US20140227364A1 - Methods of manufacturing bioactive gels from extracellular matrix material - Google Patents
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- A61L27/3633—Extracellular matrix [ECM]
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Definitions
- the present invention is related to methods of manufacturing bioactive gels from extracellular matrix material and their uses for restoration of tissues in a patient.
- Biologic scaffolds composed of extracellular matrix material (ECM) have been used for the repair of variety of tissues including the lower urinary tract, esophagus, myocardium and musculotendinous tissues, often leading to tissue-specific constructive remodeling with minimal or no scar tissue formation.
- ECM extracellular matrix material
- the present invention pertains to improved methods of manufacturing bioactive gels from ECM.
- the invention describes methods of manufacturing bioactive gels from an ECM comprising (a) providing the ECM from one or more of the group consisting of but not limited to small intestine submucosa (SIS), urinary bladder submucosa (UBS), urinary bladder matrix (UBM), porcine dermis (PD), and liver basement membrane (LBM), (b) particularizing the ECM to a particle size in the range of about 1 ⁇ m to about 1000 ⁇ m, (c) solubilizing concentrations in the range of about 0.5 to 11% weight/volume (w/v) of particularized powder in sodium hydroxide (NaOH) in the range of 0.1 to 1.0 M for periods of time ranging from about 1 hour to about 48 hours at 4° C., (d) neutralizing the solubilized ECM prepared in step (c) with equimolar hydrochloric acid (HCl) ranging from 0.1 to 1M to form the gel, and
- FIG. 1 shows the FGF-2 content (pg/mg) of gels following various solubilization conditions in NaOH according to embodiments of the invention.
- FIG. 2 shows the VEGF content (pg/mg) of gels following various solubilization conditions in NaOH according to embodiments of the invention.
- the present invention is directed to methods of manufacturing bioactive gels from ECM, i.e., gels which retain bioactivity, and can serve as scaffolds for preclinical and clinical tissue engineering and regenerative medicine approaches to tissue reconstruction.
- ECM e.g., gels which retain bioactivity
- these manufacturing methods take advantage of a new recognition that bioactive gels from ECM can be created by treating a particularized ECM in a basic environment, which when neutralized with acid provides bioactive gels.
- the ECM may be derived from native mammalian tissues including but not limited to submucosa, dermis, epithelial basement membrane, aponeurosis, fascia, tendon, ligament, smooth and skeletal muscle and treatment site-specific ECM.
- the native mammalian tissue source may be porcine, bovine, ovine, allogenic, or autogenic, for example.
- the ECM may be SIS (small intestinal submucosa), UBS (urinary bladder submucosa) or UBM (urinary bladder matrix) or liver basement membrane (LBS) described in U.S. Pat. No. 6,576,265, U.S. Pat. No. 6,579,538, U.S. Pat. No. 5,573,784, U.S. Pat. No. 5,554,389, U.S. Pat. No. 4,956,178, and U.S. Pat. No. 4,902,508, each of which are incorporated by reference herein.
- the ECM derived from any one of the above sources is particularized, i.e., the sizes of the ECM are in a range of about 1 to about 1000 ⁇ m.
- particularization of the ECM prior to subjecting the ECM to a basic environment provides homogeneity to the ECM, i.e., provides a more uniform composition in comparison to ECM from individual animals, decreasing the impact of inter-donor variability.
- the particularization of the ECM facilitates in solubilizing the matrix in a basic environment by increasing surface area to volume ratio.
- the particulate ECM product e.g., particularized UBM
- the particulate ECM product is manufactured by grinding/milling or otherwise performing a size reduction process to ECM typically originally provided in sheet form.
- the resulting particulate can be any desired range of particle size and density for example in the range of about 1-1000 microns, about 200-700 microns, about 300-600 microns, or about 400 microns.
- Alkaline compounds which could be used in accordance with the invention are metal hydroxides which include, but are not limited to, LiOH, NaOH, KOH, RbOH, and CsOH.
