Patent Application
20160243277
As disclosed herein are fast curing high porosity materials and methods of making said materials, including controlling their time to cure and their porosity in order to optimize their ex vivo, in vivo and/or in situ delivery.
The controlled delivery of a fast curing resin, particularly a resin suitable for in vivo use, remains challenging. First, the rate of polymerization and cross-linking as well as the method of delivery hampers the use of many polymeric resin materials. In addition, while some polymers offer structural benefits, they have been found unsuitable for use in vivo because of their chemical make-up or because of the complicated or time consuming manner of processing that is required prior to delivery. For example, many polymeric resins require complicated steps during or after processing and before they are suitable for delivery. Many polymeric resins have only a very short shelf-life. Many polymeric resins offer only limited structural support following polymerization and many are not specifically designed to be degradable after delivery. Many structural tissues or organs, however, require that any polymeric material incorporated therein offer adequate mechanical properties (to withstand physiologic loading, especially when tissue function is not yet restored), as well as an adequate internal morphology, preferably an interconnected network, that promotes cell growth, allows sufficient nutrient and/or waste transport and neovascularization. Unfortunately, many polymeric resins require either toxic diluents and/or high cure temperatures for cross-linking and curing, making them unsuitable for controlled in vivo delivery. Some polymeric resins, including certain gel or suspension systems, while capable of polymerizing in vivo remain difficult to control their time to cure or their porosity, or internal morphology, or mechanical strength.
Many current biodegradable polymer formulations require roughly two hours or more to cure at body temperature. In practice, however, a fast-curing material that reduces surgical time, lowers the patient's risk of infection, and rapidly stabilizes defects is preferred. Poly(methyl methacrylate) (PMMA) bone cement, which is not biodegradable, but capable of curing in about 15 minutes, is the most common injectable system used clinically to stabilize orthopedic implants. PMMA, however, does not facilitate tissue regeneration because it is highly exothermic, non-degradable, and non-porous. Previous iterations of high internal phase polymeric resin compositions relied on thermal initiation, the rate of which increases exponentially with temperature. For in situ curing, however, the physiologic temperature of the body constrains the cure temperature to 37° C. This temperature is well below the typical temperature of most thermal initiators. When some high internal phase polymeric resin compositions tried to use non-thermal initiators, the cure systems were instead found to contain toxic or non-degradable components.
There remains a need for a polymeric material that is hardenable in a controlled setting and capable of being delivered site specifically in vivo, such that it can be cured in a controlled manner and when cured is suitably biocompatible (in structure and morphology). Current polymeric systems have not been found to meet said needs, nor are they capable of being predesigned to offer desired properties upon delivery, properties that include morphologic, mechanical, and structural, as well as an ability to withstand physiologic compressive stresses.
Described herein are high internal phase resin compositions comprising biocompatible polymeric materials. The high internal phase resins when combined are capable of meeting the needs described above. The high internal phase resins described herein can be prepared to polymerize and cure in a controlled manner. The high internal phase resins when combined are capable of polymerizing and curing in an in vivo environment. In some embodiments, the high internal phase resins when combined are capable of polymerizing and curing in situ. The high internal phase resins when combined may be delivered site specifically and under controlled design conditions. The high internal phase resins when combined may be pre-selected to control the rate of polymerization or time to cure. The high internal phase resins when combined may be pre-selected to control the degree and extent of porosity. The high internal phase resins, when combined, are capable of forming a three-dimensional highly porous architecture. The formed three-dimensional architecture is a monolith. The formed three-dimensional architecture is a rigid structure having a foam body. The formed three-dimensional architecture is biocompatible, sufficiently porous and also capable of withstanding physiologic compressive stresses when fully cured. The formed three-dimensional architecture may be biodegradable. The three-dimensional architecture may contain one or more materials or active agents when formed. The formed three-dimensional architecture may be specifically fabricated and/or shaped to suit a particular in vivo environment. The formed three-dimensional architecture may be specifically fabricated to introduce one or more material or active agents into its environment.
One or more of the high internal phase resins described herein are also stable as emulsions (without fully polymerizing) enabling a long shelf-life prior to delivery or use. Said high internal phase resins may be pre-selected and tuned to control the rate of polymerization or time to cure of a final three-dimensional monolith.
In one or more forms is a first high internal phase resin composition. The first high internal phase resin is formed as a water-in-oil emulsion comprising at least a first biodegradable polymeric material in a continuous (oil) phase and an oxidizing initiator in either the continuous phase or a dispersed (water) phase. The water-in-oil high internal phase emulsion has a water volume of at least about 75% or greater. The first high internal phase resin emulsion may be designed to be stable as an emulsion without fully polymerizing at ambient or room temperature. In one or more embodiments, the first high internal phase resin is stable as an emulsion (without fully polymerizing) for at least one or more hours or two or more hours or up to about 12 hours or up to or greater than 24 hours, such as at an ambient temperature, or up to at least six months or more, such as at a temperature less than an ambient temperature. The oxidizing initiator is selected and in an amount that does not promote polymerization or delays polymerization of the first high internal phase resin. In one or more embodiments, the oxidizing initiator is selected and in an amount that is low enough to prevent polymerization or delay polymerization for at least one or more hours or two or more hours or up to about 12 hours or up to or greater than 24 hours or up to at least six months or more, such as when the first high internal phase resin emulsion at an ambient temperature, is at a sub ambient temperature, or is at a reduced temperature, such as about 4 degrees C. or less than 4 degrees C. The oxidizing initiator may be a nonaqueous free-radical oxidizing agent. The oxidizing initiator may be an aqueous free-radical oxidizing agent. Exemplary oxidizing initiators include but are not limited to peroxides, persulfates, and azo initiators. The first high internal phase resin composition may also include in the dispersed phase at least one chemical that prevents Ostwald ripening. The first high internal phase resin composition is emulsified by mechanical dispersion of the components described. The first high internal phase resin emulsion is stable in a relatively unpolymerized emulsified state at ambient or room temperature or at a temperature below ambient or room temperature.
The first biodegradable polymeric material of the first high internal phase resin emulsion is stabilized with an emulsifier in the continuous phase. The emulsifier may be an amphiphilic surfactant having a polar, water-soluble head group attached to a nonpolar, water-insoluble hydrocarbon chain. The emulsifier is selected as one lacking hydrogen bond donor sites (donors) in its hydrophilic, polar head region. In one or more embodiments, the emulsifier has a hydrophilic-lipophilic balance (HLB) in a range of between about 2 and about 9. In some embodiments, the emulsifier has a hydrophilic-lipophilic balance (HLB) in a range of between about 3 and about 5.
In a second high internal phase resin composition are components that form a water-in-oil emulsion, the components including at least a second biodegradable polymeric material in the continuous (oil) phase and a reducing agent in the dispersed (water) phase or in the continuous phase. Said water-in-oil high internal phase emulsion has a water volume of at least about 75% or greater. The second high internal phase resin emulsion may be designed to be stable as an emulsion without fully polymerizing at ambient or room temperature. In one or more embodiments, the second high internal phase resin is stable as an emulsion (without fully polymerizing) for at least one or more hours or two or more hours or up to about 12 hours or up to or greater than 24 hours, such as at an ambient temperature, or up to at least six months or more, such as at a temperature less than an ambient temperature. The reducing agent is in an amount that does not promote polymerization or delays polymerization of the second high internal phase resin. The reducing agent is selected from one or more of a hydrocarbon, metal ion, vitamin, bioactive agent and the like. The reducing agent is selected and in an amount that is low enough to prevent polymerization or delay polymerization for at least one or more hours or two or more hours or up to about 12 hours or up to or greater than 24 hours or up to at least six months or more, such as when the first high internal phase resin emulsion at an ambient temperature, is at a sub ambient temperature, or is at a reduced temperature, such as about 4 degrees C. or less than 4 degrees C. The second high internal phase resin may also include in the dispersed phase at least one chemical that prevents Ostwald ripening. The second high internal phase resin composition is emulsified by mechanical dispersion of the components described. The second high internal phase resin emulsion is stable in a relatively unpolymerized emulsified state at ambient or room temperature or at a temperature below ambient or room temperature.
