Patent Application
20140271613
The present disclosure relates to hydrogels. Specifically, the disclosure relates to multi-component hydrogels which may include bioactive agents or cells therein.
Hydrogels are three-dimensional networks which swell in the presence of aqueous solutions. In a swollen state, hydrogels are soft and rubbery, resembling living tissue.
Biocompatible cross-linked polymers having electrophilic and nucleophilic functional groups are known for their use in the formation of hydrogels. These hydrogels may be used in a variety of surgical applications. Hydrogels may be utilized, for example, as a sealant for wounds, as a glue for adhering surgical implants to tissue, as drug delivery devices, as coatings on tissue and/or medical devices, combinations thereof, and the like.
Improved hydrogels, which may be capable of delivering cells and bioactive agents, remain desirable.
The present disclosure provides therapeutic hydrogels and methods for forming the same. In embodiments, a therapeutic hydrogel of the present disclosure includes a first hydrogel precursor including a natural component such as collagen, serum, gelatin, hyaluronic acid, and combinations thereof; a second hydrogel precursor including an electrophilic functional group such as n-hydroxysuccinimides, succinimidyl succinates, succinimidyl propionates, sulfosuccinimides, isocyanates, thiocyanates, carbodiimides, benzotriazole carbonates, and combinations thereof; and cells for delivery to a patient, wherein the first hydrogel precursor and the second hydrogel precursor react to form the therapeutic hydrogel, and wherein the therapeutic hydrogel has an osmolarity from about 200 mOsm/L to about 400 mOsm/L.
As noted above, methods for forming these hydrogels are also provided. In embodiments, a method of the present disclosure includes contacting a first hydrogel precursor including a natural component with a first buffer; contacting a second hydrogel precursor including an electrophilic functional group with a second buffer; adding cells to the first hydrogel precursor, the second hydrogel precursor, or both; contacting the first hydrogel precursor and the second hydrogel precursor; and forming a therapeutic hydrogel from the first hydrogel precursor and the second hydrogel precursor, wherein the therapeutic hydrogel has an osmolarity from about 200 mOsm/L to about 400 mOsm/L.
Various embodiments of the present disclosure will be described herein with reference to the following figures, wherein:
The present disclosure provides hydrogels which include an electrophilic precursor, sometimes referred to herein as an electrophilic crosslinker, and a nucleophilic component. In embodiments, the nucleophilic component is a natural component, which may be cross-linked by the electrophilic cross-linker to form a hydrogel. In embodiments, the hydrogel may be biodegradable. The term “biodegradable” as used herein is defined to include both bioabsorbable and bioresorbable materials. By biodegradable, it is meant that the materials decompose or lose structural integrity within a clinically relevant time period, under body conditions (e.g., enzymatic degradation or hydrolysis), or are broken down (physically or chemically) under physiologic conditions in the body such that the degradation products are excretable or absorbable by the body. In embodiments, biodegradable compositions of the present disclosure may degrade over a period of time from about 1 week to about 12 months, in embodiments from about 2 weeks to about 9 months, in embodiments from about 3 weeks to about 7 months, in embodiments from about 1 month to about 6 months.
The hydrogel may be formed in situ and may be utilized as a drug delivery device or tissue scaffold, combinations thereof, and the like. In embodiments, the hydrogel may deliver and/or release biological factors/molecules and/or cells at the site of implantation. Thus, where the hydrogel of the present disclosure is utilized as a tissue scaffold, it may assist in native tissue regrowth by providing the surrounding tissue with needed nutrients and bioactive agents.
In some embodiments, as discussed further below, the hydrogel itself may include a natural component such as collagen, gelatin, hyaluronic acid, combinations thereof, and the like, and thus the natural component may be released at the site of implantation as the hydrogel degrades. The term “natural component,” as used herein, includes polymers, compositions of matter, materials, combinations thereof, and the like, which can be found in nature or derived from compositions/organisms found in nature. Natural components may also include compositions which are found in nature but can be synthesized by man, for example, using methods to create natural/synthetic/biologic recombinant materials, as well as methods capable of producing proteins with the same sequences as those found in nature, and/or methods capable of producing materials with the same structure and components as natural materials, such as synthetic hyaluronic acid, which is commercially available, for example, from Sigma Aldrich.
The components for forming hydrogels of the present disclosure on or in tissues include, for example, in situ forming materials. The in situ forming material(s) may include a single precursor or multiple precursors that form “in situ”, meaning formation occurs at a tissue in a living animal or human body. In general, this may be accomplished by having a precursor that can be activated at the time of application to create, in embodiments, a hydrogel. Activation can be through a variety of methods including, but not limited to, environmental changes such as pH, ionicity, temperature, etc. In other embodiments, the components for forming a hydrogel may be contacted outside the body and then introduced into a patient as an implant such as a (pre-formed) tissue scaffold.
The hydrogels of the present disclosure may, in embodiments, be used to deliver bioactive agents, cells, combinations thereof, and the like, to a patient. For tissue engineering and regenerative medicine strategies, it may be desirable to encapsulate cells for culture in vitro or to deliver cells in vivo. In accordance with the present disclosure, methods are provided for encapsulation of cells in a nucleophilic-electrophilic reactive hydrogel.
Formation of the hydrogels of the present disclosure may occur in the presence of cell-friendly salt solutions and buffer solutions with appropriate osmolarity, concentration, and pH. The use of such solutions allows for tailored gelation kinetics of the resulting hydrogel, while maintaining approximately neutral pH after hydrogel formation. This is important because some electrophilic groups, such as —NHS groups, may cleave from the electrophilic component during the reaction of the electrophilic and nucleophilic components. These groups are acidic, and thus may cause a drop in pH. In accordance with the present disclosure, buffers utilized in forming these hydrogels prevent the pH from dropping too low, and maintains a neutral pH (7.2±0.3) with periodic media changes (static cultures) or in a dynamic environment (bioreactors), which is necessary to maintain cell viability.
As noted above, in situ forming materials may be made from one or more precursors. The precursor may be, e.g., a monomer or a macromer. One type of precursor may have a functional group that is an electrophile or nucleophile. Electrophiles react with nucleophiles to form covalent bonds. Covalent crosslinks or bonds refer to chemical groups formed by reaction of functional groups on different polymers that serve to covalently bind the different polymers to each other. In certain embodiments, a first set of electrophilic functional groups on a first precursor may react with a second set of nucleophilic functional groups on a second precursor. When the precursors are mixed in an environment that permits reaction (e.g., as relating to pH or solvent), the functional groups react with each other to form covalent bonds. The precursors become crosslinked when at least some of the precursors can react with more than one other precursor. For instance, a precursor with two functional groups of a first type may be reacted with a crosslinking precursor that has at least three functional groups of a second type capable of reacting with the first type of functional groups.
In embodiments the hydrogel may be formed from a single precursor or multiple precursors. For example, where the hydrogel is formed from multiple precursors, for example two precursors, the precursors may be referred to as a first and second hydrogel precursor. The terms “first hydrogel precursor” and “second hydrogel precursor” each mean a polymer, functional polymer, macromolecule, small molecule, or crosslinker that can take part in a reaction to form a network of crosslinked molecules, e.g., a hydrogel. The term “functional group,” as used herein, refers to electrophilic or nucleophilic groups capable of reacting with each other to form a bond.
Electrophilic functional groups include, for example, N-hydroxysuccinimides (“NHS”), sulfosuccinimides, carbonyldiimidazole, sulfonyl chloride, aryl halides, sulfosuccinimidyl esters, N-hydroxysuccinimidyl esters, succinimidyl esters such as succinimidyl succinates and/or succinimidyl propionates, isocyanates, thiocyanates, carbodiimides, benzotriazole carbonates, epoxides, aldehydes, maleimides, imidoesters, combinations thereof, and the like. In embodiments, the electrophilic functional group is a succinimidyl ester.