- Alkaline compounds which could be used in accordance with the invention also include weak bases, such as but not limited to, ammonia (NH 3 ), pyridine (C 5 H 5 N), hydroxylamine (H 2 NOH), methylamine (NH 2 CH 3 ) and the like.
- Alkaline compounds are generally used at a concentration ranging from 0.1 Molar to 1.0 Molar, although concentrations lower that 0.1 Molar or higher than 1.0 Molar are also contemplated in an embodiment of the invention.
- Solubilization step at 4° C. i.e., digestion of the powdered ECM can extend over a period of time ranging from few minutes to several hours (e.g., 30 minutes to 48 hours) or days (e.g., 3-7 days).
- the time period required for the digestion step is determined by the size of the particularized matrix and/or the concentration of the metal hydroxide used for solubilization. For example, if the concentration of an alkaline compound, such as NaOH, is low, longer incubation, i.e., longer time period for solubilization may be required.
- the solubilized ECM i.e., the gel form
- the solubilized ECM is neutralized to a neutral pH using equimolar concentrations of an acid in volume sufficient to reach pH 6.8 to 7.4.
- Acids which aid in neutralization of the ECM gel, can be selected from weak or strong acids. Selectivity of acids for the neutralization step depends on the salts which are produced when an acids reacts with the basic environment during neutralization. For example, in an embodiment of the invention, hydrochloric acid (HCl) is used to neutralize the basic environment created by the base NaOH because the resulting salt (i.e., NaCl) is clinically acceptable.
- HCl hydrochloric acid
- gels can be subjected to various dwell periods (1-48 hours) to promote refolding of denatured bioactive components.
- Dwell periods are generally performed with or without shaking or stirring the gel in cold room (i.e., at temperature of about 4° C.). Dwell periods could extend beyond 48 hours to few days, for example, 3-7 days to promote restructuring of the gel.
- the gel may optionally be subjected to one or more steps of freeze drying cycles, one or more steps of lyophilization cycles or one or more steps of freeze drying and lyophilization cycles, to facilitate the conversion of the neutralized gel to a powder (having a neutral pH).
- the powder can be reconstituted into a gel without altering its bioactivity by mixing the powder with a liquid, such as water, which maintains the neutral pH of the gel.
- a liquid such as water
- the powder is reconstituted using water and two 3 mL syringes.
- One syringe contains the lyophilized gel, the other water, and they are mixed together via a connecter between the two syringes.
- the mixture is injected back and forth several times to achieve mixing.
- concentrations of UBM in NaOH can be tested for handling properties (i.e. injectability, tackiness, viscosity) to determine their ability to be applied using the two syringe system.
- the final consistency of all gels is foam-like, and each one adheres to the surface to which it is applied while maintaining consistency, which may be desirable in zero gravity conditions.
- ECM powder is added to increase viscosity or the bioactivity of the gel.
- UBM powder is added to a UBM gel prepared by the above methods to enhance the viscosity or the bioactivity of the gel, that is, the gel has better handling or the gel is made capable of producing higher concentration of growth factors, such as FGF-2, CTGF, or VEGF.
- ECM Powder can be added, prior to, during or after the gels are neutralized.
- any number of ECM products such as but not limited to one or more of isolated urinary bladder submucosa, small intestinal submucosa, dermis, for example, could be studied.
- UBM an ECM isolated from the urinary bladder and having epithelial basement membrane is used as an exemplary ECM.
- the invention disclosed herein is not limited to IBM and is applicable to any isolated ECM.
- gels were created using various concentrations of powder (0.5-11% w/v) and molarity of NaOH (0.1-1.0M). UBM was solubilized for various time periods (1-48 hours) in its respective concentration of UBM and NaOH at 4° C. In order to test whether the UBM could restructure after solubilization, gels were also made using various dwell periods (1-48 hours) following neutralization.