The second biodegradable polymeric material of the second high internal phase emulsion is stabilized with an emulsifier in the continuous phase. The emulsifier may be an amphiphilic surfactant having a polar, water-soluble head group attached to a nonpolar, water-insoluble hydrocarbon chain. The emulsifier is selected as one lacking hydrogen bond donor sites (donors) in its hydrophilic, polar head region. The emulsifier has, in one or more embodiments, a hydrophilic-lipophilic balance (HLB) in a range of between about 2 and about 9. In additional embodiments, the emulsifier has a hydrophilic-lipophilic balance (HLB) in a range of between about 3 and about 5.
The emulsifier stabilizing the second biodegradable polymeric material may be essentially the same as the emulsifier stabilizing the first biodegradable polymeric material. The emulsifier stabilizing the second biodegradable polymeric material may also be different from the emulsifier stabilizing the first biodegradable polymeric material.
The first and the second biodegradable polymeric material are each selected for polymerization of a porous polymer (also referred to as polyHIPE), and will include: (a) a macromonomer (macromer) having at least one reactive end group, which is biodegradable, and having a suitable viscosity for emulsion in water; and (b) reaction thermodynamics that allow polymerization and/or curing at a physiologic condition. The at least one reactive end group will crosslink the macromer at a thermal or ambient or a physiologic temperature. The molecular weight of the biodegradable polymeric material will assist in maintaining a viscosity, and preferably a controlled viscosity that replaces (and no longer requiring) a toxic diluent for macromer stabilization. The at least one reactive end group has at least one unsaturated (double) bond for undergoing free radical cross linking. In one or more embodiments, the at least one reactive end group may be an acrylate end group or a methacrylate end group.
The macromer for the first biodegradable polymeric material and the second biodegradable polymeric material will have a selected viscosity and hydrophobicity. In one or more embodiments, the hydrophobicity, determined by an octanol-water partition coefficient (LogP), is greater than 2. In some embodiments, the LogP is in a range of between 2 and 8. In some embodiments, the LogP is in a range of between 2 and 4. The macromer preferably is but is not required to have a viscosity in a range that is near that of water, which is 1 cP. In some embodiments, the viscosity is in a range of between about 0.08 cP and about 1000 cP. In some embodiments, the viscosity is not greater than 0.150 cP.
Suitable macromers are ones prepared from an ester based monomer, macromer, and/or polymer or from an anhydride based monomer, macromer, and/or polymer. A suitable macromer is represented by but is not limited to a biodegradable polymer having one or more ester linkages or a biodegradable polymer having one or more anhydride linkages. Exemplary embodiments include a biodegradable fumarate based polymer having one or more ester linkages (e.g., propylene fumarate dimethacrylate [PFDMA]), a biodegradable diglycol based polymer having one or more ester linkages (e.g., ethylene glycol dimethacrylate [EGDMA]), a butane diol dimethacrylate [BDMA]) and a biodegradable acrylic based polymer having one or more anhydride linkages (e.g., methacrylic anhydride [MA]).
In some embodiments, the first biodegradable polymeric material and the second biodegradable polymeric material are the same. In some embodiments, the first biodegradable polymeric material and the second biodegradable polymeric material are similar, having the same or similar linkages and/or end groups.
Described herein is also a third high internal phase resin composition. The third high internal phase resin composition is formed as a third emulsion by combining the first high internal phase resin emulsion previously described and the second high internal phase resin emulsion previously described. The first high internal phase resin emulsion and the second high internal phase resin emulsion are combined in a mixing chamber or apparatus, thereby providing the third high internal phase resin composition. The third high internal phase resin emulsion when formed is capable of polymerizing, serving as a template for obtaining a porous polymer from high internal phase emulsions (polyHIPE). The third high internal phase resin emulsion will undergo a redox reaction and polymerization of the continuous phase. The third high internal phase resin emulsion will undergo a free radical cross-linking of the reactive end groups. The third high internal phase emulsion may be a foam emulsion prior to curing. The third high internal phase resin emulsion may be incubated or otherwise controlled to modify the degree of crosslinking, the rate of polymerization, and the extent of porosity or three-dimensional architecture.
In one or more embodiments, the third high internal phase resin composition is formed by combining the first high internal phase resin emulsion and the second high internal phase resin emulsion in a mixing chamber for polymerization and free radical cross-linking, and thereafter introducing after combining to a mold followed by providing the molded composition either when partially or fully set or cured to a location in vivo. In additional embodiments, the first and second high internal phase resins may be combined in a mixing chamber for polymerization and free radical cross-linking, and thereafter introduced to a location in vivo. The mixing may be by mechanical dispersion or by others means for blending the first and second high internal phase emulsions. The mixing may include incubating at a temperature below or well below 100 degrees Centigrade during or upon blending. The mixing may include incubating at a more physiologic temperature below, such as between about 25 degrees and 45 degrees Centigrade. In various embodiments, blending of the first and second high internal phase emulsions in a chamber provides a third emulsion that has a foam body. The foam body has a three-dimensional architecture that is highly porous. The foam body is prepared in the absence of a blowing agent. The foam body may incubate for a period of time. Pore size and pore distribution of the foam body may be controlled by one or more of the following, as examples: mixing condition, emulsifier in the first high internal phase resin emulsion, emulsifier in the second high internal phase resin emulsion, the type and amount of polymerization and free radical cross-linking agents, and polymerization temperature. In one or more embodiments, decreasing the overall emulsifier concentration increases the average pore diameter in the foam body. In various embodiments, decreasing the rate of mixing in the mixing chamber increases the average pore diameter in the foam body. In various embodiments, increasing the temperature for making the foam body increases the average pore diameter in the foam body. In various embodiments, increasing the temperature for curing (incubating) the foam body increases the average pore diameter in the foam body. The foam body will often have an overall porosity of about or greater than 75%. In some embodiments, the foam body when fully cured (to its hardened state) will have an average pore size (as a cross-sectional diameter) in a range from at least about 2 μm to about 50 μm. In some embodiments, the foam body when fully cured (to its hardened state) will have an average compressive modulus of at or about 2-50 MPa and strength of at or about 1-10 MPa.
The first high internal phase resin emulsion and the second high internal phase resin emulsion are combined to form the third high internal phase emulsion at a selected temperature. The third high internal phase emulsion is mixed at a selected temperature. In some embodiments, the third high internal phase emulsion is incubated at a selected temperature. The combining temperature and/or the mixing temperature and/or the incubating temperature may be the same. In one or more embodiments, said temperature is a low cure temperature. In various embodiments, the temperature is 100° C. or less. In various embodiments, the temperature is a physiologic temperature. In various embodiments, the temperature is between about 15° C. and about 55° C. In various embodiments, the temperature is between about 20° C. and about 45° C. Upon combining the first and second high internal phase resin emulsions, the rate or time to cure of the third high internal phase emulsion decreases with increasing temperature.
The first and second high internal phase resin emulsions when combined as a third high internal phase emulsion promote initiator decomposition and free radical production therein. The free radical production is provided at a thermal, ambient or physiologic temperature. In one or more embodiments, cure time of the third high internal phase resin emulsion is controlled by adjusting the amount of free radical production, whereby an increase in the production (concentration) of free radicals increases the rate of polymerization (and cross-linking) and reduces the cure time or set time that follows. Free radical production may be tuned by controlling the reducing agent or the ratio of reducing agent to oxidizing initiator, whereby the rate of decomposition of the oxidizing initiator is increased in the presence of the reducing agent. The presence of the reducing agent reduces the overall concentration of an initiator required in the third high internal phase emulsions as compared with alternative high internal phase emulsions that lack a reducing agent and initiate cross-linking only in the presence of an initiator alone.
In one or more embodiments, the first high internal phase resin emulsion and the second high internal phase resin emulsion have similar components, excluding the oxidizing initiator in the first high internal phase resin emulsion and the reducing agent in the second high internal phase resin emulsion, such that only the oxidizing initiator in the first high internal phase resin emulsion is replaced by a reducing agent in the second high internal phase resin emulsion.
