Patent Application
20200016295
The present invention relates to an injectable scaffold for soft tissue repair, and in particular to such scaffolds that comprise an enzymatically crosslinked biopolymer matrix.
Three-dimensional (3-D) porous scaffold plays a crucial role in tissue engineering. It serves as a temporary template to improve cell growth and new tissue formation and provides adequate porosity for nutrients and oxygen transport. Ideally, the scaffolding materials should also be a mimic of the natural extracellular matrix (ECM) of the target tissue by integrating suitable mechanical, structural, and biological signals into scaffold. To this end, a wide range of natural biopolymer-based scaffolds have been proposed for tissue repair or regeneration during the past years, including gelatin and polysaccharides (e.g. chitosan, alginate, and hyaluronic acid).
Gelatin is a partially degraded product of collagen, which is the major protein component of natural ECM (extracellular matrix). It has been blended with other organic or inorganic biomaterials to fabricate 3-D scaffolds for various tissue engineering applications.
Polysaccharides are natural biomaterials which are inexpensive, and most of them are easily degradable. Polysaccharides are usually non-toxic, biocompatible and show a number of physico-chemical properties that make them suitable for different applications in cell therapy and tissue regeneration.
Chitosan is a polysaccharide common in the biomedical field due to its biocompatibility and low toxicity. It is a nitrogen-containing polysaccharide and related chemically to cellulose. Chitosan also promoted tissue formation and remodeling of damaged tissues in large, open wounds. It has been shown that a chitosan hydrogel interacts with fibroblast growth factor-2 in open wounds of diabetic mice, resulting in increased wound closure rate.
Alginates are linear unbranched polysaccharides. It is biodegradable and has controllable porosity. Due to their hemostatic properties, alginate and its salts are used for wound treatment in various forms such as gel or sponge. Calcium alginate can also increase cellular activity properties such as adhesion and proliferation.
Hyaluronic acid is abundant in skin, cockscomb, cartilage, and vitreous humor. This biomaterial has a high capacity of lubrication, water sorption, and water retention and influences several cellular functions such as migration, adhesion, and proliferation.
The background art does not suggest a solution for soft tissue repair, based on biocompatible materials that are non-toxic, non-inflammatory and biodegradable, yet that can form an injectable scaffold.
The present invention overcomes these deficiencies of the background art by providing an injectable scaffold made from biocompatible materials.
The biocompatible materials comprise a biopolymer, a porogen and a cross-linking agent. The biopolymer may optionally comprise a polypeptide or a polysaccharide. The polypeptide is optionally selected from the group consisting of gelatin or gelatin related variants.
The polysaccharide may optionally comprise a homoglycan or a heteroglycan.
Non-limiting examples of homoglycans include starch, β-Glucans, chitin, chitosan, cellulose and its derivatives, pullulan, dextran. Non-limiting examples of heteroglycans include alginic acid (alginate), glycosaminoglycans (heparin, heparin sulfate, chondroitin sulfate, hyaluronic acid), pectins, gaur gum, xanthan gum.
The polysaccharide is preferably selected from the group consisting of β-glucan, cellulose, alginic acid, hyaluronic acid, chitin, chitosan, heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, glycosaminoglycans, pectin, dextran, and starch.
The porogen promotes pore formation. Precipitation or particulate leaching is one of the most straightforward methods for pore formation. Each particle able to leave the gelatin matrix can be considered as a porogen. The porogen optionally is selected from the group consisting of a polysaccharide, poly(styrene), a glycol based polymer with different MW, salts (e.g. sodium chloride), supercritical carbon dioxide, and polymeric spheres with controlled size.
Non-limiting examples of the glycol based polymer include poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG).
Non-limiting examples of salts include KCl, NaCl, Na2SO4, NaHCO3, and K2HPO4, pyridinium and imidazolium salts.
The polysaccharide is optionally selected from the group consisting of β-glucan, cellulose, alginic acid, hyaluronic acid, chitin, chitosan, heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, glycosaminoglycans, pectin, dextran, and starch.
PEG may have a molecular weight range as given in the table below. PEG is optionally selected from the group consisting of PEG1450 and PEG4000.