- FIGS. 1 and 2 Data for FGF-2 and VEGF content following solubilization for each gel structure is shown in FIGS. 1 and 2 . Lower concentration gels (1-6%) are not shown but produced similar results. As shown in FIGS. 1 and 2 , FGF-2 and VEGF, particularly VEGF levels increased in these studies.
- FGF-2 and VEGF contents were measure by the standard ELISA procedure.
- gels created in the above manner can be tested in vivo in ischemic rat models, as mentioned in Appendix A and Appendix B, attached hereto with the specification.
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Abstract
The present invention is directed to methods of manufacturing bioactive gels from ECM material, i.e., gels which retain bioactivity, and can serve as scaffolds for preclinical and clinical tissue engineering and regenerative medicine approaches to tissue reconstruction. The manufacturing methods take advantage of a new recognition that bioactive gels from ECM material can be created by digesting particularized ECM material in an alkaline environment and neutralizing to provide bioactive gels.
Description
- This invention was supported by grant no. NCC 9-58 from the National Space Biomedical Research Institute through NASA. The Government has certain rights in the invention.
- The present invention is related to methods of manufacturing bioactive gels from extracellular matrix material and their uses for restoration of tissues in a patient.
- Biologic scaffolds composed of extracellular matrix material (ECM) have been used for the repair of variety of tissues including the lower urinary tract, esophagus, myocardium and musculotendinous tissues, often leading to tissue-specific constructive remodeling with minimal or no scar tissue formation.
- Although uses of ECM as scaffolds for preclinical and clinical tissue engineering and regenerative medicine approaches to tissue reconstruction are very promising, challenges remain in the process to manufacture bioactive gels from ECM, which retain their bioactivity.
- The process of manufacturing bioactive gels from ECM described thus far require the use of enzymes and are time consuming because they require aggressive purification steps, which may lead to depletion in the bioactivity of the gels and may present additional regulatory barriers to marketing.
- Thus, a need exists to manufacture bioactive gels from ECM which avoids cumbersome preparation and purification steps yet result in gels that retain the bioactivity of the original material.
- The present invention pertains to improved methods of manufacturing bioactive gels from ECM. Thus, the invention describes methods of manufacturing bioactive gels from an ECM comprising (a) providing the ECM from one or more of the group consisting of but not limited to small intestine submucosa (SIS), urinary bladder submucosa (UBS), urinary bladder matrix (UBM), porcine dermis (PD), and liver basement membrane (LBM), (b) particularizing the ECM to a particle size in the range of about 1 μm to about 1000 μm, (c) solubilizing concentrations in the range of about 0.5 to 11% weight/volume (w/v) of particularized powder in sodium hydroxide (NaOH) in the range of 0.1 to 1.0 M for periods of time ranging from about 1 hour to about 48 hours at 4° C., (d) neutralizing the solubilized ECM prepared in step (c) with equimolar hydrochloric acid (HCl) ranging from 0.1 to 1M to form the gel, and (e) optionally, freeze drying the neutralized solubilized ECM prepared in step (d), (f) lyophilizing the freeze dried ECM prepared in step (e), and (f) reconstituting the lyophilized gel in water or saline at point of use.
- The advantages provided by the methods of manufacturing bioactive gels in the above manner are that aggressive purification steps, which are deleterious to bioactivity, tedious to perform or are time consuming, are avoided.
-
FIG. 1 shows the FGF-2 content (pg/mg) of gels following various solubilization conditions in NaOH according to embodiments of the invention. -
FIG. 2 shows the VEGF content (pg/mg) of gels following various solubilization conditions in NaOH according to embodiments of the invention. - The present invention is directed to methods of manufacturing bioactive gels from ECM, i.e., gels which retain bioactivity, and can serve as scaffolds for preclinical and clinical tissue engineering and regenerative medicine approaches to tissue reconstruction. As will be described in detail below, these manufacturing methods take advantage of a new recognition that bioactive gels from ECM can be created by treating a particularized ECM in a basic environment, which when neutralized with acid provides bioactive gels.