Even further as described herein is a resulting emulsion body, provided as a three-dimensional monolith, suitable for use in vivo, ex vivo and/or in situ. The resulting emulsion body is prepared from two independently prepared high internal phase resin emulsions, one of which contains a free radical oxidizing agent and another one that contains a reducing agent. The resulting emulsion body is prepared by a redox reaction between a first high internal phase resin emulsion containing a free radical oxidizing agent in an amount that delays or prevents cross-linking and a second high internal phase resin emulsion containing a reducing agent in an amount that delays or prevents cross-linking. In one or more embodiments, the three-dimensional monolith is a foam body when cured. It may be operable for tissue replacement and stabilization. In one or more embodiments, the three-dimensional monolith when cured may be operable for stabilization as a soft tissue. In another embodiment, the three-dimensional monolith when cured may be operable for stabilization as a hard tissue. In still another embodiment, the three-dimensional monolith when cured may be operable for stabilization as an interface tissue. In yet a further embodiment, the three-dimensional monolith when cured may be operable as a catalyst support. In yet another embodiment, the three-dimensional monolith when cured may be operable as a component in a system sensitive to heat or solvents. In a further embodiment, the three-dimensional monolith may be biodegradable. In some embodiments, the three-dimensional monolith when formed will degrade at a rate complementary with tissue regeneration.
A high internal phase emulsion when formed may comprise a biodegradable polymeric material, the biodegradable polymeric material comprising: at least one end group selected from an acrylate and a methacrylate; and one or more linkages selected from an anhydride and an ester. The biodegradable polymeric material may have an octanol-water partition coefficient of between about 2 and about 8 and a viscosity of between about 0.08 cP and about 1000 cP. The biodegradable polymeric material is generally stabilized with a quantity of an emulsifier lacking hydrogen bond donors in its hydrophilic head region while having a hydrophilic-lipophilic balance in a range of between about 2 and about 9. The high internal phase emulsion further comprises a chemical to prevent Ostwald ripening, wherein the chemical to prevent Ostwald ripening is a salt. The high internal phase emulsion further comprises an oxidizing agent is in a quantity that maintains the high internal phase emulsion as an emulsion and is insufficient to initiate extensive free radical cross-linking of the biodegradable polymeric material when stored at ambient temperatures. The high internal phase emulsion further comprises an oxidizing agent is in a quantity that maintains the high internal phase emulsion as an emulsion and is insufficient to initiate extensive free radical cross-linking of the biodegradable polymeric material when stored at less than ambient temperatures. The high internal phase emulsion further comprises water. The water is generally in a volume of at least about 75% of the internal phase emulsion by volume. The high internal phase emulsion is stable as an emulsion without undergoing further polymerization when stored at a sub-ambient temperature. The octanol-water partition coefficient of the biodegradable polymeric material may be between about 2 and about 4. The viscosity of the biodegradable polymeric material may be near the viscosity of water. The oxidizing agent may be a free radical oxidizing initiator. The hydrophilic-lipophilic balance of the emulsifier may be in a range of between about 3 and about 5.
A high internal phase emulsion when formed may comprise a biodegradable polymeric material. The biodegradable polymeric material comprising: at least one end group selected from an acrylate and a methacrylate; and one or more linkages selected from an anhydride and an ester. The biodegradable polymeric material generally has an octanol-water partition coefficient of between about 2 and about 8 and a viscosity of between about 0.08 cP and about 1000 cP. The biodegradable polymeric material is stabilized with a quantity of an emulsifier lacking hydrogen bond donors in its hydrophilic head region while having a hydrophilic-lipophilic balance in a range of between about 2 and about 9. The high internal phase emulsion further comprises a chemical to prevent Ostwald ripening, wherein the chemical to prevent Ostwald ripening is in an aqueous phase. The high internal phase emulsion further comprises a reducing agent. The high internal phase emulsion further comprises water The water may be in a volume of at least about 75% of the emulsion. The high internal phase emulsion is generally stable as an emulsion without undergoing further polymerization when stored, for example at a sub-ambient temperature. The high internal phase emulsion is generally stable as an emulsion without undergoing further polymerization when stored at an ambient temperature. The octanol-water partition coefficient of the biodegradable polymeric material may be between about 2 and about 4. The viscosity of the biodegradable polymeric material may be near the viscosity of water. The reducing agent may be selected from one or more of a hydrocarbon, metal ion, vitamin, and bioactive agent. The hydrophilic-lipophilic balance of the emulsifier in a range of between about 3 and about 5.
A method of making a high internal phase emulsion may comprise combining by mechanical dispersion a first high internal phase emulsion with a second high internal phase emulsion. The first high internal phase emulsion generally comprises an oxidizing agent in a quantity insufficient to initiate extensive cross-linking in the first high internal phase emulsion when stored, for example at sub-ambient temperatures. The second high internal phase generally comprises a reducing agent.
A method of making a body using a high internal phase emulsion composition may comprise combining by mechanical dispersion a first high internal phase emulsion containing an oxidizing initiator with a second high internal phase emulsion containing a reducing agent. The method may further comprise allowing the first high internal phase emulsion and the second high internal phase emulsion to undergo a redox reaction. Upon combining the first and second emulsion a foam body is formed. The foam body generally has a porosity at or greater than 75%. In some embodiments, a rate of combining has an effect on an average pore diameter in the foam body. In some embodiments, the foam body is injectable into any of a form, a mold, and in situ, such that the foam body takes a shape of said form, said mold, or may be formed in situ. In some embodiments, the foam body has a pore size in any size or range of sizes from between about 1 micron and 300 microns. In some embodiments, the foam body cures between about 10 seconds and about 100 minutes.
A high internal phase emulsion composition is formed, the composition comprising a first high internal phase emulsion comprising at least a first biodegradable polymeric material, an oxidizing agent and water. The oxidizing agent is generally in a quantity that maintains the first high internal phase emulsion as an emulsion and delays cross-linking of the first high internal phase emulsion. The oxidizing agent is generally in a quantity that maintains the first high internal phase emulsion as an emulsion and delays cross-linking of the first high internal phase emulsion when at an ambient temperature. The water is in a volume that is at least about 75% of the first high internal phase emulsion by volume. The high internal phase emulsion composition further comprises a second high internal phase emulsion comprising at least a second biodegradable polymeric material, a reducing agent and water. The water is in a volume that is at least about 75% of the second high internal phase emulsion by volume. The reducing agent may be in a quantity that maintains the second high internal phase emulsion as an emulsion and delays cross-linking of the second high internal phase emulsion when at an ambient temperature. The high internal phase emulsion composition may further comprise one or more a bioactive component and a cell in an aqueous phase of any or both the first high internal phase emulsion and the second high internal phase emulsion. The high internal phase emulsion may further comprise a bioactive component in an organic phase of any or both the first high internal phase emulsion and the second high internal phase emulsion. The first high internal phase emulsion and the second high internal phase emulsion may be stored separately at a sub ambient temperature prior to use. The first biodegradable polymeric material and the second biodegradable polymeric material may be the same, or may each contain at least one end group selected from an acrylate and a methacrylate and one or more linkages selected from an anhydride and an ester. In some embodiments, the end groups and linkages in each biodegradable polymeric material may be similar or the same.
Various embodiments will be explained in more detail with reference to the drawings in which:
Although making and using various embodiments are discussed in detail below, it should be appreciated that as described herein are provided many inventive concepts that may be embodied in a wide variety of contexts. Embodiments discussed herein are merely representative and do not limit the scope of the invention.