Preferably the biocompatible materials feature an enzymatically crosslinked gelatin matrix and a porogen, such that the biopolymer comprises gelatin and the cross-linking agent comprises transglutaminase. Cross-linking gelatin takes advantage of the adhesive properties of gelatin, by stabilizing the gelatin matrix and rendering it thermally stable in body temperature.
More preferably said transglutaminase comprises microbial transglutaminase (mTG). Optionally transglutaminase is present at a concentration of from 0.0006 to 2 mg transglutaminase/cm3 gelatin matrix.
Optionally the gelatin is present in a weight/weight percentage of 1-15%, preferably in a weight/weight percentage of 5-10% and more preferably in a weight/weight percentage of 7-9%.
Preferably the porogen comprises one or more of PEG, alginate, chitosan or hyaluronic acid.
Preferably the porogen is PEG in an amount of 1-10%, more preferably 2-7%. Optionally and preferably if the porogen is PEG, then the porogen comprises PEG4000. More preferably, the porogen comprises PEG4000 in an amount of 1-10%, more preferably 2-7%.
If the porogen comprises alginate, chitosan or hyaluronic acid, more preferably the porogen also comprises PEG.
Some non-limiting examples of ranges of materials are given below.
Some non-limiting examples of terms and abbreviations are provided below:
The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in order to provide what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. In the drawings:
The present invention is of an injectable scaffold made from biocompatible materials.
To support cell ingrowth and facilitate the exchange of nutrients and cellular waste products, the in situ formed injectable scaffolds preferably possess highly porous networks with specified pore morphology. In this respect, parameters for at least some embodiments of the scaffold include pore size, porosity and interconnectivity of the porous network. To fabricate porous injectable scaffolds, several methods of in situ pore formation have been developed including leaching, gas forming and atomization.
As a non-limiting example demonstrated herein, the leaching method was adopted by introducing water-soluble PEG which could serve as polymeric porogen and was expected to promote the generation of porous structures.
Experimental data provided below compares different formulations of injectable scaffolds and investigates the influence of the composition on 3 main features: morphology, mechanical properties and swelling capacity. Preferred formulations meet the following criteria:
This present invention, in at least some embodiments, demonstrates a feasible strategy to fabricate porous, adhesive, injectable crosslinked gelatin scaffolds with a high adhesive strength and an interconnected porous structure in order to promote the cell adhesion capacity for tissue engineering.
According to some embodiments of the present invention, there is provided an injectable scaffold in which the cross-linking material comprises transglutaminase and the cross-linkable protein comprises gelatin.
Suitable gelatin and transglutaminase can be obtained by any of the methods known and available to those skilled in the art. Gelatin may optionally comprise any type of gelatin which comprises protein that is known in the art, preferably including but not limited to gelatin obtained by partial hydrolysis of animal tissue and/or collagen obtained from animal tissue, including but not limited to animal skin, connective tissue (including but not limited to ligaments, cartilage and the like), antlers or horns and the like, and/or bones, and/or fish scales and/or bones or other components; and/or a recombinant gelatin produced using bacterial, yeast, animal, insect, or plant systems or any type of cell culture.
According to preferred embodiments of the present invention, gelatin from animal origins preferably comprises gelatin from mammalian origins and more preferably comprises one or more of pork skins, pork and cattle bones, or split cattle hides, or any other pig or bovine source. More preferably, such gelatin comprises porcine gelatin since it has a lower rate of anaphylaxis. Gelatin from animal origins may optionally be of type A (Acid Treated) or of type B (Alkaline Treated), though it is preferably type A.
Preferably, gelatin from animal origins comprises gelatin obtained during the first extraction, which is generally performed at lower temperatures (50-60° C., although this exact temperature range is not necessarily a limitation).
The transglutaminase may optionally comprise any plant, animal, or microbe derived transglutaminase. Preferably the transglutaminase derived from Streptoverticillium mobaraensis is used.
The transglutaminase may optionally be in a composition comprising at least one other substance, such as a stabilizer or filler for example. Non-limiting examples of such materials include maltodextrin, hydrolyzed skim milk protein or any other protein substance, sodium chloride, safflower oil, trisodium phosphate, sodium caseinate or lactose, or a combination thereof preferably other than blood derived Factor XIII.