The electrophilic hydrogel precursors may have biologically inert and water soluble cores, as well as non-water soluble cores. When the core is a polymeric region that is water soluble, suitable polymers that may be used include: polyethers, for example, polyalkylene oxides such as polyethylene glycol (“PEG”), polyethylene oxide (“PEO”), polyethylene oxide-co-polypropylene oxide (“PPO”), co-polyethylene oxide block or random copolymers, and polyvinyl alcohol (“PVA”); poly(vinyl pyrrolidinone) (“PVP”); poly(amino acids); poly (saccharides), such as dextran, chitosan, alginates, carboxymethylcellulose, oxidized cellulose, hydroxyethylcellulose, hydroxymethylcellulose; hyaluronic acid; and proteins such as albumin, collagen, casein, and gelatin. In embodiments, combinations of the foregoing polymers may be utilized.
The polyethers, and more particularly poly(oxyalkylenes) or poly(ethylene glycol) or polyethylene glycol, may be utilized in some embodiments. When the core is small in molecular nature, any of a variety of hydrophilic functionalities can be used to make the first and second hydrogel precursors water soluble. For example, functional groups like hydroxyl, amine, sulfonate and carboxylate, which are water soluble, may be used to make the precursor water soluble. For example, the n-hydroxysuccinimide (“NHS”) ester of subaric acid is insoluble in water, but by adding a sulfonate group to the succinimide ring, the NHS ester of subaric acid may be made water soluble, without affecting its reactivity towards amine groups.
Each of the first and second hydrogel precursors may be multifunctional, meaning that it may include two or more electrophilic or nucleophilic functional groups, such that, for example, a nucleophilic functional group on the first hydrogel precursor may react with an electrophilic functional group on the second hydrogel precursor to form a covalent bond. At least one of the first or second hydrogel precursors includes more than two functional groups, so that, as a result of electrophilic-nucleophilic reactions, the precursors combine to form cross-linked polymeric products.
The macromolecule having the electrophilic functional group may be multi-armed. For example, the macromolecule may be a multi-armed PEG having four, six, eight, or more arms extending from a core. The core may be the same or different from the macromolecule forming the arms. For example, the core may be PEG and the multiple arms may also be PEG. In embodiments, the core may be a natural polymer.
The molecular weight (MW) of the electrophilic crosslinker may be from about 2,000 to about 100,000; in embodiments from about 10,000 to about 40,000. Multi-arm precursors may have a molecular weight that varies depending on the number of arms. For example, an arm having a 1000 MW of PEG has enough CH2CH2O groups to total at least 1000 MW. The combined molecular weight of an individual arm may be from about 250 to about 5,000; in embodiments from about 1,000 to about 3,000; in embodiments from about 1,250 to about 2,500. In embodiments, the electrophilic crosslinker may be a multi-arm PEG functionalized with multiple NHS groups having, for example, four, six or eight arms and a molecular weight from about 5,000 to about 25,000. Other examples of suitable precursors are described in U.S. Pat. Nos. 6,152,943; 6,165,201; 6,179,862; 6,514,534; 6,566,406; 6,605,294; 6,673,093; 6,703,047; 6,818,018; 7,009,034; and 7,347,850, the entire disclosures of each of which are incorporated herein by reference.
The electrophilic precursor may be a cross-linker that provides an electrophilic functional group capable of bonding with nucleophiles on another component, in embodiments a natural component. The natural component may be endogenous to the patient to which the electrophilic crosslinker is applied, or may be exogenously applied.
In embodiments, one of the precursors may be a natural component possessing nucleophilic groups. Nucleophilic groups which may be present include, for example, —NH2, —SH, —OH, —PH2, and —CO—NH—NH2.
The nucleophilic component, in embodiments a natural component, may be, for example, collagen, gelatin, blood (including serum, which may be whole serum or extracts therefrom), hyaluronic acid, proteins, albumin, other serum proteins, serum concentrates, platelet rich plasma (prp), combinations thereof, and the like. Additional suitable natural components which may be utilized or added to another natural component, sometimes referred to herein as a bioactive agent, include, for example, stem cells, DNA, RNA, enzymes, growth factors, peptides, polypeptides, antibodies, other nitrogenous natural molecules, combinations thereof, and the like. Other natural components may include derivatives of the foregoing, for example modified hyaluronic acid, dextran, other polysaccharides, polymers and/or polypeptides, including aminated polysaccharides which may be naturally derived, synthetic, or biologically derived. For example, in embodiments, hyaluronic acid may be modified to make it nucleophilic.
In embodiments, any of the above natural components may be synthetically prepared, e.g., synthetic hyaluronic acid, utilizing methods within the purview of those skilled in the art. Similarly, in embodiments the natural component could be a natural or synthetic long chain aminated polymer. The natural component may also be modified, i.e., aminated, to create a nucleophilic polymer.
The natural component may provide cellular building blocks or cellular nutrients to the tissue that it contacts in situ. For example, serum contains proteins, glucose, clotting factors, mineral ions, and hormones, which may be useful in the formation or regeneration of tissue.
In embodiments, the natural component includes whole serum. In embodiments, the natural component is autologous, i.e., collagen, serum, blood, and the like, from the body where the hydrogel is (or is to be) formed. In this manner, the person or animal in which the hydrogel is to be used may provide the natural component for use in formation of the hydrogel. In such embodiments, the resulting hydrogel is semi-autologous, including a synthetic electrophilic precursor and an autologous/endogenous nucleophilic precursor.
In embodiments, a multifunctional nucleophilic polymer, such as a natural component having multiple amine groups, may be used as a first hydrogel precursor and a multifunctional electrophilic polymer, such as a multi-arm PEG functionalized with multiple NHS groups, may be used as a second hydrogel precursor. In embodiments, the precursors may be in solution(s), which may be combined to permit formation of the hydrogel. Any solutions utilized as part of the in situ forming material system should not contain harmful or toxic solvents. In embodiments, the precursor(s) may be substantially soluble in a solvent such as water to allow application in a physiologically-compatible solution, such as buffered isotonic saline.
In embodiments, a hydrogel may be formed from a combination of collagen and/or gelatin as the natural component, with a multi-functional PEG utilized as a crosslinker. In embodiments, the collagen and/or gelatin may be placed in solution, utilizing a suitable solvent. Such solvents may also include buffers and/or buffered solutions. Such a buffer may have a pH from about 8 to about 12, in embodiments from about 8.2 to about 9. Examples of suitable buffers include, but are not limited to, borate buffers, saline solutions, Hanks Balanced Salt Solution, Dulbecco's Modified Eagle's Medium, Phosphate Buffered Saline, water, phosphate buffer, BisTris propane buffers (also referred to herein, in embodiments, as BisTris buffers and/or 1,3-Bis-[tris-(hydroxymethyl)-methyl-amino]-propane buffers), any other cellular medium, combinations thereof, and the like.
In a second solution, an electrophilic crosslinker, in embodiments a multi-arm PEG functionalized with electrophilic groups such as n-hydroxysuccinimide, may be prepared, also in a buffer. Suitable buffers include borate buffers, saline solutions, Hanks Balanced Salt Solution, Dulbecco's Modified Eagle's Medium, Phosphate Buffered Saline, water, phosphate buffer, BisTris buffers, any other cellular medium, combinations thereof, and the like. The electrophilic crosslinker, in embodiments a multi-arm PEG functionalized with n-hydroxysuccinimide groups, may be present in a solution including the above buffer at a concentration from about 0.02 grams/ml to about 0.5 grams/ml, in embodiments from about 0.05 grams/ml to about 0.3 grams/ml.
PEG-stars with degradable ester linkages and reactive succinimidyl end groups can then be cross-linked with the free amines of the nucleophilic component to form a scaffold which can be suitable for a number of regenerative medicine applications and/or delivery of bioactive agents and/or cells. These PEG-based hydrogels are designed to break apart in the presence of aqueous solutions due to the hydrolysis of the ester linkages.