High internal phase emulsions (HIPEs) are three-dimensional polymer networks characterized herein as water-in-oil emulsion systems 10 having an internal phase volume fraction 2 comprising essentially water (which may further comprise water soluble constituents and/or bioactive molecules, cells, etc.) making up 74% or more of the total emulsion volume, thus, having only a minor, continuous phase consisting of stabilized macromers 5 (e.g., polymeric materials that are functionalized and capable of undergoing further polymerization) (
Fabrication of biodegradable HIPEs and polyHIPEs and their compositions have been described in U.S. patent application Ser. No. 13/651,362 (the entirety of which is incorporated herein by reference). Improvements to said biodegradable HIPEs and polyHIPEs and methods of making are now described. The improvements include controlling one or more of the following: free radical production, time to cure, average pore diameter, overall porosity and morphology, hardness, and various combinations thereof.
As described herein are various biodegradable HIPE formulations stable when prepared as an emulsion without polymerizing, though still containing a polymerizing initiator.
Additionally, as described herein are various biodegradable HIPE formulations, and biodegradable polyHIPE formulations provided therefrom, containing a reduced amount of a polymerization initiator (e.g., free radical initiator) as compared to the amounts provided in formulations described in U.S. patent application Ser. No. 13/651,362 or other comparative biodegradable HIPE formulations, and biodegradable polyHIPE formulations provided therefrom.
Also as described herein are various biodegradable polyHIPE formulations that, though containing a reduced amount of a polymerization initiator (e.g., free radical initiator), will, when formed, polymerize faster, having an increased time to cure as compared with alternative polyHIPE formulations that contain a higher amount of polymerization initiator (e.g., free radical initiator). The polyHIPEs described herein provide a number of advantages: they are injectable, cure quickly and in situ, cure in a solvent free environment and, due to their low cure temperatures permit adequate flow site specifically followed by formation into rigid bodies or foam-like bodies, all at a physiologic temperature.
The injectable polyHIPEs described herein may cure in a same time period as PMMA but with the advantages that the polyHIPEs will stabilize a bone defect and may also be loaded with cells prior to injection to provide a temporary matrix that supports tissue regeneration. In addition, polyHIPE grafts described herein remain stable in storage for a much longer period of time, by being capable of storage for up to and more than 6 months, and will still cure rapidly after injection, meaning the described polyHIPE materials are more suitable as an off-the-shelf graft for both emergency and/or scheduled procedures.
The formulations described herein are capable of creating tunable, biodegradable polyHIPEs not previously described yet still capable of polymerizing and curing under physiologic conditions. The polyHIPE formulations described herein have a reduced time to cure (cure faster) once formed. The polyHIPE formulations described herein are suitable for various in vivo, in situ and ex vivo applications that can take advantage of a highly porous polymer. The polymer chemistry chosen for the polyHIPE formulation may be further tuned to alter and manipulate structural hardness (e.g., compressive strength) when so desired.
The HIPEs as described herein are biodegradable and, as fabricated, can remain stable as an emulsion, thereby capable of delaying or suspending polymerization. Such emulsions may be stored for extended periods of time (months, years) prior to use. The HIPEs are generally prepared from a biodegradable polymeric material comprising a functionalized macromer capable of undergoing further polymerization.
The macromer used for the HIPEs described herein is either an ester based monomer, macromer, and/or polymer or an anhydride based monomer, macromer, and/or polymer and will have at least one reactive end group, which is biodegradable, a suitable hydrophobicity (determined by an octanol-water partition coefficient [LogP]) and viscosity for emulsion in water, and may be polymerized and/or cured at or near physiologic conditions. In some embodiments, the macromer itself may be prepared via a two-step reaction, such as that described in U.S. patent application Ser. No. 13/651,362, which includes: (i) backbone synthesis, and (ii) functionalization.
The at least one reactive end group of the macromer described herein is one that crosslinks at a thermal temperature or a lower temperature (e.g., ambient temperature, physiologic temperature) and of a low molecular weight that maintains a low viscosity for the macromer, hence acting to replace and no longer requiring addition of a toxic diluent. The at least one reactive end group has at least one unsaturated (double) bond for undergoing free radical cross linking. The carbon-carbon double bond in the at least one reactive end group allows thermal decomposition to occur in the presence of the redox initiators, to be described further. In one or more embodiments, the at least one reactive end group generally includes an acrylate end group or a methacrylate end group.
The hydrophobicity of the macromer described herein is defined by a LogP at about or greater than 2. It may also be defined by a LogP from between about 2 and about 8. It may also be defined by a LogP from between about 2 and about 4. A host of suitable macromers may be identified using available tools, including online services, such as one provided by Molinspiration Cheminformatics. For example, Molinspiration Cheminformatics provides model predictions of the LogP for more than 12,000 molecules or compounds, generally calculated from the sum of non-overlapping molecular fragments after fitting calculated LogP with experimental LogP values.
The LogP value of various representative compounds suitable under the definition provided herein are provided in TABLE 1, including PFDMA, which is propylene fumarate dimethacrylate; BDMA, which is butane diol dimethacrylate; EGDMA, which is ethylene glycol dimethacrylate; and MA, which is methacrylic anhydride. These suitable and representative macromers are compared to ones that are already used by alternative methods to prepare a stable HIPE, such as styrene and divinyl benzene.
The viscosity of the macromer described herein is defined as being in a range between about 0.08 cP and about 1000 cP (at an ambient temperature). In some embodiments, the initial viscosity is near that of water, which is near 1 cP (at about ambient temperature or about 20 degrees C.). In some embodiment, the viscosity is not greater than 0.150 cP (at an ambient temperature).
The macromer as described herein may also be characterized as a biodegradable polymeric material having one or more ester linkages or a biodegradable polymeric material having one or more anhydride linkages. Exemplary embodiments include but are not limited to a biodegradable fumarate based polymer having one or more ester linkages (e.g., propylene fumarate dimethacrylate [PFDMA]), a biodegradable glycol based polymer having one or more ester linkages (e.g., ethylene glycol dimethacrylate [EGDMA]), a hydroxy (e.g., diol) based polymer having one or more ester linkages (e.g., butane diol dimethacrylate [BDMA]) and a biodegradable acrylic based polymer having one or more anhydride linkages (e.g., methacrylic anhydride [MA]).
The macromer when used for the HIPEs described is stabilized in the continuous phase by an emulsifier. The emulsifier may be an amphiphilic surfactant having a polar, water-soluble head group attached to a nonpolar, water-insoluble hydrocarbon chain. The emulsifier is selected as one lacking hydrogen bond donor sites (donors) in its hydrophilic, polar head region. The emulsifier has, some embodiments, a hydrophilic-lipophilic balance (HLB) in a range of between about 2 and about 9. In some embodiments, the emulsifier has a hydrophilic-lipophilic balance (HLB) in a range of between about 3 and about 5.
In the embodiments described, more than one independently prepared HIPE is used for fabrication of a polyHIPE. Each HIPE is a high internal phase resin emulsion formed as a water-in-oil emulsion comprising a stabilized biodegradable polymeric material in its continuous (oil) phase and at least a redox polymerizing initiator; the redox polymerizing initiator may be in the dispersed (water) phase (when aqueous) or the continuous phase (when non-aqueous). The redox initiator is, in a first high internal phase resin emulsion, an oxidizing initiator (oxidant) and is, in second high internal phase resin emulsion, a reducing initiator (reductant). Thus, a high internal phase resin emulsion will either have an oxidizing initiator or a reducing agent. In one or more embodiments, decomposition is not initiated in either the first high internal phase resin emulsion or the second high internal phase resin emulsion when prepared as described herein. In some embodiments, decomposition is delayed in either the first high internal phase resin emulsion or the second high internal phase resin emulsion when prepared as described herein.