Although the optimal pH for activity of crude transglutaminase is 6.0, it also functions with high activity in the range of pH 5.0 to pH 8.0. Therefore, a composition according to the present invention for implant fixation preferably has a pH value in a range of from about 5 to about 8.
Scaffold 100 also preferably comprises an adhesive section 106, which may also comprise a support section from collage with an adhesive layer, such as a cross-linked gel layer as described herein. Adhesive section 106 may comprise the cross-linked gel as described herein, optionally without a further support section from collagen. Adhesive section 106 is optionally from 0.25 to 2 cm in length, preferably 1 cm in length.
The width of scaffold 100 is optionally from 0.5 cm to 5 cm, preferably 1-3 cm and more preferably 2.5 cm.
The total length of scaffold 100 is optionally from 1 cm to 10 cm, preferably 3-7 cm and more preferably 5 cm.
The syringes (gelatin and enzyme components) were placed in a water bath at 35° C. for 2 hours. The components were mixed with a volumetric ratio of 1:1 using a metal applicator and a static mixer. Curing occurred at 37° C. for 30 min, afterward saline was added to the gels. For the formulations containing alginate, a saline solution containing 50 mM CaCl2) was used.
The formulations that have been tested are:
The enzyme and gelatin syringes were mixed with a volumetric ratio of 1:1 using a three way stopcock. 0.3-0.4 g of the mixture were applied on a collagen sheet (in the appropriate area) and a second collagen sheet was immediately placed above. 6 repeats of each formulation were tested. After application, the samples were placed in a petri dish, between two wet gauzes, and placed at 37° C. for 30 minutes. Finally, the gauzes were removed and 20 mL saline 0.9% were added to each dish plate (saline 0.9%+50 mM CaCl2 was used for the samples containing alginate). The samples were kept at 37° C. overnight.
The collagen sheets were held by the Instron's grips and extension was performed at a rate of 0.4 mm/sec (the test started after a preload of 0.2 N). From the results, the maximum load (N) and the extension
were calculated.
The method and procedure are descripted in the report DHF-DR-0170.
Once the gels were prepared as descripted in the section 1.4, they were washed in saline, for 24 hours at 37° C. In parallel, a trypsin or collagenase solution was prepared:
First, 5 mL of 0.1 mg/mL trypsin were prepared by dissolving 0.5 gr trypsin in 5 mL HCl 1 mM. This solution was diluted 100-fold in phosphate buffer (50 mM, pH 7.4) to obtain a final trypsin concentration of 12 U/mL.
First, a 0.5M HEPES solution, a 110 mM CaCl2 solution and a 4 M NaCl solution were prepared. Then, 10 mL of the HEPES solution, 0.91 mL of the CaCl2 solution and 3.75 mL of the NaCl solution were mixed and the final volume was completed to 100 mL with water (final concentrations—0.05M HEPES, 0.15M NaCl, 1 mM CaCl2). A 15 U/mL collagenase solution was prepared by dissolving 5.2 mg collagenase in the precedent buffer solution.
Saline was replaced by the prolytic enzyme solution. The absorbance of the solution was measured at 220 nm (in order to assess the gelatin concentration) at fixed intervals. A clear enzyme solution at the same concentration was used as blank. After the total dissolution of the hydrogel, the maximal gelatin absorbance was measured and the degradation percent was calculated for each time point, as follows:
The porous structures of LifeSeal and hydrogel containing 8% gelatin (final gelatin content) are presented in
The porous structures of the swollen scaffolds were visualized using secondary electron (SE) imaging in an environmental scanning electron microscope (ESEM, FEI Quanta 200). The void diameters were estimated by analyzing the low magnification SEM images. The fracture surfaces were generated using a scalpel.
The scaffolds were casted in glass vials and weighted (˜1 gr) on an analytical balance. Curing occurred at 37° C. for 30 minutes, followed with addition of 15 mL saline and incubation at 37° C., with horizontal shaking at 120 rpm. After 24 hours, the hydrogels were weighted and the swelling percent was calculated as follows:
Where, Wf and Wi are the final and initial weights of the hydrogel, respectively.