The two components may be introduced in situ, wherein the electrophilic groups on the multi-arm PEG crosslink the amine nucleophilic components of the natural component, such as collagen and/or gelatin. The ratio of natural component to electrophilic component (i.e., collagen:PEG) may be from about 0.1:1 to about 100:1, in embodiments from about 1:1 to about 10:1.
The natural components, e.g., collagen, gelatin, and/or hyaluronic acid, may together be present at a concentration of at least about 1.5 percent by weight of the hydrogel, in embodiments from about 1.5 percent by weight to about 20 percent by weight of the hydrogel, in other embodiments from about 2 percent by weight to about 10 percent by weight of the hydrogel. In certain embodiments, collagen may be present from about 0.5 percent to about 7 percent by weight of the hydrogel, in further embodiments, from about 1 percent to about 4 percent by weight of the hydrogel. In another embodiment, gelatin may be present from about 1 percent to about 20 percent by weight of the hydrogel, in further embodiments, from about 2 percent to about 10 percent by weight of the hydrogel. In yet another embodiment, hyaluronic acid and collagen combined as the natural component(s) may be present from about 0.5 percent to about 8 percent by weight of the hydrogel, in further embodiments, from about 1 percent to about 5 percent by weight of the hydrogel. It is also envisioned that the hyaluronic acid may not be present as a “structural” component, but as more of a bioactive agent. For example, hyaluronic acid may be present in solution/gel in concentrations as low as 0.001 percent by weight of the solution/gel and have biologic activity.
The electrophilic crosslinker may be present in amounts of from about 0.5 percent by weight to about 20 percent by weight of the hydrogel, in embodiments from about 1.5 percent by weight to about 15 percent by weight of the hydrogel.
Hydrogels may be formed either through covalent, ionic or hydrophobic bonds. Physical (non-covalent) crosslinks may result from complexation, hydrogen bonding, desolvation, Van der Waals interactions, ionic bonding, combinations thereof, and the like, and may be initiated by mixing two precursors that are physically separated until combined in situ, or as a consequence of a prevalent condition in the physiological environment, including: temperature, pH, ionic strength, combinations thereof, and the like. Chemical (covalent) crosslinking may be accomplished by any of a number of mechanisms, including: free radical polymerization, condensation polymerization, anionic or cationic polymerization, step growth polymerization, electrophile-nucleophile reactions, combinations thereof, and the like.
In some embodiments, hydrogels of the present disclosure may include biocompatible multi-precursor systems that spontaneously crosslink when the precursors are mixed, but wherein the two or more precursors are individually stable for the duration of the deposition process. In other embodiments, in situ forming hydrogels may include a single precursor that crosslinks with endogenous materials and/or tissues.
The crosslinking density of the resulting hydrogel may be controlled by the overall molecular weight of the crosslinker and natural component and the number of functional groups available per molecule. A lower molecular weight between crosslinks, such as 600 daltons (Da), will give much higher crosslinking density as compared to a higher molecular weight, such as 10,000 Da. Elastic gels may be obtained with higher molecular weight natural components with molecular weights of more than 3000 Da.
The crosslinking density may also be controlled by the overall percent solids of the crosslinker and natural component solutions. Increasing the percent solids increases the probability that an electrophilic group will combine with a nucleophilic group prior to inactivation by hydrolysis. Yet another method to control crosslink density is by adjusting the stoichiometry of nucleophilic groups to electrophilic groups. A one to one ratio may lead to the highest crosslink density, however, other ratios of reactive functional groups (e.g., electrophile:nucleophile) are envisioned to suit a desired formulation.
As noted above, the natural component containing nucleophilic functional group(s) and the electrophilic functional precursor react to form a hydrogel. The reaction may occur in situ or outside of the body for future implantation therein or thereon. In embodiments, a hydrogel of the present disclosure may be produced by combining precursors that crosslink quickly after application to a surface, e.g., on a tissue of a patient, to form an in situ forming material.
The hydrogel thus produced may be bioabsorbable, so that it does not have to be retrieved from the body. Absorbable materials are absorbed by biological tissues and disappear in vivo at the end of a given period, which can vary, for example, from one day to several months, depending on the chemical nature of the material. Absorbable materials include both natural and synthetic biodegradable polymers, as well as bioerodible polymers.
In embodiments, one or more precursors having biodegradable linkages present in between functional groups may be included to make the hydrogel biodegradable or absorbable. In some embodiments, these linkages may be, for example, esters, which may be hydrolytically degraded in physiological solution. The use of such linkages is in contrast to protein linkages that may be degraded by proteolytic action. A biodegradable linkage may also optionally form part of a water soluble core of one or more of the precursors. Alternatively, or in addition, functional groups of precursors may be chosen such that the product of the reaction between them results in a biodegradable linkage. For each approach, biodegradable linkages may be chosen such that the resulting biodegradable biocompatible crosslinked polymer degrades or is absorbed in a desired period of time. Generally, biodegradable linkages may be selected that degrade the hydrogel under physiological conditions into non-toxic or low toxicity products.
The gels of the present disclosure may degrade due to hydrolysis or enzymatic degradation of the biodegradable region, whether part of the natural component or introduced into a synthetic electrophilic crosslinker. The degradation of gels containing synthetic peptide sequences will depend on the specific enzyme and its concentration. In some cases, a specific enzyme may be added during the crosslinking reaction to accelerate the degradation process. In the absence of any degradable enzymes, the crosslinked polymer may degrade solely by hydrolysis of the biodegradable segment. In embodiments in which polyglycolate is used as the biodegradable segment, the crosslinked polymer may degrade in from about 1 day to about 30 days depending on the crosslinking density of the network. Similarly, in embodiments in which a polycaprolactone based crosslinked network is used, degradation may occur over a period of time from about 1 month to about 8 months. The degradation time generally varies according to the type of degradable segment used, in the following order: polyglycolate<polylactate<polytrimethylene carbonate<polycaprolactone. Thus, it is possible to construct a hydrogel with a desired degradation profile, from a few days to months, using a proper degradable segment.
Where utilized, the hydrophobicity generated by biodegradable blocks such as oligohydroxy acid blocks or the hydrophobicity of PPO blocks in PLURONIC™ or TETRONIC™ polymers utilized to form the electrophilic crosslinker may be helpful in dissolving small organic drug molecules. Other properties which will be affected by incorporation of biodegradable or hydrophobic blocks include: water absorption, mechanical properties and thermosensitivity.
Other applications may require different characteristics of the hydrogel system. Generally, the materials should be selected on the basis of exhibited biocompatibility and lack of toxicity.
As noted above, in embodiments a branched multi-arm PEG, sometimes referred to herein as a PEG star, may be included to form a hydrogel of the present disclosure. A PEG star may be functionalized so that its arms include biofunctional groups such as amino acids, peptides, antibodies, enzymes, drugs, combinations thereof, or other moieties such as bioactive agents in its cores, its arms, or at the ends of its arms. The biofunctional groups may also be incorporated into the backbone of the PEG, or attached to a reactive group contained within the PEG backbone. The binding can be covalent or non-covalent, including electrostatic, thiol mediated, peptide mediated, or using known reactive chemistries, for example, biotin with avidin.
Amino acids incorporated into a PEG star may be natural or synthetic, and can be used singly or as part of a peptide. Sequences may be utilized for cellular adhesion, cell differentiation, combinations thereof, and the like, and may be useful for binding other biological molecules such as growth factors, drugs, cytokines, DNA, antibodies, enzymes, combinations thereof, and the like. Such amino acids may be released upon enzymatic degradation of the PEG star.
These PEG stars may also include functional groups as described above to permit their incorporation into a hydrogel. The PEG star may be utilized as the electrophilic crosslinker or, in embodiments, be utilized as a separate component in addition to the electrophilic crosslinker described above. In embodiments, the PEG stars may include electrophilic groups that bind to nucleophilic groups. As noted above, the nucleophilic groups may be part of a natural component utilized to form a hydrogel of the present disclosure.