In a first high internal phase resin emulsion, components will include a stabilized macromer, an oxidizing initiator, a chemical preventing Ostwald ripening and water. Water will be in an amount of about or greater than 75% (v/v). The at least one chemical that prevents Ostwald ripening may be a salt or an electrolyte. Only a small amount of the chemical preventing Ostwald ripening is typically required, generally about 1 to about 5% (v/v). The oxidizing initiator is selected as one that generates free radicals by thermal or ambient decomposition. The oxidizing initiator is a free radical oxidizing agent, such as, but not limited to a peroxide, a persulfate, or an azo compound initiator. Representative examples include but are not limited to azobis-isobutyronitrile (AIBN) and benzoyl peroxide (BPO). The organic and soluble free radical initiators are useful for polyHIPEs introduced in vivo because these do not appear to leach components from the scaffold that effect cell growth and viability (data not shown). The oxidizing initiator is either a non-aqueous free radical oxidizing agent or an aqueous free radical oxidizing agent. The quantity of oxidizing initiator in the first HIPE composition is considered to be low, in an amount that is insufficient to initiate extensive free radical cross-linking of a macromer chain (of unsaturated double bonds of the one or more end groups). The amount will vary from between about 1 wt. % to about 10 wt. % or about 15 wt. % (based on the total weight in the organic phase) but will be less than what would be required were the high internal phase resin emulsion to undergo polymerization to form a polyHIPE. The reduced concentration of the oxidizing initiator described herein, not found in previous HIPES, ensures stability of the first high internal phase resin emulsion and delays and/or prevents polymerization at a physiologic or ambient temperature. As such, these HIPEs may be stored without fully polymerizing. The HIPEs described herein are, thus, suitably stable as an emulsion at a physiologic or an ambient temperature without setting (curing). These HIPEs may be stored at a temperature that is a physiologic or ambient or may be frozen for later use as depicted in box 18,
This first high internal phase resin emulsion is prepared as illustrated in
In an example of a first HIPE, a macromer (comprising 1 g propylene fumarate dimethacrylate [PFDMA]) was initially blended in a mixer (a dual asymmetric centrifugal mixer) with a stabilizer lacking hydrogen bond donor sites at its polar head region (20 wt. % polyglycerol polyricinoleate [PGPR]), to form a stabilized macromer. The stabilized macromer was emulsified with a non-aqueous initiator (0.5 wt. % benzoyl peroxide [BPO]) and a chemical that prevents Ostwald ripening (1% (v/v) calcium chloride) in deionized water (3 g) by an additional blending in the dual asymmetric centrifugal mixer (500 rpm). The emulsion prepared was a high internal phase emulsion. This emulsion did not set at physiologic temperature (e.g., about 37° C.) when evaluated for 72 hours.
In a second high internal phase resin emulsion, components will include a stabilized macromer, a reducing agent, a chemical preventing Ostwald ripening and water. Water will be in an amount of about or greater than 75% (v/v). The at least one chemical that prevents Ostwald ripening may be a salt or an electrolyte. Only a small amount of the chemical preventing Ostwald ripening is typically required, generally about 1 to about 5% (v/v). The reducing agent may be a hydrocarbon, inorganic compound, biological agent, vitamins, metal ion, and the like. The quantity of the reducing agent is in an amount that is insufficient to initiate free radical cross-linking of a macromer chain (of unsaturated double bonds of the one or more end groups). The amount will generally vary from between about 0.5 wt. % to about 10 wt. %. The low concentration of the reducing agent ensures stability of the second high internal phase resin emulsion and delays and/or prevents its polymerization at a physiologic or ambient temperature. As such, these HIPEs may be stored without fully polymerizing and are, thus, suitably stable as an emulsion at a physiologic or ambient temperature without setting (curing). These HIPEs may be stored at a temperature that is a physiologic or ambient or may be frozen for later use as depicted in box 28,
This second high internal phase resin emulsion is prepared as illustrated in
The two independently prepared first and second high internal phase resin emulsions are combined to fabricate a polyHIPE via a redox reaction (
The making of representative polyHIPEs is outlined in
Decomposition and polymerization of the final emulsion provides a three-dimensional architecture that is in the form of a foam body. The foam body is a porous monolith. Decomposition may occur at a thermal temperature, at a physiologic temperature (e.g., temperature at about or less than about 40° centigrade) or an ambient temperature. The extent of polymerization is generally dependent on the amount of the redox initiators and the reaction temperature (box 312,
The porosity and pore size is generally dependent on the rate of mixing when forming the emulsion (box 312,
In an example, a polyHIPE was formed from a PFDMA macromer contained in the first HIPE and in the second HIPE, in which the final formed product had an open pore morphology, many of which were, on average, approximately 20 micrometers in diameter, with an about 75% overall porosity and an average compressive modulus of about 30 MPa and strength of about 5 MPa. Complete polymerization was obtained within a few seconds after forming the emulsion at a physiologic temperature (between 37 and 40° C.). In general, a polyHIPE when formed will exhibit at least about 75% or greater porosity. Additionally, a polyHIPE described herein when formed may have an average pore size ranging at least from about 4 μm to at least about 29 μm.
As illustrated in
The ability to synthesize a fully biodegradable polyHIPE without a toxic diluent that can also rapidly cure at a selected cure rate by controlling the amount of emulsifier, amount or redox initiators, rate of mixing (for emulsification) and/or polymerization temperature is an important adaptation of emulsion templating that is described herein. The improved polyHIPEs described herein are suitable for injection and/or for molding. The improved polyHIPEs offer sufficient strength to be structural and supportive. Said HIPEs and polyHIPEs may be stored for weeks, months or at least a year (i.e. frozen and later thawed) without notable changes in architecture or strength or, for HIPEs, without notable changes in their ability to polymerize when combined as described herein (data not shown). The polyHIPEs when fully formed and cured also exhibit sufficient mechanical strength and modulus to withstand physiological loading, which is necessary to promote and restore tissue function without causing deleterious stress-shielding effects.
Another advantage of the methods of compositions described herein is that either or both initial HIPEs (the first high internal phase emulsion composition or the second high internal phase emulsion composition) may be prepared in batches, in which the type and/or concentration of emulsifier or the type and/or concentration of initiator are specifically altered. Thus any number of combinations may be introduced together to provide finely tunes polyHIPE products with selected pore size, range of pore sizes, or hierarchical pore size distributions (e.g., larger pores surrounded by smaller, monodisperse pores).
Yet, another advantage, particularly in a commercial setting, is the ability to provide a vast repertoire of polyHIPEs simply by adjusting the ratio of a first high internal phase emulsion composition to a second high internal phase emulsion composition, which will affect the rate of polymerization or time to cure without changing initial HIPE fabrication parameters. When one of the first high internal phase emulsion composition or the second high internal phase emulsion composition contains an additive or biologic constituent (either in its organic or aqueous phase), the final concentration in the polyHIPE can be readily altered by again simply changing the ratio of either or both of the starting HIPEs. Further modifications may be made to polyHIPEs prepared ex vivo by modifying the cure temperature.
Previously described PFDMA macromers have been used to fabricate polyHIPEs using a large amount of a free radical oxidizing initiator alone, including either 30 wt. % ammonium persulfate (APS) or 5 wt. % BPO; such polyHIPEs were found to take at least 2 hours to set for 1 g of PFDMA polyHIPE (Moglia R S, Holm J L, Sears N A, Wilson C J, Harrison D M, and Cosgriff-Hernandez E. Biomacromolecules 2011; 12(10):3621-3628). The resulting PFDMA macromer had a sufficiently low viscosity (about 125 cP) and hydrophobicity to permit HIPE formation. These PFDMA macromers also had a single fumarate unit with two terminal methacrylate groups. The average functionalization was calculated to be greater than 80%. In some embodiments, functionalization was calculated to be at or about 83%. The methacrylate and fumarate groups provided sites for radical crosslinking and polyHIPE fabrication.
In a first example, propylene fumarate dimethacrylate (PFDMA) polyHIPEs were prepared using a similarly prepared PFDMA macromer as described in Moglia et al. The PFDMA macromer has a general structure as depicted in
In a second example, ethylene glycol dimethacrylate (EGDMA) polyHIPEs were prepared using an EGDMA macromer having a general structure as depicted in
Combinations of the first HIPE and the second HIPE were incubated together and molded in a manner similar to what was described in the first example to form porous polyHIPEs. These redox formed polyHIPEs were evaluated for their time to cure and compared to the time to cure of the first HIPEs and the second HIPES (when uncombined, therefore containing either only an oxidizing initiator or only a reducing initiator/agent). It was found that the time to cure for the redox formed polyHIPEs could be controlled. Not only was the cure time dramatically reduced when redox initiators were used, but by altering the amount of redox initiators, the time to cure could be quickly and easily manipulated. Thus, fast curing polyHIPEs were created that could cure in less than comparative times (which were at least two hours). As described herein, cure times were less than two hours. They could be designed to cure for much less than two hours. For example, the cure times could be for less than 1 hour, or less than thirty minutes, or less than about 20 minutes to as little as 30 seconds. Some representative time to cure values for the first HIPEs (alone), the second HIPEs (alone) and the combinations are provided in TABLE 2.