- In accordance with the inventive methods, the ECM may be derived from native mammalian tissues including but not limited to submucosa, dermis, epithelial basement membrane, aponeurosis, fascia, tendon, ligament, smooth and skeletal muscle and treatment site-specific ECM. The native mammalian tissue source may be porcine, bovine, ovine, allogenic, or autogenic, for example. For example, the ECM may be SIS (small intestinal submucosa), UBS (urinary bladder submucosa) or UBM (urinary bladder matrix) or liver basement membrane (LBS) described in U.S. Pat. No. 6,576,265, U.S. Pat. No. 6,579,538, U.S. Pat. No. 5,573,784, U.S. Pat. No. 5,554,389, U.S. Pat. No. 4,956,178, and U.S. Pat. No. 4,902,508, each of which are incorporated by reference herein.
- In accordance with the inventive method, the ECM derived from any one of the above sources is particularized, i.e., the sizes of the ECM are in a range of about 1 to about 1000 μm. In one embodiment, particularization of the ECM prior to subjecting the ECM to a basic environment provides homogeneity to the ECM, i.e., provides a more uniform composition in comparison to ECM from individual animals, decreasing the impact of inter-donor variability. In another embodiment, the particularization of the ECM facilitates in solubilizing the matrix in a basic environment by increasing surface area to volume ratio.
- The particulate ECM product, e.g., particularized UBM, is manufactured by grinding/milling or otherwise performing a size reduction process to ECM typically originally provided in sheet form. The resulting particulate can be any desired range of particle size and density for example in the range of about 1-1000 microns, about 200-700 microns, about 300-600 microns, or about 400 microns.
- Basic environment is provided by solutions of alkaline compounds. Alkaline compounds which could be used in accordance with the invention are metal hydroxides which include, but are not limited to, LiOH, NaOH, KOH, RbOH, and CsOH. Alkaline compounds which could be used in accordance with the invention also include weak bases, such as but not limited to, ammonia (NH3), pyridine (C5H5N), hydroxylamine (H2NOH), methylamine (NH2CH3) and the like. Alkaline compounds are generally used at a concentration ranging from 0.1 Molar to 1.0 Molar, although concentrations lower that 0.1 Molar or higher than 1.0 Molar are also contemplated in an embodiment of the invention.
- Solubilization step at 4° C. (i.e., digestion) of the powdered ECM can extend over a period of time ranging from few minutes to several hours (e.g., 30 minutes to 48 hours) or days (e.g., 3-7 days). In an embodiment of the invention it is contemplated that the time period required for the digestion step is determined by the size of the particularized matrix and/or the concentration of the metal hydroxide used for solubilization. For example, if the concentration of an alkaline compound, such as NaOH, is low, longer incubation, i.e., longer time period for solubilization may be required. After the solubilization step, the solubilized ECM (i.e., the gel form) is neutralized to a neutral pH using equimolar concentrations of an acid in volume sufficient to reach pH 6.8 to 7.4. Acids, which aid in neutralization of the ECM gel, can be selected from weak or strong acids. Selectivity of acids for the neutralization step depends on the salts which are produced when an acids reacts with the basic environment during neutralization. For example, in an embodiment of the invention, hydrochloric acid (HCl) is used to neutralize the basic environment created by the base NaOH because the resulting salt (i.e., NaCl) is clinically acceptable.
- Following neutralization, optionally, gels can be subjected to various dwell periods (1-48 hours) to promote refolding of denatured bioactive components. Dwell periods are generally performed with or without shaking or stirring the gel in cold room (i.e., at temperature of about 4° C.). Dwell periods could extend beyond 48 hours to few days, for example, 3-7 days to promote restructuring of the gel.