In some embodiments a biofunctional group may be included in a PEG star by way of a degradable linkage, including an ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biofunctional group. In this case, the ester groups may hydrolyze under physiological conditions to release the biofunctional group.
In embodiments an in situ forming material may also include an initiator. An initiator may be any precursor or group capable of initiating a polymerization reaction for the formation of the in situ forming material.
As briefly described above, the hydrogel precursor(s) may be dissolved to form a solution prior to use, with the solution being delivered to the patient. As used herein, a solution may be homogeneous, heterogeneous, phase separated, or the like. In other embodiments, the precursor(s) may be in an emulsion. Solutions suitable for use in accordance with the present disclosure include those that may be used to form implants in lumens or voids. Where two solutions are employed, each solution may contain one precursor of an in situ forming material which forms upon contact. The solutions may be separately stored and mixed when delivered to tissue.
Generally, aqueous solutions of crosslinkers may be mixed with natural components to produce a crosslinked hydrogel. The reaction conditions for forming a crosslinked polymeric hydrogel will depend on the nature of the functional groups. In embodiments, reactions are conducted in buffered aqueous solutions at a pH of about 5 to about 12. Buffers include, for example, borate buffers including sodium borate buffer, saline solutions, phosphate buffered saline (PBS), Hanks Balanced Salt Solution, Dulbecco's Modified Eagle's Medium, water, phosphate buffers, triethanol amine buffer, BisTris propane buffers, any other cellular medium, combinations thereof, and the like. In some embodiments, organic solvents such as ethanol or isopropanol may be added to improve the reaction speed or to adjust the viscosity of a given formulation.
When the electrophilic crosslinker is synthetic (for example, based on polyalkylene oxide), it may be desirable to use molar equivalent quantities of the reactants. In some cases, molar excess of an electrophilic crosslinker may be added to compensate for side reactions such as reactions due to hydrolysis of the functional group.
When choosing the electrophilic crosslinker and natural component, at least one of the precursors may have more than two functional groups per molecule and at least one degradable region, if it is desired that the resultant biocompatible crosslinked polymer be biodegradable.
Formulations may be prepared that are suited to make precursor crosslinking reactions occur in situ. In general, this may be accomplished by having a precursor that can be activated at the time of application to a tissue to form a crosslinked hydrogel. Activation can be made before, during, or after application of the precursor to the tissue, provided that the precursor is allowed to conform to the tissue's shape before crosslinking and associated gelation is otherwise too far advanced. Activation includes, for instance, mixing precursors with functional groups that react with each other. Thus, in situ polymerization includes activation of chemical moieties to form covalent bonds to create an insoluble material, e.g., a hydrogel, at a location where the material is to be placed on, within, or both on and within, a patient. In situ polymerizable polymers may be prepared from precursor(s) that can be reacted such that they form a polymer within the patient. Thus precursor(s) with electrophilic functional groups can be mixed or otherwise activated in the presence of precursors with nucleophilic functional groups.
With respect to coating a tissue, and without limiting the present disclosure to a particular theory of operation, it is believed that reactive precursor species that crosslink quickly after contacting a tissue surface may form a three dimensional structure that is mechanically interlocked with the coated tissue. This interlocking contributes to adherence, intimate contact, and continuous coverage of the coated region of the tissue. In other embodiments, where electrophilic precursors are used, such precursors may react with free amines in tissue, thereby serving as a means for attaching the hydrogel to tissue.
The crosslinking reaction leading to gelation can occur, in some embodiments within a time from about 1 second to about 5 minutes, in embodiments from about 3 seconds to about 1 minute; persons of ordinary skill in these arts will immediately appreciate that all ranges and values within these explicitly stated ranges are contemplated. For example, in embodiments, the in situ gelation time of hydrogels according to the present disclosure is less than about 20 seconds, and in some embodiments, less than about 10 seconds, and in yet other embodiments less than about 5 seconds.
As noted above, the electrophilic crosslinker may be introduced by itself, whereby it crosslinks endogenous natural components. Alternatively, natural components may be removed from a patient or obtained from other sources, and then introduced along with the electrophilic crosslinker to form a hydrogel of the present disclosure, such as, for example, plasma. The precursor(s) may be placed into solution prior to use, with the solution being delivered to the patient. One may use a syringe for delivery of a single precursor, i.e., an electrophilic crosslinker, or a dual syringe or similar device to apply more than one precursor solutions, such as those described in U.S. Pat. Nos. 4,874,368; 4,631,055; 4,735,616; 4,359,049; 4,978,336; 5,116,315; 4,902,281; 4,932,942; 6,179,862; 6,673,093; and 6,152,943.
In other embodiments, the precursors may be combined to create a film or foam, which then may be implanted into the patient. Methods for creating films and foams are within the purview those skilled in the art, which include but are not limited to spraying, film casting, lyophilization techniques, etc.
As noted above, the hydrogels of the present disclosure may be utilized as tissue adhesives or sealants. The hydrogel may also be used to correct a tissue defect. As used herein, a “tissue defect” may include any breakdown of tissue from a normal, healthy state. This breakdown may be due to internal factors such as degenerative disease, or external factors such as injury. Any variation from the normal structure of a tissue may be a “tissue defect.” In some embodiments, where utilized to correct a tissue defect, a hydrogel of the present disclosure may be referred to as a tissue scaffold.
The hydrogel compositions may be utilized for cartilage repair, tendon repair, ligament repair, treatment of osteoarthritis, bone fillers, to repair soft tissue defects, as a filler for soft/hard tissue interfaces, and/or for general organ or tissue repair. General organ or tissue repair may include, for example, gastrointestinal, thoracic, cardiac, neural, or other organ repair.
In embodiments, where utilized for cartilage repair, a method for using a hydrogel of the present disclosure may include identifying a cartilage defect and then cleaning out the cartilage defect. Once the defect has been cleaned, a hydrogel composition of the present disclosure may be introduced into the defect. As noted above, in embodiments, at least one hydrogel precursor may be introduced therein. In embodiments, one of the precursors, sometimes referred to as a first hydrogel precursor, may be a natural component possessing nucleophilic groups. As noted above, the natural component may be endogenous or exogenously applied. A second precursor possessing electrophilic functional groups may also be applied to the defect utilizing any method or apparatus described above or as within the purview of those skilled in the art. The first hydrogel precursor and the second hydrogel precursor may then be allowed to react to form a hydrogel, which may act as a tissue scaffold at the site of the cartilage defect.
The hydrogel may be porous or smooth. In certain embodiments, the hydrogel may be porous. The term “porous” as used herein means that the hydrogel may possess defined openings and/or spaces which are present as a surface characteristic or a bulk material property, partially or completely penetrating the hydrogel. Pores may be created using methods within the purview of those skilled in the art including, but not limited to, processes such as sintering, leaching of salt, sugar or starch crystals. Porous materials may have an open-cell structure, where the pores are connected to each other, forming an interconnected network. Conversely, the hydrogel may be closed cell, where the pores are not interconnected.
The hydrogel may be coated with or include additional bioactive agents. The term “bioactive agent”, as used herein, is used in its broadest sense and includes any substance or mixture of substances that have clinical use. Consequently, bioactive agents may or may not have pharmacological activity per se, e.g., a dye.
Alternatively a bioactive agent could be any agent which provides a therapeutic or prophylactic effect, a compound that affects or participates in tissue growth, cell growth, cell differentiation, an anti-adhesive compound, a compound that may be able to invoke a biological action such as an immune response, or could play any other role in one or more biological processes. It is envisioned that the bioactive agent may be applied to the hydrogel in any suitable form of matter, e.g., films, powders, liquids, gels and the like.
As noted above, in embodiments that include a multi-arm PEG or PEG star, the bioactive agent may be incorporated into the core of the PEG, the arms of the PEG, or combinations thereof. In embodiments, the bioactive agent may be attached to a reactive group in the PEG chain. The bioactive agent may be bound covalently, non-covalently, i.e., electrostatically, through a thiol-mediated or peptide-mediated bond, or using biotin-avidin chemistries and the like.