The redox formed polyHIPEs (ones prepared with a combination of a first HIPE and second HIPE) were analyzed by scanning electron microscopy when fully cured. For SEM sampling, circular specimens were sectioned into quarters, fractured at the center of the quarter, sputter-coated with gold, and imaged using FE-SEM (JEOL JSM-7500F). Images at 250× were used to determine the average pore size when the pores were 25-100 μm. Higher magnification (500×, 1000×) images were utilized to determine the average pore size when the pores were less than 25 μm. Each section was imaged in a raster pattern yielding five images. Measurements were made on at least the first 10 pores along the image median to minimize user bias. Averages pore sizes for each polyHIPE composition are shown (n=150). A statistical correction was calculated to account for non-perfect spherical pores, h2=R2−r2, where R is the void diameter's equatorial value, r is the diameter value measured from the micrograph, and h is the distance from the center. The average diameter values were multiplied by this correction factor resulting in a more accurate description of pore diameter. Representative SEM images are provided in which the average pore size was found to increase with increasing concentration of redox initiator pairs (see
For mechanical testing, redox formed polyHIPEs were mechanically tested with an Instron 3300, equipped with a 1000-N load cell. Generally, three specimens were taken from each sample. The data was then averaged from three sections for each sample tested (n=9). The test specimens were cut into flat rectangular shapes (9×9×3 mm) and compressed at 50 μm/s. Calculations were generally in accordance with ASTM method D1621-04a to determine the compressive modulus. A straight edge and computer software were used to determine the linear region of the stress-strain curve by extending a line from the steepest slope of the curve to the zero-load axis. The point at which this line crossed the axis was determined to be where strain equaled zero and all data points were shifted accordingly. The elastic modulus was equal to the slope of the line in the linear region, as outlined in ASTM D1621-04a. On average, the compressive modulus was dependent on initiator concentration and macromer chemistry. A maximum compression modulus was about 46 MPa and a maximum strength was about 5 MPa.
In a third example, butanediol dimethacrylate (BDMA) polyHIPEs were prepared using a BDMA macromer having a general structure as depicted in
In a fourth example, methacrylic anhydride (MA) polyHIPEs were prepared using a MA macromer as depicted in
Representative systems and methods are further described below for fabrication of injectable polyHIPEs prepared from biodegradable macromers, said polyHIPEs capable of being stored for months at a time and then still able to cure rapidly in situ. For these examples, two separate but near-identical HIPEs (1 and 2) are used, as depicted in
In the below examples, three materials are prepared: ethylene glycol dimethacrylate (EGDMA), butanediol dimethacrylate (BDMA), and propylene fumarate dimethacrylate (PFDMA). The effects of redox concentration and ratio on cure time, pore architecture, and compressive modulus and strength were evaluated in relation to use in orthopedic applications, and demonstrated the applicability of fabricating the described rapid-curing polyHIPEs that have long shelf-lives and subsequent use as tissue engineered bone grafts.
PFDMA was synthesized in a two-step process. First, propylene oxide was added drop wise to a solution of fumaric acid and pyridine in 2-butanone (2.3:1.0:0.033 mol) and refluxed at 75° C. for 18 hours. Residual propylene oxide and 2-butatone were removed by distillation and the product redissolved in dichloromethane. Residual acidic byproducts and water were removed with washing, and the product dried under vacuum to yield the diester bis (1,2 hydroxypropyl) fumarate product. The diester was then endcapped with methacrylate groups using methacryloyl chloride in the presence of triethylamine. The molar ratios of the diester, methacryloyl chloride, triethylamine were 1:2.1:2.1, respectively. Hydroquinone was added to the diester to inhibit crosslinking during synthesis at a molar ratio of 0.008:1. The reaction was maintained below −10° C. to reduce undesirable side reactions and stirred vigorously under a nitrogen blanket. The macromer was neutralized overnight with 2 M potassium carbonate. Residual triethylamine and methacrylic acid were removed with an aluminum oxide column (7 Al2O3:1 TEA). The integration ratio of methacrylate protons to fumarate protons in the 1H NMR spectra was used to confirm >90% functionalization for all macromers prior to polyHIPE fabrication. (300 MHz, CdCl3) δ 1.33 (dd, 3H, CH3), 1.92 (s, 3H, CH3), 4.20 (m, 2H, —CH2), 5.30 (m, 1H, —CH), 5.58 (s, 1H, —C═CH2), 6.10 (s, 1H, —C═CH2), 6.84 (m, 2H, —CH═CH−).
EGDMA and BDMA (purchased from Sigma Aldrich) were purified to remove inhibitors prior to use. The macromers were filtered through an aluminum oxide column to remove monomethyl ether hydroquinone. The purified products were stored at 4° C. under a nitrogen blanket until used for HIPE fabrication.
HIPEs were fabricated using a FlackTek Speedmixer DAC 150 FVZ-K. Briefly, a macromer was mixed with 10 wt. % PGPR 4125 and benzoyl peroxide (varied from 0.5-5.0 wt. %) prior to emulsification. A second mixture consisting of macromer, 10 wt. % PGPR, and a varied amount of trimethylaniline (TMA, 0.5-5.0 wt. %) was also combined prior to emulsification. Once both were thoroughly mixed, an aqueous solution of calcium chloride (1 wt. %) was then added to the organic phases (75% v) in 3 additions and mixed at 500 rpm for 2.5 minutes each. HIPEs were placed in double barrel syringe and the two components mixed upon injection using a static mixing head (5 mL syringe with 3 cm straight mixer, Sulzer Mixpac K-System). HIPEs were then placed in a 37° C. bath to initiate polymerization (approximately 10 minutes). (See also
PolyHIPE cure times were characterized using an Anton Paar MCR 301 rheometer. HIPEs were injected through a mixing head to facilitate redox initiation directly onto the 37° C. plate. Storage and loss moduli were measured every 15 seconds using a parallel-plate configuration with a 1 mm gap and 0.5% strain. Work time was presented as the onset of increasing storage modulus and set time was presented as the tan 6 minimum, which corresponds to storage modulus yielding.
PolyHIPEs were dried in vacuo for about 24 hours to remove water prior to characterization of pore architecture. Average pore and interconnect size of each composition was determined using scanning electron microscopy (SEM, JEOL 6500). Circular specimens from three separate polyHIPE specimens were sectioned into quarters and fractured at the center. Each specimen was coated with gold and imaged in a rastor pattern yielding five images. Pore size measurements were completed on the first ten pores that crossed the median of each 500× magnification micrograph. Average pore sizes for each polyHIPE composition were reported (n=450). A statistical correction was calculated to account for the random fracture plane through spherical voids and pores, 2/√{square root over (3)}. Average diameter values were multiplied by this correction factor resulting in a more accurate pore diameter description.
PolyHIPE compressive properties were measured using an Instron 3300 equipped with a 1000-N load cell. ASTM D1621-04a was utilized to determine the compressive modulus and strength of the polyHIPEs. Each polyHIPE specimen was sectioned into three discs (approximately 15 mm diameter, 5 mm thick) using an Isomet® saw (registered with Illinois Tool Works Inc. Corporation, Glenview, Ill.) and compressed at a strain rate of 50 μm/s. The compressive modulus was calculated from the slope of the linear region after correcting for zero strain and the compressive strength was identified as the stress at the yield point or 10% strain, whichever occurred first. Reported moduli and strength data were averages of 9 specimens for each composition tested.
