing it until a precipitate forms; ix. once the polymer precipitate forms, removing methanol in the upper phase, liquidizing the frozen polymer precipitate at room temperature, and repeating the process twice by adding fresh methanol to the bottle; x. following methanol washing, removing the remaining methanol completely using a rotary evaporator, and storing the methacrylated 3-arm polycaprolactone (3PCLMA); xi. for HIPE preparation, mixing the oil phase components, pre-polymer (3PCLMA) and surfactant (PGPR), in a glass bottle with a magnetic stirrer;xii. while stirring continues, adding the internal phase containing LAP dropwise at a rate of 1 drop per second, and stirring the mixture to obtain a stable emulsion.

12. The method for preparing an emulsion composition according to claim 11, characterized in that it comprises the steps of: i. dissolving 3-arm polycaprolactone (~300 g / mol) in 0.133 mol 3PCL and 50–500 mL dichloromethane (DCM) and forming a mixture; ii. adding 0.1-1 mol triethylamine (TEA) and 50-500 mL DCM to the resulting mixture; iii. stirring the mixture with a magnetic stirrer and placing it in an ice bath; iv. dissolving 0.1–1 mol methacrylic anhydride (MAAn) in 10–150 mL DCM and adding it dropwise to the system in the ice bath using a dropping funnel; v. after MAAn addition is completed, bringing the mixture to room temperature and allowing it to polymerize by stirring for 24-150 hours at 100-1000 rpm; vi. after the polymerization is complete, performing washing processes to remove residual TEA, MAAn, and salts from the 3PCLMA pre-polymer; vii. removing DCM from the system using a rotary evaporator after washing operations; viii. after removing DCM, transferring the polymer into a bottle for methanol washing, then filling the bottle with methanol, and shaking and storing it at -80°C until a precipitate forms; ix. once the polymer precipitate forms, removing methanol in the upper phase, liquidizing the frozen polymer precipitate at room temperature, and repeating the process twice more with adding fresh methanol to the bottle; x. following methanol washing, removing the remaining methanol completely using a rotary evaporator, and storing the methacrylated 3-arm polycaprolactone (3PCLMA) at -20^°C;xi. for HIPE preparation, mixing the oil phase components, pre-polymer (3PCLMA) and surfactant 1-30% by weight of the pre-polymer (PGPR), in a glass bottle using a magnetic stirrer at 100-1000 rpm for 1-10 minutes; xii. while stirring continues, adding the internal phase containing 0.01-10% (w / v) LAP dropwise at a rate of 1 drop per second, and stirring the mixture for an additional 1-10 minutes to obtain a stable emulsion.

13. The method according to claim 12, characterized in that it comprises the steps of: i. dissolving 3-arm polycaprolactone (~300 g / mol) in 0.133 mol 3PCL and 100 mL of dichloromethane (DCM) and forming a mixture, ii. adding 0.8 mol of triethylamine (TEA) and 150 mL of DCM to the resulting mixture, iii. stirring the mixture with a magnetic stirrer and placing the resulting mixture in an ice bath, iv. dissolving 0.8 mol of methacrylic anhydride (MAAn) in 150 mL of DCM, then adding it to the system in the ice bath dropwise using a dropping funnel, v. once the addition of MAAn is completed, bringing the mixture to room temperature and allowing it to polymerize by stirring continously at 380 rpm for 68 hours, vi. after the polymerization is completed, performing washing processes to remove residual TEA, MAAn, and salts from the 3PCLMA pre-polymer, vii. removing DCM from the system using a rotary evaporator after washing process, viii. after removing DCM, transferring the polymer into a bottle for methanol washing, then filling the bottle with methanol, and shaking and storing it at -80°C until a precipitate forms, ix. once the polymer precipitate forms, removing methanol in the upper phase, liquidizing the frozen polymer precipitate at room temperature, and repeating the process twice by adding fresh methanol to the bottle,x. following methanol washing, removing the remaining methanol completely using a rotary evaporator, and storing the methacrylated 3-arm polycaprolactone (3PCLMA) at −20°C, xi. for HIPE preparation, mixing the oil phase components, 0.4 g of the pre- polymer (3PCLMA) and surfactant 5% by weight of the pre-polymer (0.02 g) (PGPR), in a glass bottle using a magnetic stirrer at 380 rpm for 2 minutes, xii. while stirring continues, adding 1.1 mL of internal phase containing 0.1% (w / v) LAP dropwise at a rate of 1 drop per second and stirring the mixture for an additional 2 minutes to obtain a stable emulsion.

14. An emulsion composition prepared by a method according to any of claims 11- 13.

15. The emulsion composition according to claim 14, characterized in that it has 1– 99% internal phase volume.

16. An emulsion composition according to claim 15 for use as a bone graft.

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