Examples of classes of bioactive agents, which may be utilized in accordance with the present disclosure include, for example, anti-adhesives, antimicrobials, analgesics, antipyretics, anesthetics, antiepileptics, antihistamines, anti-inflammatories, cardiovascular drugs, diagnostic agents, sympathomimetics, cholinomimetics, antimuscarinics, antispasmodics, hormones, growth factors, muscle relaxants, adrenergic neuron blockers, antineoplastics, immunogenic agents, immunosuppressants, gastrointestinal drugs, diuretics, steroids, lipids, lipopolysaccharides, polysaccharides, platelet activating drugs, clotting factors and enzymes. It is also intended that combinations of bioactive agents may be used.
Anti-adhesive agents can be used to prevent adhesions from forming between the hydrogel and the surrounding tissues opposite the target tissue. In addition, anti-adhesive agents may be used to prevent adhesions from forming between the coated implantable medical device and the packaging material. Some examples of these agents include, but are not limited to hydrophilic polymers such as poly(vinyl pyrrolidone), carboxymethyl cellulose, hyaluronic acid, polyethylene oxide, poly vinyl alcohols, and combinations thereof.
Suitable antimicrobial agents which may be included as a bioactive agent include, for example, triclosan, also known as 2,4,4′-trichloro-2′-hydroxydiphenyl ether, chlorhexidine and its salts, including chlorhexidine acetate, chlorhexidine gluconate, chlorhexidine hydrochloride, and chlorhexidine sulfate, silver and its salts, including silver acetate, silver benzoate, silver carbonate, silver citrate, silver iodate, silver iodide, silver lactate, silver laurate, silver nitrate, silver oxide, silver palmitate, silver protein, and silver sulfadiazine, polymyxin, tetracycline, aminoglycosides, such as tobramycin and gentamicin, rifampicin, bacitracin, neomycin, chloramphenicol, miconazole, quinolones such as oxolinic acid, norfloxacin, nalidixic acid, pefloxacin, enoxacin and ciprofloxacin, penicillins such as oxacillin and pipracil, nonoxynol 9, fusidic acid, cephalosporins, and combinations thereof. In addition, antimicrobial proteins and peptides such as bovine lactoferrin and lactoferricin B may be included as a bioactive agent.
Other bioactive agents, which may be included as a bioactive agent include: local anesthetics; non-steroidal antifertility agents; parasympathomimetic agents; psychotherapeutic agents; tranquilizers; decongestants; sedative hypnotics; steroids; sulfonamides; sympathomimetic agents; vaccines; vitamins; antimalarials; anti-migraine agents; anti-parkinson agents such as L-dopa; anti-spasmodics; anticholinergic agents (e.g., oxybutynin); antitussives; bronchodilators; cardiovascular agents, such as coronary vasodilators and nitroglycerin; alkaloids; analgesics; narcotics such as codeine, dihydrocodeinone, meperidine, morphine and the like; non-narcotics, such as salicylates, aspirin, acetaminophen, d-propoxyphene and the like; opioid receptor antagonists, such as naltrexone and naloxone; anti-cancer agents; anticonvulsants; anti-emetics; antihistamines; anti-inflammatory agents, such as hormonal agents, hydrocortisone, prednisolone, prednisone, non-hormonal agents, allopurinol, indomethacin, phenylbutazone and the like; prostaglandins and cytotoxic drugs; chemotherapeutics, estrogens; antibacterials; antibiotics; anti-fungals; anti-virals; anticoagulants; anticonvulsants; antidepressants; antihistamines; and immunological agents.
Other examples of suitable bioactive agents, which may be included in the hydrogel include, for example, viruses and cells, including stem cells; peptides, polypeptides and proteins, as well as analogs, muteins, and active fragments thereof; immunoglobulins; antibodies; cytokines (e.g., lymphokines, monokines, chemokines); blood clotting factors; hemopoietic factors; interleukins (IL-2, IL-3, IL-4, IL-6); interferons (β-IFN, α-IFN and γ-IFN); erythropoietin; nucleases; tumor necrosis factor; colony stimulating factors (e.g., GCSF, GM-CSF, MCSF); insulin; anti-tumor agents and tumor suppressors; blood proteins such as fibrin, thrombin, fibrinogen, synthetic thrombin, synthetic fibrin, synthetic fibrinogen; gonadotropins (e.g., FSH, LH, CG, etc.); hormones and hormone analogs (e.g., growth hormone); vaccines (e.g., tumoral, bacterial and viral antigens); somatostatin; antigens; blood coagulation factors; growth factors (e.g., nerve growth factor, insulin-like growth factor); bone morphogenic proteins; TGF-B; protein inhibitors; protein antagonists; protein agonists; nucleic acids, such as antisense molecules, DNA, RNA, RNAi; oligonucleotides; polynucleotides; and ribozymes.
The precursors and/or the hydrogel formed from the precursors may contain visualization agents to improve their visibility during surgical procedures. Visualization agents may be selected from a variety of non-toxic colored substances, such as dyes, suitable for use in implantable medical devices. Suitable dyes are within the purview of those skilled in the art and may include, for example, a dye for visualizing a thickness of the hydrogel as it is formed in situ, e.g., as described in U.S. Pat. No. 7,009,034. In some embodiments, a suitable dye may include, for example, FD&C Blue #1, FD&C Blue #2, FD&C Blue #3, FD&C Blue #6, D&C Green #6, methylene blue, indocyanine green, other colored dyes, and combinations thereof. It is envisioned that additional visualization agents may be used such as fluorescent compounds (e.g., flurescein or eosin), x-ray contrast agents (e.g., iodinated compounds), ultrasonic contrast agents, and MRI contrast agents (e.g., Gadolinium containing compounds).
The visualization agent may be present in either a crosslinker or natural component solution. The colored substance may or may not become incorporated into the resulting hydrogel. In embodiments, however, the visualization agent does not have a functional group capable of reacting with the crosslinker or natural component.
The visualization agent may be used in small quantities, in embodiments less than 1% weight/volume, and in other embodiments less that 0.01% weight/volume and in yet other embodiments less than 0.001% weight/volume concentration.
In embodiments, the bioactive agent may be encapsulated by the hydrogel. For example, the hydrogel may form polymer microspheres around the bioactive agent.
Where the hydrogel is utilized as a tissue scaffold, it may be desirable to include bioactive agents which promote wound healing and/or tissue growth, including colony stimulating factors, blood proteins, fibrin, thrombin, fibrinogen, hormones and hormone analogs, blood coagulation factors, growth factors, bone morphogenic proteins, TGF-13, IGF, combinations thereof, and the like.
As the hydrogel hydrolyzes in situ, the bioactive components and any added bioactive agents are released. This may provide nutrients from the natural components, as well as bioactive agents, to the surrounding tissue, thereby promoting growth and/or regeneration of tissue.
In other embodiments, a method of cell encapsulation is disclosed. The method includes creating a first solution, such as a cell solution, which may be combined with a second solution, such as an electrophilic multi-arm polyethylene glycol (PEG) solution in about a 1:1 ratio (hereinafter a third solution). The third solution may be co-injected into tissue with a nucleophilic component. In embodiments, the nucleophilic component may include a solution including a natural component such as collagen, serum, gelatin, hyaluronic acid and combinations thereof. The term solution as used herein includes suspensions, emulsions, slurries, and the like.
In yet other embodiments, a method of cell encapsulation comprises co-injecting into a pre-shaped mold the third solution and the natural component. The mold may be pre-formed to mimic or replicate specific tissue structures such as organs or grafts. The molds may also be shaped to yield an implant or hydrogel which may be used to fill tissue spaces or voids.
In alternate embodiments, the third solution may be injected into a scaffold or tissue containing the natural component.
Suitable multi-arm PEGs include those disclosed herein, such as 4, 6, and 8-arm PEGs. Additionally, it should be understood that the multi-armed PEGs include electrophilic functional groups. Suitable electrophilic functional groups include those described above.