- In the inventive methods, once the gel is neutralized it may optionally be subjected to one or more steps of freeze drying cycles, one or more steps of lyophilization cycles or one or more steps of freeze drying and lyophilization cycles, to facilitate the conversion of the neutralized gel to a powder (having a neutral pH). The powder can be reconstituted into a gel without altering its bioactivity by mixing the powder with a liquid, such as water, which maintains the neutral pH of the gel. In addition, preservation of bioactivity of ECMs through lyophilization is well known.
- In one embodiment, the powder is reconstituted using water and two 3 mL syringes. One syringe contains the lyophilized gel, the other water, and they are mixed together via a connecter between the two syringes. The mixture is injected back and forth several times to achieve mixing. Various concentrations of UBM in NaOH can be tested for handling properties (i.e. injectability, tackiness, viscosity) to determine their ability to be applied using the two syringe system. The final consistency of all gels is foam-like, and each one adheres to the surface to which it is applied while maintaining consistency, which may be desirable in zero gravity conditions.
- In one embodiment, to the solubilized and neutralized ECM gel, ECM powder is added to increase viscosity or the bioactivity of the gel. For example, UBM powder is added to a UBM gel prepared by the above methods to enhance the viscosity or the bioactivity of the gel, that is, the gel has better handling or the gel is made capable of producing higher concentration of growth factors, such as FGF-2, CTGF, or VEGF. ECM Powder can be added, prior to, during or after the gels are neutralized.
- For the following exemplifications, any number of ECM products such as but not limited to one or more of isolated urinary bladder submucosa, small intestinal submucosa, dermis, for example, could be studied. In the following exemplifications, UBM, an ECM isolated from the urinary bladder and having epithelial basement membrane is used as an exemplary ECM. However the invention disclosed herein is not limited to IBM and is applicable to any isolated ECM.
- In an exemplification, gels were created using various concentrations of powder (0.5-11% w/v) and molarity of NaOH (0.1-1.0M). UBM was solubilized for various time periods (1-48 hours) in its respective concentration of UBM and NaOH at 4° C. In order to test whether the UBM could restructure after solubilization, gels were also made using various dwell periods (1-48 hours) following neutralization.
- Gels created in the above manner were tested in vitro for growth factor FGF-2, CTGF, VEGF) content. Data for FGF-2 and VEGF content following solubilization for each gel structure is shown in
FIGS. 1 and 2 . Lower concentration gels (1-6%) are not shown but produced similar results. As shown inFIGS. 1 and 2 , FGF-2 and VEGF, particularly VEGF levels increased in these studies. - In one study it was found that using 7.0% w/v UBM to various range of NaOH with 24 hours of solubilizing at 4° C. and no dwell period had significantly influenced the FGF-2 and VEGF contents in the gel. Experimentally, FGF-2 and VEGF contents were measure by the standard ELISA procedure.
- In another exemplification, gels created in the above manner can be tested in vivo in ischemic rat models, as mentioned in Appendix A and Appendix B, attached hereto with the specification.
Claims (5)
1. A method of manufacturing a bioactive gel from an extracellular matrix material comprising (a) providing an ECM material from the group consisting of small intestine submucosa (SIS), urinary bladder submucosa (UBS), urinary bladder matrix (UBM) and liver basement membrane (LBM), (b) particularizing the ECM to a particle size in the range of about 1 μm to about 100 μm, (e) solubilizing concentrations in the range of about 0.5 to 11% w/v of particularized powder in NaOH in the range of 0.1 to 1.0 M for periods of time ranging from about 1 hour to about 48 hours, (d) neutralizing the solubilized ECM prepared in step (c) with equimolar hydrochloric acid ranging from 0.1M to 1.0M to form the gel, and (e) optionally, freeze drying the neutralized solubilized ECM prepared in step (d), (f) lyophilizing the freeze dried ECM prepared in step (e) and (f) reconstituting the lyophilized gel in water.
2. The method of claim 1 wherein the ECM is UBM.
3. The method of claim 1 wherein the ECM is SIS.
4. The method of claim 1 wherein the ECM is UBS.
5. The method of claim 1 wherein the ECM is LBM.
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