Gel fraction was measured gravimetrically to evaluate the extent of network formation. After curing for about 24 hours, polyHIPE samples were sectioned into about 15 mm by about 1 mm discs. Mass was recorded for each specimen after vacuum drying for 48 hours, incubating in 100× dichloromethane at 20° C. for about 48 hours, and vacuum drying again until a constant mass was achieved. The final weight divided by the initial weight was assessed as the gel fraction.
For storage analysis, uncured PFDMA HIPEs were stored at 4° C. for up to six months and sampled each month to determine the impact of storage on polyHIPE architecture and mechanical properties. After a sample was removed, it was thawed for 60 minutes then injected through a syringe and cured for 48 hours prior to characterization, as described above.
Investigation of macromer cytocompatibility was performed initially prior to seeding cells directly on polyHIPE sections. The in vitro cytocompatibility of BDMA, EGDMA, and PFDMA was assessed using a modified ISO 10993-5 extraction dilution test. All macromers were further purified by washing in deionized water at a volume ratio of 1:100 and incubated in vacuum for about 24 hours prior to the experiment. Bone marrow-derived human mesenchymal stem cells (hMSCs) were obtained as Passage 1 in a cryovial from the Center for the Preparation and Distribution of Adult Stem Cells. Cells were cultured in growth media containing 16.5% fetal bovine serum (Atlanta Biologicals), 1% L-glutamine (Life Technologies) and Minimum Essential Media α (MEM α, Life Technologies) to about 80% confluency and utilized at Passages 5 and 6. Cells were trypsinized with 0.25% Trypsin-EDTA (Life Technologies) and seeded at a density of about 40,000 cells/cm2 in a 96 well plate and allowed to adhere for about 24 hours. On the following day, about 100 μl of each macromer was incubated in about 300 μl growth media supplemented with 1 vol. % penicillin-streptomycin (Life Technologies) in a 48 well plate to mimic the ratio of organic to aqueous phase in the HIPE. After about 10 minute incubation at about 37° C., 5% CO2, the supernatant above the macromers was aspirated and diluted to about 10× and about 100× solutions. This time frame was selected based on the cure rates determined previously for these macromers which approximates the maximum extraction of unreacted macromer prior to cure. Extracted and diluted media (1, 10, and 100×) was then added to cells and cultured for an additional time, generally about 24 hours. Viability was assessed utilizing the Live/Dead® assay kit (registration with Molecular Probes Corporation, Eugene, Oreg.). The analysis used a plate reader (Tecan Infinite M200Pro) with excitation/emission wavelengths of 485/528 and 528/620 for calcein-AM and ethidium homodimer-1 dyes, respectively. Viability was normalized to cells on tissue culture polystyrene.
The viability of hMSCs directly seeded on cured polyHIPEs was assessed to illustrate cytocompatibility of quick-curing redox foams. PolyHIPEs were fabricated as stated above and sectioned into about 500 μm thick wafers using an Isomet® saw. Specimens were sterilized for about 3 hours in about 70% ethanol, subjected to a wetting ladder, washed 4 times with PBS, and incubated overnight in MEM α supplemented with 40 v/v % FBS at about 5% CO2, 37° C. Cells were seeded at about 25,000 cells/cm2 (for EGDMA and BDMA) and about 100,000 cells/cm2 (for PFDMA) in growth media supplemented with 1 vol. % penicillin-streptomycin and cultured for about 3 hours and about 24 hours. Cell viability was assessed as described above. Rastor imaging (5 images per specimen) was conducted on four specimens (n=20) utilizing a fluorescent microscope (Nikon Eclipse TE2000-S). Cells were manually counted to quantify viability.
Data described and/or shown are provided as a mean±standard deviation for each composition. A Student's t-test was performed to determine any statistically significant differences between compositions. All tests were carried out at a 95% confidence interval (P<0.05).
Prior to curing, all HIPEs flowed like viscous fluids but were rheologically similar to gels (E′>E″), as would be expected for HIPEs. Their moduli remained relatively constant before curing began, indicating that the emulsions were stable without significant phase separation. Work time was defined by ISO1997 as the “period of time, measured from the start of mixing, during which it is possible to manipulate a dental material without an adverse effect on its properties” and set time is accepted as the point at which a polymer network is formed. While previous cure times for PFDMA have been approximately 2 hours (with 5 wt. % BPO), the previous cure times for both EGDMA and BDMA were over 10 hours to set with thermal initiation alone. As described herein, using a reducing agent TMA in combination with BPO, it was found that both work and set time for all materials were reduced from 2+ hours to only minutes. This corresponded to an order of magnitude increase in rate as compared with thermal initiation alone. Increasing total redox initiator concentration from 0.5 wt. % to 5 wt. % was also found to decrease both work and set times for all materials, as depicted in
Additional rheological and gel fraction data were analyzed to investigate the impact of redox initiation concentration on network formation in candidate polyHIPEs. In each material, an induction period was evident prior to an increase in modulus that was dependent on macromer chemistry (EGDMA<BDMA<PFDMA). The induction period appeared to be primarily responsible for the difference in cure times, as depicted in
Gel fraction was used to compare an extent of network formation in polyHIPEs after 24 hours of curing. Network formation ranged from 78% to 92% for all compositions, depicted in TABLES 3 and 4. As expected, increasing initiator concentration correlated with increased gel fraction. PFDMA gel fraction increased the most, from 78% to 86%. BDMA gel fraction also increased significantly with higher initiator concentrations (86% to 92%). EGDMA gel fractions increased from 86% to 89%. Both EGDMA and BDMA had significantly higher gel fractions than the corresponding PFDMA polyHIPEs, likely due to steric hindrance and reduced radical diffusion associated with its higher molecular weight. Additionally, it is possible that highly crosslinked microgels, when formed, begin to sterically hinder further crosslinking, increasing network defects and free-ends. It should be noted that PGPR was not removed from the specimens prior to DCM incubation and this should account for approximately 9% of specimen mass. FTIR spectroscopy of the extract solutions showed the presence of PGPR, but the concentration was not quantified (data not shown). Assuming all of the PGPR was removed with the DCM, gel fractions were actually between 94% and 100%. Overall, these polyHIPEs showed excellent network formation that was further enhanced at higher initiator concentration. In TABLES 3 and 4: *=P<0.001 compared to EGDMA 1.0 and 5.0 wt. % pore sizes; †=P<0.05 compared to BDMA 0.5 and 1.0 wt. % pore sizes; ‡=P<0.01 compared to PFDMA 1.0 and 5.0 wt. % pore sizes; and =P<0.01 compared to 0.5:1.0 and 5.0:1.0 TMA:BPO pore sizes.