Further, buffer concentrations of the natural component solution and/or electrophilic component solution may vary as a function of the multi-arm PEG MW and the number of arms or branches on the multi-arm PEG. Sodium borate buffer ranges are from about 0.075M to about 0.5M, in embodiments from about 0.175M to about 0.235M. BisTris propane buffers may be at a concentration from about 0.05M to about 0.3M, in embodiments from about 0.1M to about 0.21M.
Suitable cells which may be encapsulated by hydrogels of the present disclosure and delivered therefrom include those derived from tissue layers, tissues, organs, organ systems, solid and/or hollow, circulatory/bodily fluid-based, etc., including, but not limited to, one or more of the following: endo or epithelium; stratum; adventitia; blood cells (red or white); venous or arterial origin; tissue or blood based inflammatory/immune cells including macrophage/monocytes, T-cells, killer cells, giant cells, neutrophils/polymorphonuclear cells, and the like; lymphatic derived cells; bone derived cells (osteoblasts, osteoclasts, osteoprogenitor); bone marrow aspirate or derived cells; stem cells/progenitor cells/adult stem cells; heart/myocardium; blood vessel/artery; umbilical/fetal tissue/organs/cells; kidney/renal cells; fat; liver; pancreas; bladder; ureter; periosteum; fascia; lung; GI tract cells such as esophageal, small/large intestine, colon, rectum, and stomach; neural cells including neurons and glial cells such as (central and/or peripheral nervous system) neurons, astrocytes, schwann cells, oligodendrocytes, microglia, olfactory ensheathing cells, dorsal root ganglion cells, neural stem cells and progenitors, brain, cord, or nerve-derived, and optic nerve/retina; eye including various layers of cornea, retina, iris, optic nerve, ciliary muscle, and strabismus; connective/soft tissue including abdominal wall, pelvic floor, cartilage, meniscus, ligament, tendon, joint capsule, muscle, skin and dermal cells, hair follicles and combinations thereof.
In embodiments, cells for delivery with the hydrogels of the present disclosure include, for example, chondrocytes for cartilage implants, hepatocytes for liver implants, adipose cells for breast reconstruction and other body contouring, osteoblasts for bone repair, stem cells for any tissue engineering or regenerative medicine application, tenocytes for tendon repair, fibroblasts for soft tissue repair, combinations thereof, and the like.
The aforementioned cells may be a whole population from one or more of the above, or a selected population from one or more of the above. Selected populations may be obtained by a variety of sorting or selection processes, including surface markers, adhesion methods, density methods, genetic methods, size methods, proteomic methods, or any other method used to differentiate cell populations.
In accordance with the present disclosure, hydrogels for delivering cells may have an osmolarity from about 200 milliosmoles (mOsm)/liter (L) to about 400 mOsm/L, in embodiments from about 260 mOsm/L to about 300 mOsm/L.
Following the methods of the present disclosure, hydrogels may be formed that are suitable for delivering cells, the hydrogels having a high loading of viable cells. In embodiments, hydrogels of the present disclosure may have cells therein at a loading from about 500 cells/ml to about 500,000,000 cells/ml, in embodiments from about 1,000,000 cells/ml to about 100,000,000 cells/ml, with the cells remaining viable therein indefinitely.
The following Examples are being submitted to illustrate embodiments of the present disclosure. These Examples are intended to be illustrative only and are not intended to limit the scope of the present disclosure. Also, parts and percentages are by weight unless otherwise indicated.
A hydrogel is prepared as follows. Collagen is dissolved in borate buffer (pH 8.75) using a sonicator at about 37° C. for approximately 5 minutes to yield a final concentration of from about 3 to about 6% by weight. The collagen solution is then centrifuged to remove gas/bubbles. Separately, a multi-arm PEG is dissolved in Hanks buffer (at a neutral pH) to yield a final concentration of from about 4 to about 40% by weight. The two solutions are then simultaneously sprayed in situ at a 1:1 ratio to yield a final gel concentration of from about 1.5 to about 3% by weight collagen, and from about 2.5 to about 20% by weight PEG.
A hydrogel is prepared as set forth in Example 1, except at a 10:1 ratio collagen:PEG, to yield a final gel concentration of from about 0.5 to about 4% by weight multiarm PEG and from about 3 to about 6% by weight collagen.
A hydrogel is prepared as follows. Gelatin is dissolved at a concentration of from about 5 to about 15% by weight in borate buffer at a pH of about 8.25. Separately, a multi-arm PEG is dissolved in Hanks buffer at a concentration of from about 5 to about 20% by weight. Solutions are simultaneously sprayed in situ at a 1:1 ratio, yielding a final gel concentration of from about 2.5 to about 7.5% by weight gelatin, and from about 2.5 to about 10% by weight multi-armed PEG.
A hydrogel is prepared as follows. Hyaluronic acid is dissolved in Hanks buffer at neutral pH, at up to about 2% by weight. A multi-arm PEG is next added to the hyaluronic acid solution at a concentration of from about 5 to about 20% by weight. In a separate vessel, collagen is dissolved in a borate buffer (pH 8.75) at a final solution concentration of from about 3 to about 6% by weight. The 2 solutions are then sprayed at a 1:1 ratio in situ. The final gel concentration is about 1% by weight hyaluronic acid, from about 1.5 to about 3% by weight collagen, and from about 2.5 to about 10% by weight multi-arm PEG.
The components of the gels of Examples 1, 3 and 4 are summarized below in Table 1.
A first cell solution was created. Cells were trypsinized, neutralized trypsin, and counted. Cells were then spun down and resuspended in Hank's buffer salt solution to create a first cell solution at a concentration of 4×106 cells/mL.
A second solution was created by dissolving multi-aimed PEG (200 mg/mL) in Hank's buffer salt solution.
The first solution and second solution were mixed at about a 1:1 ratio to create a third solution.
Separately, gelatin was solubilized at 10 wt % in 0.235M Sodium Borate buffer at a pH of about 8.25. The resultant gelatin solution was vortexed, sonicated and spun down to remove bubbles.
The third solution and the gelation solution were loaded into a dual barrel syringe and co-injected. (The third solution and the gelatin being disposed in separate barrels in the dual barrel syringe.)
It should be noted that while the examples listed above are formed in situ, the materials may be formed ex vivo and then utilized as an implant in situ.
An in situ forming injectable hydrogel solution was prepared using a two-component system including a solution of aminated biological macromolecules, in this case collagen, and a solution of reactive succinimidyl ester multi-armed PEG.
A 0.075M borate buffer was adjusted to a pH of 8.75 using 1N sodium hydroxide (from JTBaker-Mallinckrodt, NJ). Various amounts of bovine collagen (from Covidien-Sofradim, France) were dissolved in the borate buffer using a vortex mixer and sonicator at 37° C. to obtain 3 weight %, 4.5 weight %, and 6 weight % aqueous solutions. The collagen solutions were then spun at 1500 rpm for 2 minutes to remove bubbles.
Eight-armed PEG-stars, having a molecular weight of 15,000 g/mol with reactive succinimidyl ester (glutarate) end groups (abbreviated 8a15 kSG (from JenKemUSA, TX)) were dissolved in Hank's balanced solution (Hank's) (from Sigma-Aldrich, MO) using a vortex mixer and sonicator to obtain 50 mg/mL and 100 mg/mL aqueous solutions.
A solution including four-armed PEG-stars, having a molecular weight of 20,000 g/mol with reactive succinimidyl ester (glutarate) end groups (abbreviated as 4a20 kSG (from JenKemUSA, TX)) was similarly prepared. The PEG was dissolved in Hank's balanced solution (Hank's) (from Sigma-Aldrich, MO) using a vortex mixer and sonicator to obtain a 100 mg/mL aqueous solution.
An in situ forming injectable hydrogel solution was prepared using a two-component system including a solution of aminated biological macromolecules, in this case gelatin, and a solution of reactive succinimidyl ester multi-armed PEG.