Collagen sheets were wiped off using 70% IPA wipes, both sides of collagen were wiped well. They were placed in a beaker and covered with water, then washed in an ultrasonic bath for 3 cycles of 30 minutes each, replacing water between cycles. The washed collagen was kept in 0.9% saline until used.
The collagen sheets were cut into samples of 5 cm×2.5 cm. A mark of 2 cm was done to indicate where the Instron's top grip will be placed. A mark of 1 cm was done to indicate the area of scaffold application.
3.3.2 Addition of Polysaccharides (Chitosan. Alginate and Hyaluronic Acid)
The morphologies of 8% crosslinked gelatin-based hydrogels containing chitosan, hyaluronic acid and alginate are shown in
The morphologies of 8% crosslinked gelatin-based hydrogels containing PEG1450 or PEG4000 from enzyme and gelatin components are presented in
The structures of 8% based-gelatin containing PEG as porogen (in enzyme component) and hyaluronic acid or alginate are shown in
The swelling results of crosslinked-gelatin based scaffolds as a function of formulation are presented in Table 1. Overall, the swelling capacity of such hydrogels ranges from −5% to 69%. The parameters that influence the swelling capacity are: the gelatin content, presence and type of polysaccharides and porogen. The swelling capacity increases as the gelatin content decreases (the crosslink density and the chains mobility are improved, consequently the swelling capacity increases). The swelling capacity increases with the porogen (PEG) concentration. Chitosan decreases the swelling of the scaffolds. It may be related to the non-porous morphology of such hydrogels. Hyaluronic acid improves significantly the swelling capacity, probably due to the ability of HA to attract water molecules. Alginate does not seem to significantly affect the swelling capacity of such hydrogels.
The results of the mechanical properties assessed by lap-shear tests are summarized in Table 2. The maximum load ranges from 0.16 N to more than 9 N, as a function of the formulation. Some parameters seem to influence the adhesive strength of the hydrogels: chitosan seems to strengthen the matrix by up to 45%. This may be attributed to the abundancy of amino-sugar moieties in chitosan molecules which can participate in the crosslinking reaction of gelatin, increasing the crosslinking density and consequently the adhesive strength. In addition, the positively charged chitosan molecules may form electrostatic interactions with negatively charged gelatin moieties and consequently contribute to the stabilization of hydrogel and its strength. Hyaluronic acid does not seem to affect the mechanical properties, but crosslinked alginate increases the maximum load by more than three fold.
Finally, the presence of porogen (PEG1450 or PEG4000) significantly weakens the gelatin matrix (by up to 3 fold, when it is placed in the enzyme component). The combination of porogen and crosslinked alginate allows to reach the maximum load value of the basic formulation (8% gelatin).
Cell viability results are shown in
3.7.1 Influence of Type of Enzyme (mTG Vs. PEG-mTG) and Gelatin Concentration
The type of enzyme and the gelatin concentration may affect the degradation profile of such scaffolds since they influence the crosslinking density. The results of this study are presented in
The effects of porogen and gelatin concentration on the enzymatic degradation profile of gelatin-based hydrogels in collagenase solution are shown in
3.7.3 Influence of Chitosan The effects of chitosan on the enzymatic degradation profile of gelatin-based hydrogels in trypsin solution are shown in
The main aim was to find gelatin-based formulations that meet the criteria to be potential injectable adhesive scaffolds for soft tissue repair (high adhesive strength, appropriate morphology, incorporation of bioactive molecules, enzymatically degradable). The results of the current study are summarized in Table 3 and in
Injectable, adhesive, porous and degradable gelatin scaffolds were successfully developed. The degradation rate by proteolytic enzyme is affected by crosslink density and matrix porosity. The incorporation of PEG molecules with different molecular weights allows to control the porous morphology and the pore size of the gelatin matrices. The addition of alginate and chitosan has a double effect—they reinforce the gelatin scaffolds and, as polysaccharides, may play an important role in would healing promotion of cell viability and proliferation. The 2D culture results demonstrate a well proliferation ability of cells on the enzymatically crosslinked gelatin-based scaffolds.
These materials are suitable injectable scaffolds for soft tissue repair according to at least some embodiments of the present invention.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.
| Number | Date | Country | |
|---|---|---|---|
| 62696904 | Jul 2018 | US |