27 ± 12 *
5 ± 3 ‡
The relationship between the rapid, redox-initiated cure and polyHIPE micro-architecture was examined. First, it was found that desirable pore size and interconnection was retained. In the representative examples, EGDMA polyHIPEs possessed the largest pore diameters, almost double the size of BDMA and quadruple the size of PFDMA pores at each initiator concentration. Traditionally, pore size has been used as a marker of emulsion stability with smaller pore size indicating enhanced stability and reduced droplet coalescence prior to the gel point. Here, increased pore size was found to correlate with decreasing HIPE viscosity: PFDMA (11.0 Pa*s), BDMA (0.464 Pa*s) and EGDMA (0.343 Pa*s). Pa*s is the viscosity measure in Pascal seconds. Despite differences between materials, scanning electron micrographs revealed that average pore and interconnect diameter were generally not affected by redox initiator concentration for most tested materials (
With use of a double-delivery system, such as a double-barrel device, or a similar type mechanism or device, two HIPEs may be stored separately until needed. This extends shelf-life of the described HIPEs, especially since said HIPEs may be stored at reduced temperatures. HIPEs can be stored at least at 4° C., or at lower temperatures. In an example, various PFDMA HIPE samples were stored at about 4° C. and removed and cured (polymerized to form polyHIPEs) at various time points, including 1 month (
The polyHIPEs described herein set within several minutes, and were further characterized on sections taken, generally after a 24 hour cure time. Compressive modulus and strength were found to increase as redox initiator concentration increased for all representative materials tested, as depicted in
Representative compressive loading curves are presented for various representative materials at differing redox initiator concentration (see
Overall, it was found that these porous materials, when formed as a monolith, had compressive properties that approached that of cancellous bone when matched by density. Thus, said materials may be used to mechanically stabilize a defect and elicit the appropriate mechanical cues to regenerate a tissue such as bone. Since some studies have shown that the mechanical properties required to trigger bone formation may be much lower than those of fully matured bone tissue, these materials may be suitably manipulated as desired. It was also found that with the described redox initiator system used herein, there was a rapid maturation of mechanical properties as compared to thermal initiation alone (
The ratio of reductant component to oxidant component was further investigated. In short, as expected, increasing the relative amount of TMA to BPO resulted in decreased work and set times, e.g., from 90 to 30 seconds (work time) and 2.5 to 1 minute (set time) (
The above findings demonstrate that polyHIPE work and set time may be tuned independently from other polyHIPE properties (compressive modulus/strength, pore or interconnect diameter, architecture) with small variations in the reductant:oxidant ratio. Thus, when the described polyHIPE compositions are provided as a device, or as a tissue engineered graft, there is the opportunity to tune physical properties, to adjust for cytocompatibility and to optimize work and set time to meet clinical demands and/or preferences.
The described compositions did not reveal cytotoxic effects. For example, in studies analyzing the surfactant PGPR, there was no apparent direct cellular exposure of the surfactant at any of the concentrations described (>95% viability). Assessment of hMSC viability was also performed after exposure to extract media from unreacted macromers and direct seeding onto cured polyHIPE grafts. This provides an assessment as to whether there is an effect from exposure of the described macromer to cells prior to crosslinking in situ. Cell viability of hMSCs after exposure to PFDMA extract media was high (>80%)(
Cells delivery via the described polyHIPE composition delivery system is also disclosed. This allows viable cells (e.g., autologous hMSCs) to be delivered to a desired physiologic site (e.g., defect or damage site), promoting healing and reducing cell or immune rejection, often found when allogeneic tissues are delivered.
Cells are encapsulated in the polyHIPE upon injection, meaning that upon curing, the cells remain encapsulated in the rigid, highly porous foam when said polyHIPE is fully cured. This overcomes problems associated with mechanical weakness in typical hydrogel systems. The injectable polyHIPEs when formed create foam bodies that serve as a protective substrate for delivery of cells or other bioactive or biologic materials. Furthermore, the rigid foam bodies, as shown above, can withstand physiological loading conditions.
To ensure encapsulation and maintenance of cell viability it has been found that the described injectable polyHIPEs offer both adequate viscosity for proper mixing with a cell suspension and a quick cure time, which reduced time for diffusion of unreacted macromer and radicals into the cell-laden aqueous phase or the diffusion of cells away from the emulsion. As depicted in
In one example, a BDMA HIPE with 0.5 wt. % BPO and 2.5 wt. % ferrocene was fabricated with basal media as the aqueous phase and loaded in the larger side of a 10:1 double barrel syringe. Cells (hMSCs) at a fifth passage were trypsinized and loaded into the smaller unit in a suspension to ensure a 10:1 ratio. This head was of a larger diameter and length to ensure proper mixing of the HIPE components with the cell suspension. It is possible that a further decrease in the viscosity of the organic phase may allow the use of a 1:1 double barrel while maintaining successful mixing of the two phases.
A representative micrograph of hMSC viability in sections of the BDMA polyHIPE structure when formed is shown in
The pore architecture after cell encapsulation is illustrated in
PolyHIPE compositions that are cytocompatible and can be manipulated to polymerize rapidly, as desired, and methods for making said polyHIPE compositions are disclosed. These polyHIPE compositions polymerize via a redox reaction, are fast curing, and can be suitably controlled to optimize cure time, porosity, pore shape, and/or hardness. Said polyHIPE compositions are capable of curing ex vivo or in vivo, are deliverable to a specific site, and are biodegradable. The polyHIPE compositions may be used in vivo and are also amenable for use in other applications requiring a porous polymeric material.
A low temperature polymerization as described herein allows the compositions to form in vivo or ex vivo, and when formed to be applicable for use in vivo or ex vivo. Moreover, the compositions described herein may incorporate one or more additional components prior to polymerization, including highly sensitive components (biologic or non-biologic materials), including but not limited to cells, growth factors, enzymes, and proteins. It is also understood that while the preparations and polymerizations described herein may occur at physiologic temperatures, they may also occur at other temperatures, even at a temperature that is up to or higher than 100° C.
As described, the combined initiators described herein (redox-initiator) when combined with the biodegradable HIPEs described herein and formed via a double delivery system provide suitable polyHIPE compositions for use as a tissue scaffold, replacement, and/or graft. The combined initiators described herein reduced work and set times for the described polyHIPEs from hours to minutes, matching current cement products, such as PMMA. These reductions in cure times were achieved with lower total concentration of the initiator, which may be, in part, for the enhanced cytocompatibility of the described polyHIPEs.
Compressive modulus and strength may be manipulated with adjusting redox initiator concentration with minimal impact on pore architecture. Further modulation of the reductant:oxidant ratio decoupled set time from compressive modulus and strength allowing for increased tunability when forming an ex vivo, in vivo or in situ scaffold. The compressive modulus and strength values for the described formed rigid foam structures are higher than was is found in typical hydrogel matrices. Said values are comparable with values shown to be suitable for promoting healing.
Use of a double delivery system for introducing polyHIPEs ex vivo, in vivo or in situ, as described herein, also permits emulsions to be prepared and stored well in advance of use. Preparation and storage may be many months, and likely even a year prior to use. Storage may be at an ambient temperature. In some embodiments, storage is preferably at sub ambient temperatures, and reduced temperatures (e.g., at refrigerated temperatures, such as at or below 4° C.) to prevent possible slow curing. The double delivery system described herein also allows for curing at a rate that is less than two hours, such as in about 100 minutes, or less than about 100 minutes. Rapid curing may also be less than about 90 minutes, or less than about 60 minutes, or may be less than about 50 minutes, or may be less than about 45 minutes, or may be less than about 40 minutes, or may be less than about 30 minutes, or at or less than about 25 minutes, or at or less than about 20 minutes, at or less than about 15 minutes, or at or less than about 10 minutes, or at or less than about 5 minutes, or at or less than about 4 minutes, or at or less than about 3 minutes, or at or less than about 2 minutes, or at or less than about 1 minutes, or at or less than about 30 seconds, or any range there between. The in situ delivery of the described polyHIPEs provides an on-demand structure without sacrificing porosity or mechanical properties.
Morphology of the described polyHIPEs are well suited for cell adhesion and cell growth. Said morphology and architecture also translate into a support scaffold that permits physiologic loading.
In one or more embodiments, polymerizable HIPEs are injectable in situ for use in filling irregular bone defects. Said polyHIPEs when fully polyermized serve as rigid support structure having various degrees of porosity. Said structure as porous scaffolds may potentially improve tissue healing.
As described are HIPE compositions that remain stable without initiating polymerization or are capable of being prepared so that polymerization is significantly delayed (as compared with HIPE compositions comprised of similar macromers for polymerization). The HIPEs are prepared from starting biodegradable material (e.g., macromer) that forms a resulting biodegradable polymeric material with a specific and appropriate viscosity and hydrophobicity for emulsification when stabilized with an emulsifier. The macromer will also include certain structural features (e.g., ester or anhydride linkages; hydrogen bond acceptor sites; unsaturated end groups).
In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measured cannot be used or used to an advantage.
While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims.
This application claims the benefit of U.S. Provisional Patent Application No. 61/881,406 filed Sep. 23, 2013, the entirety of which is incorporated herein by reference.
The disclosed was made with government support under 1R21AR057531 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US14/57025 | 9/23/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61881406 | Sep 2013 | US |