A 0.075M borate buffer was adjusted to a pH of 8.25 using 1N sodium hydroxide solution. Various amounts of bovine Type-B gelatin (from Sigma-Aldrich, MO) were dissolved in the borate buffer using a vortex mixer and sonicator at 37° C., to obtain 5 weight %, 10 weight %, and 15 weight % solutions. The gelatin solutions were then spun at 1500 rpm for 2 minutes to remove bubbles.
PEG-star solutions, based upon the eight-armed PEG-stars described above in Example 6, having a molecular weight of 15,000 g/mol with reactive succinimidyl ester (glutarate) end groups, were prepared as described in Example 6 above, except at concentrations of 100 mg/mL and 200 mg/mL.
Additional PEG-star solutions were prepared, using four-armed PEG-stars as described above in Example 6, having a molecular weight of 20,000 g/mol with reactive succinimidyl ester (glutarate) end groups (abbreviated as 4a20 kSG (from JenKemUSA, TX)). As in Example 6 above, the PEG was dissolved in Hank's balanced solution (Hank's) (from Sigma-Aldrich, MO) using a vortex mixer and sonicator to obtain a 100 mg/mL aqueous solution.
An in situ forming injectable hydrogel solution was prepared using a two-component system including a solution of aminated biological macromolecules, in this case collagen/hyaluronic acid, and a solution of reactive succinimidyl ester multi-armed PEGs.
Collagen solutions of 4.5 weight % and 6 weight % were prepared as described above in Example 6.
Hyaluronic acids (HA) (from LifeCore Medical, MN) having molecular weights of 74 kDa and 910 kDa were dissolved in Hank's using a vortex mixer to obtain 0.5 weight % and 2 weight % aqueous solutions, for each molecular weight (74 kDa and 910 kDa) of HA.
PEG-star solutions, based upon the eight-armed PEG-stars described above in Example 6, having a molecular weight of 15,000 g/mol with reactive succinimidyl ester (glutarate) end groups, were prepared as described in Example 6 above, except the PEG-stars were dissolved in each of the solutions of HA (74 kDa and 910 kDa) of HA in Hank's at a concentration of 100 mg/mL.
The solutions prepared in Examples 6, 7, and 8 above were combined with a 1:1 dual-barrel static mixer system (from Medmix, Switzerland). The free amine component was loaded in one barrel of the dual-barrel mixer and the PEG-star component was loaded into the other barrel of the dual barrel chamber. The two solutions were then passed through a static mixer tip that ensured consistent and uniform mixing. The free amine solution reacted with the PEG-star solution, which cross-linked the biological macromolecules.
The hydrogels were prepared by injecting the mixed solution in custom rectangular DELRIN® molds. The solutions were allowed to set under normal ambient conditions for 10 minutes and the mold was carefully opened to reveal a slab of hydrogel with a uniform thickness of 3 mm. Cylindrical discs (6.75 mm diameter) were cut from the hydrogel slabs using a trephine punch.
The cylindrical hydrogel samples were then placed in Dulbecco's Phosphate Buffered Saline (PBS) at 37° C. to mimic in vivo pH and temperature conditions. The PEG arms containing the ester linkages underwent hydrolysis in the presence of the PBS. The hydrogels were qualitatively observed every week until the hydrogels could not maintain a cylindrical shape.
The degradation of the collagen-based hydrogels formed of the 8-arm PEGs of Example 6 was first evaluated, with the results set forth in
The degradation of the collagen-based hydrogels formed of the 4-arm PEGs of Example 6 (concentration of 50 mg/mL) was evaluated, with the results set forth in
The degradation of collagen-based hydrogels formed of the 4-arm PEGs of Example 6 (concentration of 100 mg/mL) was next evaluated, with the results set forth in
The degradation of the gelatin-based hydrogels formed of the 8-arm PEGs of Example 7 was evaluated, with the results set forth in
The degradation of gelatin-based hydrogels formed of the 4-arm PEGs of Example 7 was evaluated, with the results set forth in
The degradation of the collagen/HA-based hydrogels of Example 8 was evaluated, with the results set forth in
A summary of the formulations that were prepared and tested are set forth below in Table 2. In all cases, the solvent for the multi-arm PEG was Hank's Solution.
Based upon the above observations, several conclusions may be drawn:
the in situ forming hydrogels of the present disclosure were shown to have a range in degradation time;
4.5% Collagen with 2% 74 kDa HA showed to have the shortest degradation time, with an average degradation time of 7.66 weeks;
10% Gelatin with 100 mg/mL PEG-star showed to have the longest degradation time, with an average degradation time of 17.66 weeks;
the component type and concentration may be manipulated to control degradation time; and
the PEG type and concentration did affect the rate of degradation.
Experiments with L929 mouse fibroblast cells were performed where cells were seeded on the hydrogels set forth in Table 2 above, and also encapsulated within the hydrogels. Static and bioreactor experiments were first performed to determine the effect of culture conditions on the L929 cell populated hydrogels formed by reacting the 8-arm PEG with 6% collagen or 15% gelatin. Gels were cultured in a spinner flask bioreactor or in 48-well plates for T0, T3day and T7day, after which cell viability and number were evaluated and compared to cell-only controls.
A standard culture media was prepared for L929 cells (a murine aneuploid fibrosarcoma cell line used to assay TNF-alpha and TNF-beta, commercially available from Sigma-Aldrich). The culture medium included Eagle's Minimum Essential Medium (EMEM), 10% fetal bovine serum (FBS), and 1% Penicillin/Streptomycin. The cells were placed in the culture medium.
After cells reached about 80% confluence (cells covering the entire flask and being in direct contact with one another) the media was removed from the flasks. The cells were rinsed with 5 mL 1×PBS (−/−). About 3 mL trypsin was then added to each flask. The flask contents were incubated for about 5 minutes, after which cells were checked for detachment. Incubation continued until all cells were detached and not clumped (no more than 10 minutes incubation). The trypsin was then neutralized using 3 mL of standard media.
The cells were then centrifuged for about 5 minutes at about 1500 rpm. The media was aspirated, and the cells resuspended in 2 mL of Hanks Buffer and counted using a hemocytometer. About 7 mL of cell solution was prepared with 4×106 cells/mL in Hanks Buffer (44×106 cells in 11 mL of Hanks buffer). Gels were prepared as described above in Example 9. Briefly, 15 weight % gelatin was solubilized in 0.235M Sodium Borate buffer at a pH of 8.25. The cells were resuspend cells in Hanks at twice the desired concentration.
An 8 arm PEG, having succinimidyl glutarate functional groups and a molecular weight of about 20000 (abbreviated (8a20 kSG PEG) was dissolved at twice the desired concentration (100 mg/mL). The result was 2 mL of a 200 mg/mL 8a20 kSG PEG solution in HANKS buffer. Equal volumes of the cell suspension was mixed with the PEG solution to obtain desired concentration. A 1 mL aliquot (4×106 cells/mL) of the above cell solution was added to the PEG solution.
Dual barrel syringes were loaded with the gelatin and cells/PEG mixture for mixing at a 1:1 ratio so that the concentration of cells was 2×106 cells/mL, and the PEG concentration was 100 mg/mL. The final gel concentrations were 1×106 cells/mL and 50 mg/mL PEG.
The solution was injected into the mold as described above in Example 9 and allowed to solidify for about 5-7 minutes. Half of the mold was then removed, and samples were cut and placed in Petri dishes containing media including To samples. The gels were threaded onto skewers, one bioreactor at a time, for a total of 3 bioreactors (one for each time point). Collagen gels were made the same way, using 6 weight % collagen in 5 mL 0.175 M borate buffer, pH 8.75.
A PEG gel was prepared with PEG and gelatin as noted above, without cells. About 4 mL of a 100 mg/mL PEG solution was prepared in HANKS buffer. The syringes were loaded with PEG and gelatin solutions as described above and injected into molds.
At days 3 and 7, 3 gels and cells in 3 wells (per time point) were examined as part of a Live/Dead assay and visualized with a microscope. Briefly, to prepare live/dead solutions, about 10 mL of 1×PBS (++) was added to a 15 mL conical tube and add 20 μL ethidium dimer (EthD) and 5 μL calcein AM Calcein AM (Calcein acetoxymethyl ester, a non-fluorescent, cell permeable compound, which converts to the strongly green fluorescent calcein when hydrolyzed by intracellular esterases in live cells) were added thereto. The media was removed from the samples and rinsed with PBS. The PBS was removed and enough live/dead stain added to cover the gels. The plates were covered with foil to protect from light. The plates were incubated for about 30 minutes at room temperature. They were then imaged with an inverted fluorescent microscope.
Initial samples were prepared of the other collagen and gelation gels as well as 4-arm PEG instead of 8-arm PEG.
Initial live/dead imaging showed the following trends:
Some other observations for the different hydrogel formulations were qualitatively made based on in vitro live/dead imaging of 3T3 fibroblasts encapsulated within these gels:
To prepare cells for the PicoGreen standard curve, 1×106 cells were transferred to a 1.5 mL Eppendorf tube. To prepare cells for 0 hour time point study, 1×105 cells were transferred into each of 6 Eppendorf tubes, and centrifuged at 1500 rpm for 5 minutes. The media was then aspirated and pellets were frozen at −80° C.
6 cellular and 6 acellular gels were transferred from each test group to Eppendorf tubes and flash frozen with liquid nitrogen. They were then transferred to a freezer at −80° C. freezer for the PicoGreen assay.
At days 3 and 7, the culture media was removed from the Pico Green samples and cell only samples, rinsed 1× with PBS++, flash frozen with liquid nitrogen, and transferred to −80° C. freezer for the PicoGreen assay.
For matrix digestion, all frozen hydrogel samples and cell pellet were lyophilized overnight. The matrix was digested in Eppendorf tubes in a 250 μL proteinase K solution. Briefly, a 1 liter Tris-HCl buffer was prepared, which included 0.05M Tris-HCl (7.88 g/L) and 1 mM CaCl2 (147 mg/L), dissolved in 900 mL distilled water. The pH was adjusted to 6.5 with 1N NaOH. Additional distilled water was added to obtain the desired volume (1 liter).
A proteinase K stock solution was prepared by resuspending 50 mg proteinase K powder in 5 mL of the above Tris-HCl buffer. The stock solution was stored in 1 mL aliquots in a −20° C. freezer until needed. To prepare the proteinase K working solution, the proteinase K stock solution was diluted with the above Tris-HCl buffer, 100 times.
The hydrogel samples and cell pellet were recovered from the lyophilizer. About 250 μL proteinase K working solution was added to each Eppendorf tube with lyophilized hydrogel. About 500 μL proteinase K working solution was added to each Eppendorf tube with cell pellet. The solutions were placed into a 60° C. incubator for 16-24 hours.
To perform the Pico Green Assay, a Quant-iT PicoGreen dsDNA Assay Kit (from Invitrogen P11496) was used. The PicoGreen dye reagent (1 mL solution DMSO) was light sensitive and combined with a second component designated 20×TE buffer (25 mL of 200 mM Tris-HCL, 20 mM EDTA, pH 7.5)). The reagent was added to the digested matrix in Eppendorf tubes described above, with a digested cell pellet in Eppendorf tube (106 cells in 500 μL proteinase K solution) also being tested. About 20 mL of 1×TE solution was prepared by adding 1 mL 20×TE to 19 mL of nuclease-free water. The PicoGreen working solution was prepared by diluting PicoGreen dye reagent with 1×TE buffer, 200×. 100 μL needed per well. Cell standards were prepared in opaque wells using the 106 digested cells suspension. About 200 μL of the cell suspension was added to the first well, with about 100 μL of 1×TE solution added to the next 10 wells. A 2× serial dilution was performed leaving the last well blank, and repeated in triplicate. A summary of the assay is set forth below in Table 3.
Samples were diluted 2-fold in 1×TE solution. A 50 μL digested sample was added to 50 μL 1×TE solution in opaque wells. About 100 μL PicoGreen working solution was added to each well (samples and standards). The samples were incubated at room temperature for 2-5 minutes, away from light. A fluorescence reading was then taken on a microplate reader using Softmax Pro software
Optimal results using these formulations were found, with cell viability and proliferation deteriorating past 3 days due to high osmolarity and pH conditions within the hydrogel.
A standard culture media was prepared for L929 cells (a murine aneuploid fibrosarcoma cell line used to assay TNF-alpha and TNF-beta, commercially available from Sigma-Aldrich). The culture medium included Eagle's Minimum Essential Medium (EMEM), 10% fetal bovine serum (FBS), and 1% Penicillin/Streptomycin.
The L929 cells were thawed at a temperature of about 37° C. and transferred into flasks. The media was changed after about 24 hours after thawing was complete and then changed about every 3-4 days thereafter.
After cells reached about 80% confluence (cells covering the entire flask and being in direct contact with one another) the media was removed from the flasks. The cells were rinsed with 5 mL 1×PBS (−/−). About 3 mL trypsin was then added to each flask. The flask contents were incubated for about 5 minutes, after which cells were checked for detachment. Incubation continued until all cells were detached and not clumped (no more than 10 minutes incubation). The trypsin was then neutralized using 3 mL of standard media.
The cells were then centrifuged for about 5 minutes at about 1500 rpm. The media was aspirated, and the cells resuspended in 2 mL of media and counted using a hemocytometer.
A cell solution was prepared with approximately 8×106 cells/mL in media, which was then spun down in the centrifuge and aspirated to obtain a concentrated cell pellet. The concentrated cell pellet was at about twice the desired final concentration.
PEG-stars having NHS arms were dissolved at twice the desired final concentration in a 0.1M BisTris propane buffer at pH 7.5. More specifically, about 2 mL of a 100 mg/mL PEG solution was prepared in 0.1 M BisTris buffer. The cell pellet described above was resuspended in the PEG solution.
Gels were prepared as follows. Gelatin was solubilized at twice the desired final concentration in 0.21M BisTris buffer. More specifically, about 10 wt % gelatin was dissolved in 2 mL of 0.21 M BisTris buffer at the required pH of from about 7.8-7.9. The solubilized gelatin was kept warm at a temperature of about 37° C. until ready.
Dual barrel syringes were loaded with the gelatin solution in one barrel and the cells/PEG solution in the other barrel at a 1:1 mixing ratio to create a final hydrogel. When mixed together, the two buffers utilized equaled a neutral pH and cellular osmolarity (2290 mOsm).
The solutions were co-injected into a mold and allowed to solidify for about 5-7 minutes. After that time, half of the mold was removed, samples were cut into a desired size, and placed in respective well plates.
The final gel concentrations were about 2×106 cells/mL and 50 mg/mL PEG, 5% gelatin.
Similar gels were prepared as summarized below with collagen. The concentrations and buffer pH of the selected precursor solutions were as follows:
To prepare the cells for the PicoGreen standard curve, 1×106 cells were transferred to a 1.5 mL Eppendorf tube, and centrifuged at 1500 rpm for 5 minutes. The media was aspirated and the pellet was frozen at −80° C. The Eppendorf tubes were labeled for histological analysis and PicoGreen assay as described above in Example 10.
Buffers for use with the PEG-star and biopolymer components were evaluated for gelation time, pH, and osmolarity. Buffers were chosen to maintain consistency throughout material formulations, while having adequate cell viability, pH and desired gelation characteristics.
The buffers utilized with the various biopolymers and PEG-stars, including amounts, are set forth in
In view of the foregoing, the BisTris system was deemed better for cell loading than the borate buffer system.
While the above description contains many specifics, these specifics should not be construed as limitations on the scope of the present disclosure, but merely as exemplifications of preferred embodiments thereof. Those skilled in the art will envision many other possible variations that are within the scope and spirit of the present disclosure. cm What is claimed is:
