Decellularized tendon matrix method and its use

Abstract

Methods for producing decellularized tendon matrix (DTM) and DTM hydrogels are provided. These compositions and hydrogels are useful for repairing tendon injuries, and in some cases can be used by injection, arthroscopic procedures, or as an adjunct to traditional surgical repair.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is an international application claiming the benefit of U.S. Provisional Application No. 62 / 782,903, filed December 20, 2018, and U.S. Provisional Application No. 62 / 890,865, filed August 23, 2019, which are incorporated by reference in their entireties.

[0002] The invention described herein generally relates to decellularized tendon matrices and methods of making and using decellularized tendon matrices. [Background technology]

[0003] Regenerative medicine is an emerging field that has identified many applications for extracellular matrix materials. Tendons are fibrous connective tissues that connect muscle to bone. The junction between muscle and tendon is called the myotendinous junction or tendon-muscle insertion point, and the junction between tendon and bone is called the osteotendinous junction. This is also known as the tendon insertion or enthesopathy, and disease at this site is known as enthesopathy. This latter junction, i.e., the junction between tendon and bone where tendon collagen fibrils insert into the bone matrix, is a common site of tendon injury. These injuries generally result from overuse of the site, intrinsic tendon degeneration (tendinopathy), or traumatic injury.

[0004] Tendon injuries result in well-characterized cellular and tissue changes that alter the tendon's biomechanical properties (e.g., Non-Patent Document 1). Injury due to overuse, intrinsic degeneration, or trauma to the tendon site can manifest as a tendon rupture. Tears are classified by severity, from a Grade 1 (the least severe rupture) to a Grade 2 (moderate to severe rupture) and finally a Grade 3 (complete rupture). They are also classified in other ways, such as partial or complete rupture, in various anatomical regions of the body, such as the rotator cuff, Achilles tendon, quadriceps tendon, and biceps tendon.

[0005] Ruptures generally require surgical intervention. In some embodiments, the present invention provides methods of making compositions useful for repairing tendon injuries, including ruptures.

[0006] Additionally, the compositions of the present invention induce tissue regeneration that accelerates tendon regeneration, tendon healing, or reconstitution of native tendon insertion into bone. The methods of the present invention maintain endogenous growth factors present in the extracellular matrix, providing compositions for tendon regeneration, healing, and / or repair. Summary of the Invention

[0007] In one aspect, the present invention provides a method of making a composition comprising tendon tissue digested with matrix metalloproteinase (MMP) and / or collagenase, an antimicrobial agent, and a sterile aqueous carrier solution.

[0008] In another aspect, the present invention provides a decellularized tendon matrix (DTM) composition prepared by a process comprising: (i) comminuted a tendon tissue sample; (ii) decellularizing the comminuted tendon tissue sample; (iii) grinding; (iv) digesting; (v) arresting and neutralizing; (vi) washing; and (vii) freeze-drying.

[0009] In one aspect, the present invention provides a method for preparing a decellularized tendon matrix that retains growth factors.

[0010] In some embodiments, the present disclosure provides a decellularized tendon matrix (DTM) composition comprising tendon tissue digested with matrix metalloproteinase (MMP). In some embodiments, the present disclosure provides a decellularized tendon matrix (DTM) composition comprising tendon tissue digested with collagenase. In some embodiments, the composition comprises a collagen digest. In some embodiments, the composition further comprises an antimicrobial agent. In some embodiments, the composition further comprises a sterile aqueous carrier solution. In some embodiments, the decellularized tendon matrix (DTM) is rich in protein and maintains at least 50% of the growth factors present in the shredded tendon tissue. In some embodiments, the composition is moldable. In some embodiments, the composition is capable of substantially adhering to anatomical topography.

[0011] In some embodiments, the present disclosure also provides a method for producing a decellularized tendon matrix (DTM) composition, the method comprising one or more steps selected from the steps of comminuted a tendon tissue sample, decellularizing the comminuted tendon tissue sample, grinding, digesting, arresting and neutralizing, washing, and lyophilizing. In some embodiments, the method comprises digestion with a matrix metalloproteinase (MMP) selected from the group consisting of MMP-2, MMP-9, MMP-14, or a combination thereof. In some embodiments, the method comprises digestion with a collagenase as described herein. In some embodiments, the method comprises decellularizing with a DNase as described herein.

[0012] The present disclosure also provides a decellularized tendon matrix (DTM) composition prepared by a process including one or more steps selected from the steps of: comminuted a tendon tissue sample, decellularizing the comminuted tendon tissue sample, digesting, and freeze-drying. In some embodiments, the present disclosure provides a decellularized tendon matrix (DTM) composition prepared by a process including one or more steps selected from the steps of: comminuted a tendon tissue sample, decellularizing the comminuted tendon tissue sample, grinding, digesting, arresting, neutralizing, washing, and freeze-drying. In some embodiments, the decellularization step includes exposing the comminuted tendon tissue sample to a solution containing one or more components selected from a chaotropic salt, a nonionic surfactant, a zwitterionic surfactant, a cationic surfactant, an anionic surfactant, or a combination thereof. In some embodiments, the decellularization step includes exposing the comminuted tendon tissue sample to DNase, RNase, or a combination thereof. In some embodiments, the decellularization step includes exposing the comminuted tendon tissue sample to DNase. In some embodiments, the digestion step comprises digestion with a solution comprising a matrix metalloproteinase (MMP). In some embodiments, the matrix metalloproteinase (MMP) is selected from MMP-2, MMP-9, MMP-14, or a combination thereof. In some embodiments, the terminating and / or neutralizing step comprises terminating and / or neutralizing with a solution comprising one or more protease inhibitors selected from TAPI-0, TAPI-1, TAPI-2, marimastat, phosphoramidon, luteolin, PMSF, pepstatin A, leupeptin, E-64, sodium orthovanadate, or a combination thereof.

[0013] The present disclosure also provides a method of stimulating tendon regeneration, comprising one or more steps selected from resuspending a DTM composition described herein in a pharmaceutically acceptable carrier and applying the resuspended DTM composition to a tendon site requiring stimulated tendon regeneration. In some embodiments, the resuspended DTM composition is moldable. In some embodiments, the resuspended DTM composition has a putty-like consistency. In some embodiments, the resuspended DTM composition is a gel. In some embodiments, the resuspended DTM composition is a paste. In some embodiments, the resuspended DTM composition is thixotropic. In some embodiments, the resuspended DTM composition is viscoelastic. In some embodiments, the resuspended DTM composition is injectable. In some embodiments, the resuspended DTM composition is spreadable.

[0014] The present disclosure also provides a decellularized tendon matrix (DTM) hydrogel comprising a resuspended DTM composition described herein and one or more of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and a PEG-N-hydroxysuccinimide (NHS) ester. In some embodiments, the hydrogel is moldable. In some embodiments, the hydrogel has a putty-like consistency. In some embodiments, the hydrogel is a paste. In some embodiments, the hydrogel is thixotropic. In some embodiments, the hydrogel is viscoelastic. In some embodiments, the hydrogel is injectable. In some embodiments, the hydrogel is smearable.

[0015] The present disclosure also provides a soft cast decellularized tendon matrix (DTM) body prepared by a process including one or more of the following steps: resuspending a decellularized tendon matrix (DTM) composition described herein in a physiological buffer; mixing the DTM composition with a PEG-N-hydroxysuccinimide (NHS) ester to form a soft hydrogel; transferring the soft hydrogel to a three-dimensional mold; curing the polymerization reactants; and inactivating the polymerization reactants.

[0016] The present disclosure also provides a decellularized tendon matrix (DTM) hydrogel comprising the resuspended DTM composition described herein, and further comprising 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and a water-soluble coupling agent selected from N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (sulfo-NHS) together with the (EDC) coupling agent.

[0017] The present disclosure also provides a method for treating tendon rupture and / or stimulating tendon regeneration in a subject, the method comprising one or more of obtaining a decellularized tendon matrix (DTM) composition comprising tendon tissue digested with matrix metalloproteinase (MMP) or collagenase, resuspending the DTM composition in a pharmaceutically acceptable carrier, and applying the resuspended DTM composition to a tendon site requiring stimulated tendon regeneration.

[0018] The present disclosure also provides a decellularized tendon matrix produced from a natural tendon, the decellularized tendon matrix comprising greater than 90% by weight of TGF-β found in a natural tendon. In some embodiments, the decellularized tendon matrix comprises greater than 95% by weight of TGF-β found in a natural tendon. In some embodiments, the decellularized tendon matrix comprises greater than 99% by weight of TGF-β found in a natural tendon. In some embodiments, the decellularized tendon matrix of any of claims 18-20 comprises less than 5% by weight of the cellular material found in a natural tendon. In some embodiments, the decellularized tendon matrix described herein comprises less than 2% by weight of the cellular material found in a natural tendon. In some embodiments, the decellularized tendon matrix described herein comprises less than 1% by weight of the cellular material found in a natural tendon. In some embodiments, the decellularized tendon matrix described herein comprises less than 0.1% by weight of the cellular material found in a natural tendon. In some embodiments, the decellularized tendon matrix described herein is substantially free of TGF-β-producing cells. In some embodiments, the decellularized tendon matrix described herein comprises less than 5% by weight of the DNA found in a natural tendon. In some embodiments, the decellularized tendon matrix described herein contains less than 2% by weight of the DNA found in native tendons. In some embodiments, the decellularized tendon matrix described herein contains less than 1% by weight of the DNA found in native tendons. In some embodiments, the decellularized tendon matrix described herein contains less than 0.1% by weight of the DNA found in native tendons. In some embodiments, the decellularized tendon matrix described herein is substantially free of DNA.

[0019] The present disclosure also provides methods for producing a decellularized tendon matrix (DTM) composition from a tendon, the method comprising one or more of decellularizing the tendon to produce a decellularized tendon, contacting the decellularized tendon with an enzyme solution containing a matrix metalloproteinase (MMP) to produce a digested decellularized tendon, lyophilizing the digested decellularized tendon to produce a lyophilized tendon, and reconstituting the lyophilized tendon to produce a decellularized tendon matrix. In some embodiments, the method comprises contacting the tendon with a DNase solution. In some embodiments, the DNase solution comprises about 10 to about 100 units of DNase per milliliter of solvent, about 25 to about 75 units of DNase per milliliter of solvent, about 40 to about 60 units of DNase per milliliter of solvent, about 40 to about 60 units of DNase per milliliter of solvent, or about 50 units of DNase per milliliter of solvent. In some embodiments, decellularization comprises contacting the tendon with about 4 milliliters to about 50 milliliters of DNase solution per gram of tendon. In some embodiments, decellularization comprises contacting the tendon with about 5 milliliters to about 10 milliliters of DNase solution per gram of tendon. In some embodiments, decellularization comprises contacting the tendon with about 10 milliliters to about 50 milliliters of DNase solution per gram of tendon. In some embodiments, contacting is for about 1 hour, optionally on a shaker. In some embodiments, decellularization further comprises washing the tendon with phosphate-buffered saline. In some embodiments, decellularization further comprises filtering the tendon. In some embodiments, lyophilization comprises freezing the digested, decellularized tendon at minus 80°C for at least about 30 minutes. In some embodiments, the method further comprises filtering through a 70 micrometer strainer using centrifugation at about 1500 to about 2500 g for about 1 minute to about 15 minutes. In some embodiments, the MMP comprises collagenase. In some embodiments, the collagenase is selected from the group consisting of collagenase type I, collagenase type III, and combinations thereof.In some embodiments, the concentration of collagenase type I in the enzyme solution is about 2 milligrams per milliliter. In some embodiments, the concentration of collagenase type III in the enzyme solution is about 1 milligram per milliliter. In some embodiments, the decellularized tendon is contacted with about 10 milliliters to about 50 milliliters of enzyme solution per gram of tendon. In some embodiments, the decellularized tendon is contacted with about 5 milliliters to about 10 milliliters of enzyme solution per gram of tendon. In some embodiments, the decellularized tendon is contacted with the enzyme solution for about 24 hours. In some embodiments, the decellularized tendon is contacted with the enzyme solution for about 12 hours. In some embodiments, the decellularized tendon is contacted with the enzyme solution for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours. In some embodiments, the decellularized tendon is contacted with the enzyme solution at about 37° C. In some embodiments, reconstitution comprises mixing about 2 microliters to about 5 microliters of solvent with about 1 milligram of lyophilized tendon. [Brief explanation of the drawings]

[0020] [Figure 1] Figures 1A and 1B show the characterization of native patellar and Achilles tendons for DNA content (Figure 1A) and protein content (Figure 1B) before processing. Measurements represent tendons from a total of six donors.

[0021] [Figure 2] Figures 2A and 2B show native TGF-β concentrations based on tendon type and location. TGF-β3 (Figure 2A) and TGF-β1 (Figure 2B) concentrations found in native tendon samples (before treatment) are shown.

[0022] [Figure 3]Figure 3 shows a comparison of decellularization using DNase and detergents. DNA content of both patellar and Achilles tendons is measured in native tendons, tendons treated with 50 U of DNase for 1 hour, tendons treated with 50 U of DNase for 2 hours, and tendons treated with conventional decellularization methods using SDS or EDTA.

[0023] [Figure 4] Figure 4 shows the total protein content of tendons using various enzyme reagents to digest tendon samples, including C-1 collagenase I, C-3 collagenase III, both C-1 collagenase I and C-3 collagenase III, and pepsin.

[0024] [Figure 5] Figure 5 shows the TGF-β concentration in the tendons before and after processing into decellularized tendon matrix. Native tendons were measured by averaging the concentrations across the proximal, mid-substance, and distal portions of both the patellar and Achilles tendons.

[0025] [Figure 6] Figures 6A and 6B show that decellularized tendon matrix processing promotes elastic properties with the ability to stretch (Figure 6A) from an unstretched shape (Figure 6B) without tearing. DTM is storage-stable as a sterile, lyophilized powder and can be reconstituted into a putty or injection solution. This image shows a DTM putty that can be produced by resuspending lyophilized DTM at 3-5 ul / mg. This putty is moldable / stretchable upon surgical application to the area where repair is desired.

[0026] [Figure 7]Figures 7A-7C show that DNAse treatment effectively decellularizes tendon tissue. Tendons were decellularized for 1 hour using various concentrations of DNAse (10 U, 50 U, and 100 U). 1x PBS was used as a no-decellularization control. DNA concentration was determined using a DNEasy kit (Qiagen). The data show that as little as 50 U of DNAse is effective in decellularizing tissue.

[0027] [Figure 8] Figure 8 shows that DNAse treatment is as effective as standard detergent methods in decellularizing tendons. 50 U of DNAse was compared to conventional detergent, 1% SDS, and 0.1% EDTA. 50 U of DNAse was tested at 0.5, 1, and 2 hours, whereas the standard SDS and EDTA protocol requires 24 hours of decellularization. DNA concentration was determined using the DNEasy kit (Qiagen, n=3). All values ​​were normalized to "no decellularization." Tukey's HSD multiple comparison post-hoc test showed no significant differences between DNAse treatment or DNAse decellularization at each time point compared to SDS and EDTA.

[0028] [Figure 9-1] Figures 9A-9H show that the Achilles tendon matrix has a higher protein content than the patellar tendon. Achilles and patellar tendons were divided into one-third sections, consisting of the proximal end, midcenter / middle, and distal end of the tendon. (A-D) Total protein in native tendons was measured using a BCA protein quantification kit (Thermo Scientific). (E-H) TGF-β was measured using a TGF-β magnetic bead panel Milliplex kit (Millipore Sigma, #TGFBMAG-64K-03). ANOVA showed no statistically significant differences between tendon regions, so the entire tendon can be used for processing. Comparing the two different tendons, (D) there was no difference in total protein (P = 0.93), but (H) TGF-β was statistically higher in the Achilles tendon than in the patellar tendon (P = 0.0045). [Figure 9-2] Figures 9A-9H show that the Achilles tendon matrix has a higher protein content than the patellar tendon. Achilles and patellar tendons were divided into one-third sections, consisting of the proximal end, midcenter / middle, and distal end of the tendon. (A-D) Total protein in native tendons was measured using a BCA protein quantification kit (Thermo Scientific). (E-H) TGF-β was measured using a TGF-β magnetic bead panel Milliplex kit (Millipore Sigma, #TGFBMAG-64K-03). ANOVA showed no statistically significant differences between tendon regions, so the entire tendon can be used for processing. Comparing the two different tendons, (D) there was no difference in total protein (P = 0.93), but (H) TGF-β was statistically higher in the Achilles tendon than in the patellar tendon (P = 0.0045). [Figure 9-3] Figures 9A-9H show that the Achilles tendon matrix has a higher protein content than the patellar tendon. Achilles and patellar tendons were divided into one-third sections, consisting of the proximal end, midcenter / middle, and distal end of the tendon. (A-D) Total protein in native tendons was measured using a BCA protein quantification kit (Thermo Scientific). (E-H) TGF-β was measured using a TGF-β magnetic bead panel Milliplex kit (Millipore Sigma, #TGFBMAG-64K-03). ANOVA showed no statistically significant differences between tendon regions, so the entire tendon can be used for processing. Comparing the two different tendons, (D) there was no difference in total protein (P = 0.93), but (H) TGF-β was statistically higher in the Achilles tendon than in the patellar tendon (P = 0.0045). [Figure 9-4]Figures 9A-9H show that the Achilles tendon matrix has a higher protein content than the patellar tendon. Achilles and patellar tendons were divided into one-third sections, consisting of the proximal end, midcenter / middle, and distal end of the tendon. (A-D) Total protein in native tendons was measured using a BCA protein quantification kit (Thermo Scientific). (E-H) TGF-β was measured using a TGF-β magnetic bead panel Milliplex kit (Millipore Sigma, #TGFBMAG-64K-03). ANOVA showed no statistically significant differences between tendon regions, so the entire tendon can be used for processing. Comparing the two different tendons, (D) there was no difference in total protein (P = 0.93), but (H) TGF-β was statistically higher in the Achilles tendon than in the patellar tendon (P = 0.0045).

[0029] [Figure 10] Figure 10 shows that filtration effectively removes collagenase activity. Decellularized tendons were treated with collagenase to improve the form factor of DTM. The 100 kDa filter was highly effective in removing collagenase activity from the final product. ANOVA showed a significant difference between groups (F(4,22) = 18.06, p < 0.0001). Importantly, no significant difference in collagenase activity was observed between the native and 100 kDa filtered samples.

[0030] [Figure 11]Figure 11 shows that DTM retains higher bioactivity than the standard method for decellularizing tendons with pepsin. Tendons were digested after decellularization using a solution containing collagenase type 1 (92.5 g tendon / 1 g collagenase 1) and collagenase type 3 (185 g tendon / 1 g Collagenase 3) or a previously published method (Farnebo et al. 2014, PMID: 24341855). ANOVA showed a significant difference between groups (F(3,11) = 5.056, p = 0.0193). Tukey's HSD post-hoc analysis showed significantly less TGF-β with pepsin (P = 0.0249).

[0031] [Figure 12-1] Figures 12A-12C show the difference in proliferation of cells plated on different surfaces. Tissue culture plates were left untreated (control, "TC-treated") or coated with collagen or DTM. Primary tenocytes (ZenBio#TEN-F) were plated at 20,000 cells / well, and cell viability was quantified using Presto Blue (Thermo Fisher Scientific) 48 hours after plating (Figure 12A) or 7 days after plating (Figure 12B). Significantly different proliferation rates were observed (Figure 12C). (ANOVA = F(3,26) = 10.6, p < 0.0001). [Figure 12-2] Figures 12A-12C show the difference in proliferation of cells plated on different surfaces. Tissue culture plates were left untreated (control, "TC-treated") or coated with collagen or DTM. Primary tenocytes (ZenBio#TEN-F) were plated at 20,000 cells / well, and cell viability was quantified using Presto Blue (Thermo Fisher Scientific) 48 hours after plating (Figure 12A) or 7 days after plating (Figure 12B). Significantly different proliferation rates were observed (Figure 12C). (ANOVA = F(3,26) = 10.6, p < 0.0001).

[0032] [Figure 13]Figures 13A-13F show differences in cell morphology and / or proliferation when plated on different surfaces. Tissue culture plates were left untreated (control, "TC-treated") or coated with collagen or DTM. Primary tenocytes (ZenBio#TEN-F) were plated at 20,000 cells / well. Live cell images were captured by time-lapse video over a 3-day period, revealing significantly different cell morphologies and proliferation rates among the various surface treatments (Figure 6). Still images from live cell imaging taken at 48 hours demonstrate that tenocytes adhere and proliferate more rapidly, with increased focal adhesions and a more natural cell morphology on DTM (F) compared to standard tissue culture (D) or collagen-coated plates (E).

[0033] [Figure 14A] Figure 14A shows an image of the surgical application of the DTM. The DTM can be a putty or an injectable solution. In this case, the putty was placed over the larger tuberosity, and the supraspinatus muscle was surgically attached to immobilize the DTM. [Figure 14B] Figure 14B shows an image of the surgical application of the DTM. The DTM can be a putty or an injectable solution. In this case, the putty was placed over the larger tuberosity, the supraspinatus muscle was surgically attached, and the DTM was immobilized. [Figure 14C] Figure 14C shows an image of the surgical application of the DTM. The DTM can be a putty or an injectable solution. In this case, the putty was placed over the larger tuberosity, the supraspinatus muscle was surgically attached, and the DTM was immobilized.

[0034] [Figure 15] Figure 15 shows normalized TGFb content across four samples from four different donors across two processing steps. For each donor, the first column represents the amount of TGFb in native tendon, the second column represents the amount of TGFb in decellularized tendon, and the third column represents the amount of TGFb in digested tendon.

[0035] Detailed Description of the Invention Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications mentioned herein are incorporated by reference in their entirety.

[0036] definition As used herein, the terms "co-administration," "co-administer," "administered in combination," "administered in combination," "simultaneous," and "concurrent" include the administration of two or more active pharmaceutical ingredients. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Concurrent administration in separate compositions and administration in which both agents are present in a composition are preferred.

[0037] The term "in vivo" refers to an event that takes place inside a subject's body.

[0038] The term "in vitro" refers to an event that occurs outside a subject's body. In vitro assays include cell-based assays in which live or dead cells are used, and can also include cell-free assays in which intact cells are not used.

[0039] As used herein, the terms "treatment," "treat," "alleviate," and "ameliorate" are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results, including, but not limited to, therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to the eradication or amelioration of the underlying disorder being treated. Therapeutic benefit is also achieved by the eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the patient, even though the patient may still be afflicted by the underlying disorder.

[0040] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds described herein sufficient to achieve the intended use, including, but not limited to, disease treatment. A therapeutically effective amount may vary depending on the intended use (in vitro or in vivo), or the subject and condition being treated (e.g., the subject's weight, age, and sex), the severity of the condition, the method of administration, etc., and can be readily determined by one of ordinary skill in the art. The term also applies to a dose that elicits a specific response in target cells (e.g., reduced platelet adhesion and / or cell migration). The specific dose may vary depending on the particular compound selected, the dosing regimen followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system that carries the compound.

[0041] As used herein, a "therapeutic effect" includes a therapeutic benefit and / or a prophylactic benefit. A prophylactic effect includes delaying or eliminating the onset of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or ameliorating the progression of a disease or condition, or any combination thereof.

[0042] As used herein, "donor" refers to a mammalian source of tendon connective tissue. The donor can be human or other animal source, including cadaveric tendon tissue. "Allogeneic" donor tissue is donor tissue from a non-genetically identical member of the same species, for example, harvested from one human subject, and the resulting composition then administered to another human subject. Tendon connective tissue can be harvested from a donor of another species for use in the methods herein to generate a decellularized tendon matrix composition. Such a composition is a "xenogenic" decellularized tendon matrix composition. Preferred xenogenic sources are porcine, equine, bovine, ovine, canine, and rodent. Regardless of source, xenogenic tendon tissue can be fresh or frozen tissue from a cadaveric donor. Preferred allograft sources are the Achilles tendon and patellar tendon. These tendons are readily available and relatively large in size. They are also widely used in autograft and allograft applications for the reconstruction of torn or damaged ligaments and tendons.

[0043] As used herein, "decellularization" refers to the typical (at least 80%), near complete (at least 95%), or essentially complete (at least 99%) removal of the cellular components of tendon connective tissue.

[0044] As used herein, "matrix metalloproteinase" refers to proteins of the matrix metalloproteinase (MMP) family. Matrix metalloproteinases (MMPs) comprise a large family of zinc-dependent endoproteinases that can degrade all extracellular matrix (ECM) components. The term encompasses both the apo and activated forms of each MMP family member. The term encompasses MMP-2, MMP-9, MMP-14, homologs, derivatives, and fragments thereof. Fanjul-Fernandez et al. summarize the mammalian MMP family in a review article, Biochim. Biophys. Acta 1803:3-19 (2010).

[0045] Various growth factors are known in the art, including IGF-1 (insulin-like growth factor 1, or somatomedin C), TGF-β (transforming growth factor beta), PDGF (platelet-derived growth factor), VEGF (vascular endothelial growth factor (VEGF) (also known as vascular permeability factor (VPF)), bFGF (basic fibroblast growth factor, or fibroblast growth factor 2 (FGF2)), GDF-5 (growth differentiation factor 5), GDF-6 (growth differentiation factor 6), GDF-7 (growth differentiation factor 7), and HGF (hepatocyte growth factor or scatter factor). Without being bound by theory, the above non-limiting list of growth factors is known in the art to be present in the extracellular matrix of tendons.

[0046] The phrase "pharmaceutically acceptable" means those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0047] "Pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inactive ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as conventional pharmaceutically acceptable carriers or pharmaceutically acceptable excipients are incompatible with the DTM components, their use in the therapeutic compositions of the present invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be included in the compositions and methods described.

[0048] When ranges are used herein to describe physical or chemical properties, such as, for example, molecular weight or chemical formula, all combinations and subcombinations of ranges, as well as specific embodiments within those ranges, are intended to be included. The use of the term "about" when referring to a numerical value or numerical range means that the stated numerical value or numerical range is an approximation within experimental variation (or within statistical experimental error), and that the numerical value or numerical range may vary. The variation is typically 0% to 15%, preferably 0% to 10%, and more preferably 0% to 5% of the stated numerical value or numerical range. The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") includes embodiments, such as, for example, any composition, method, or process embodiment that "consists of" or "consists essentially of" the described features.

[0049] The terms "sequence identity," "percent identity," and "percent sequence identity" in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are identical, or that have the same specified percentage of nucleotide or amino acid residues, when compared and aligned for maximum correspondence (introducing gaps, if necessary) without considering conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to align amino acid or nucleotide sequences. Suitable programs for determining percent sequence identity include, for example, the BLAST suite of programs available from the BLAST website of the U.S. government's National Center for Biotechnology Information. Comparison between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. ALIGN, available from DNASTAR, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign are other publicly available software programs that can be used to align sequences. Those skilled in the art can determine appropriate parameters for maximal alignment with a particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0050] For the avoidance of doubt, it is understood that a particular feature (e.g., integer, property, value, use, disease, formula, compound, or group) described in connection with a particular aspect, embodiment, or example of the invention is applicable to any other aspect, embodiment, or example described herein, unless incompatible. Accordingly, such features may be used in combination with any of the definitions, claims, or embodiments defined herein, where appropriate. All features disclosed herein (including the accompanying claims, abstract, and drawings), and / or all steps of any disclosed method or process, may be combined in any combination, except for combinations in which at least some of the features and / or steps are mutually exclusive. The invention is not limited to the details of the disclosed embodiments. The invention extends to any novel one or novel combination of features, or any novel combination of steps of any disclosed method or process, disclosed herein (including the accompanying claims, abstract, and drawings).

[0051] Method for producing decellularized tendon matrix One goal of embodiments of the present disclosure is to develop a gentle and specific decellularization and digestion protocol to produce DTM that maintains growth factors, particularly TGF-β, in the matrix. Traditionally, detergents are harsh and can remove or denature proteins and cellular material.

[0052] Typical digestion techniques for decellularized matrices use common proteinases, most commonly pepsin. Pepsin indiscriminately cleaves all proteins into small polypeptides. In this application, an enzyme specific for the degradation of collagen is used to break down tendons into smaller parts that can then form self-assembling peptides. Collagen, primarily type I collagen, forms the structural framework of tendons. By specifically cleaving collagen, the tendons are digested while maintaining the biological activity of bound growth factors.

[0053] Collagenase is an endopeptidase that digests native collagen fibrils in the triple-helical structure typically found in tendons. Collagenase cleaves the bond between a neutral amino acid (X) and glycine in the frequently occurring Pro-X-Gly-Pro sequence in collagen. Bacterial collagenases, such as those produced by Clostridium histolyticum, attack almost all types of collagen and can degrade both water-insoluble native collagen and water-soluble denatured collagen. The ability of clostridial collagenase to digest native triple-helical collagen types I, II, and III by multiple cleavages of the triple helix is ​​a major distinguishing factor. Clostridial collagenases are extremely large metalloproteases, a family of proteases that contain a zinc-containing motif at the center of their active site (Gonzales and Robert-Baudouy 1996).

[0054] Matrix metalloproteinases (MMPs) also have the ability to cleave collagen fibers in very specific sequences. Interstitial collagen types I, II, and III are highly resistant to proteolytic attack due to their triple helical structure, but can be degraded by MMP collagenases at specific sites. MMP-2 and MMP-9 are closely related at the structural level and exhibit collagenase activity against types I and III collagen, and are classical 3 / 4 and 1 / 4 MMP-1, MMP-8, MMP-13, and MT-MMP also have some limited collagenase activity.

[0055] In one aspect, the present invention provides a method for producing a composition comprising tendon tissue digested with a matrix metalloproteinase (MMP), an antimicrobial agent, and a sterile aqueous carrier solution. In some embodiments, the matrix metalloproteinase (MMP) is selected from the group consisting of MMP-2, MMP-9, MMP-14, or a combination thereof. In one aspect, the MMP is designed to be constitutively active. Those skilled in the art will appreciate that other MMPs can be used. Collagenase, gelatinase, stromelysin, and membrane-type MMP (MT-MMP) can be used. In certain embodiments, collagenase can be used to decellularize tendons and / or digest the decellularized tendons. As described herein, collagenase can degrade triple-helical fibrous collagens into characteristic ¾ and ¼ fragments. These collagens are the main components of bone, cartilage, and dentin. Collagenase includes collagenase type 1, collagenase type 2, collagenase type 3, collagenase type 8, collagenase type 13, collagenase type 14, and collagenase type 18. Non-limiting examples of one or more MMPs that can be used include MMP1 (interstitial collagenase, CLG, CLGN), MMP2 (gelatinase-A, 72 kDa gelatinase), MMP3 (stromelysin 1, CHDS6, MMP-3, SL-1, STMY, STMY1, STR1), MMP7 (matrilysin, PUMP1, MMP-7, MPSL1, PUMP-1), MMP8 (neutrophil collagenase, CLG1, HNC, MMP-8, PMNL-CL), MMP9 (gelatinase-B, 92 kDa gelatinase, CLG4B, GELB, MANDP2, MM P-9), MMP10 (stromelysin 2, SL-2, STMY2), MMP11 (stromelysin 3, SL-3, ST3, STMY3), MMP12 (macrophage metalloelastase, HME, ME, MME, MMP-12), MMP13 (collagenase 3, CLG3, MANDP1, MMP-13), MMP14 (MT1-MMP, MMP-14, MMP-X1, MT-MMP, MT-MMP1, MT1-MMP, MT1MMP, MTMMP1, WNCHRS), MMP15 (MT2-MMP, MT2-MMP, MTMMP2, SMCP-2,MMP-15, MT2MMP), MMP16 (MT3-MMP, C8orf57, MMP-X2, MT-MMP2, MT-MMP3, MT3-MMP), MMP17 (MT4-MMP, MT4-MMP, MMP-17, MT4MMP, MTMMP4), MMP18 (collagenase 4, xcol4, Xenopus collagenase), MMP19 (RASI-1 (sometimes called stromelysin-4), MMP18, RASI-1, CODA), MMP20 (enamelysin, AI2A2, MMP-20), MMP21 (X-MMP, MMP-21, HTX7), MMP23A (CA-MMP), MM Examples of MMPs include P23B (MIFR, MIFR-1, MMP22), MMP24 (MT5-MMP, MMP-24, MMP25, MT-MMP5, MT-MMP5, MT5-MMP, MT5MMP, MTMMP5), MMP25 (MT6-MMP, MMP-25, MMP20, MMP20A, MMPL1, MT-MMP6, MT-MMP6, MT6-MMP, MT6MMP, MTMMP6), MMP26 (matrilysin-2, endometase), MMP27 (MMP-22, C-MMP, MMP-27), and MMP28 (epilysin, epilysin, MMP28, MMP-25, MMP-28).

[0056] The concentration of collagenase used to enzymatically digest the decellularized tendon can vary depending on the particular collagenase used. In certain embodiments, collagenase type 1 can be used to enzymatically digest the decellularized tendon. In certain embodiments, collagenase type 3 can be used to enzymatically digest the decellularized tendon. The concentrations of collagenase used to enzymatically digest the decellularized tendon are about 0.1 milligram (mg) per milliliter (mL), about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, about 1.1 mg / mL, about 1.2 mg / mL, about 1.3 mg / mL, about 1.4 mg / mL, about 1.5 mg / mL, about 1.6 mg / mL, about 1.7 mg / mL, about 1.8 mg / mL, about 1.9 mg / mL, about 2.0 mg / mL, about 2.1 mg / mL, The collagenase concentration can be about 2.2 mg / mL, about 2.3 mg / mL, about 2.4 mg / mL, about 2.5 mg / mL, about 2.6 mg / mL, about 2.7 mg / mL, about 2.8 mg / mL, about 2.9 mg / mL, about 3.0 mg / mL, about 3.1 mg / mL, about 3.2 mg / mL, about 3.3 mg / mL, about 3.4 mg / mL, about 3.5 mg / mL, about 3.6 mg / mL, about 3.7 mg / mL, about 3.8 mg / mL, about 3.9 mg / mL, about 4.0 mg / mL, about 5.0 mg / mL, about 6.0 mg / mL, about 7.0 mg / mL, about 8.0 mg / mL, about 9.0 mg / mL, or about 10.0 mg / mL. In certain embodiments, the collagenase concentration used to enzymatically digest the decellularized tendon is about 1.0 mg / mL. In other embodiments, the concentration of collagenase used to enzymatically digest the decellularized tendon is about 2.0 mg / mL.

[0057] The antibacterial agent is suitable for use in parenteral formulations, for example, alkyl alcohols or aryl alcohols such as benzyl alcohol, chlorobutanol, or 2-ethoxyethanol. Aminoaryl acid esters, for example, methyl, ethyl, propyl, or butyl paraben, and combinations thereof, are also suitable. Alkyl and aryl acids may also be suitable, for example, benzoic acid or sorbic acid, biguanides, for example, chlorhexidine, or phenols, for example, phenol or 3-cresol. In some embodiments, a combination of chemically compatible antibacterial agents is used.

[0058] In one aspect, the present invention provides a decellularized tendon matrix (DTM) composition prepared by a process comprising: (i) comminuted a tendon tissue sample; (ii) decellularizing the comminuted tendon tissue sample; (iii) digesting; and (iv) freeze-drying.

[0059] In one aspect, the present invention provides a decellularized tendon matrix (DTM) composition prepared by a process comprising: (i) comminuted a tendon tissue sample; (ii) decellularizing the comminuted tendon tissue sample; (iii) grinding; (iv) digesting; (v) arresting and neutralizing; (vi) washing; and (vii) freeze-drying.

[0060] In some cases, prior to decellularization, grinding, digestion, lyophilization, and / or washing, the tendon matrix can be present in an amount of about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% by weight of the isolated tendon tissue.

[0061] In some cases, prior to decellularization, grinding, digestion, lyophilization, and / or washing, the tendon matrix can be present in an amount of about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 50% to about 55%, 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 60% to about 65%, 70% to about 90%, about 70% to about 80%, about 70% to about 75%, 80% to about 90%, about 80% to about 85%, or about 85% to about 90% by weight of the isolated tendon tissue.

[0062] In some cases, prior to decellularization, grinding, digestion, freeze-drying, and / or washing, the tendon matrix can be present in an amount of less than about 90% by weight, less than about 85% by weight, less than about 80% by weight, less than about 75% by weight, less than about 70% by weight, less than about 65% by weight, less than about 60% by weight, less than about 55% by weight, less than about 50% by weight, less than about 45% by weight, less than about 40% by weight, less than about 35% by weight, less than about 30% by weight, less than about 25% by weight, less than about 20% by weight, less than about 15% by weight, or less than about 10% by weight of the isolated tendon tissue.

[0063] In some cases, prior to decellularization, grinding, digestion, lyophilization, and / or washing, the tendon matrix can be present in an amount of about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% by volume of the isolated tendon tissue.

[0064] In some cases, prior to decellularization, grinding, digestion, lyophilization, and / or washing, the tendon matrix can be present in an amount of about 50% to about 90% by volume, about 50% to about 80% by volume, about 50% to about 70% by volume, about 50% to about 60% by volume, about 50% to about 55% by volume, 60% to about 90% by volume, about 60% to about 80% by volume, about 60% to about 70% by volume, about 60% to about 65% by volume, 70% to about 90% by volume, about 70% to about 80% by volume, about 70% to about 75% by volume, 80% to about 90% by volume, about 80% to about 85% by volume, or about 85% to about 90% by volume of the isolated tendon tissue.

[0065] In some cases, prior to decellularization, grinding, digestion, freeze-drying, and / or washing, the tendon matrix can be present in an amount of less than about 90% by volume, less than about 85% by volume, less than about 80% by volume, less than about 75% by volume, less than about 70% by volume, less than about 65% by volume, less than about 60% by volume, less than about 55% by volume, less than about 50% by volume, less than about 45% by volume, less than about 40% by volume, less than about 35% by volume, less than about 30% by volume, less than about 25% by volume, less than about 20% by volume, less than about 15% by volume, or less than about 10% by volume of the isolated tendon tissue.

[0066] In some cases, after decellularization, grinding, digestion, freeze-drying, and / or washing, the tendon matrix can be present in an amount of about 40% by weight, about 45% by weight, about 50% by weight, about 55% by weight, about 60% by weight, about 65% by weight, about 70% by weight, about 75% by weight, about 80% by weight, about 85% by weight, about 90% by weight, about 95% by weight, or about 99% by weight of the decellularized, grinded, digested, freeze-dried, and / or washed tendon tissue.

[0067] In some cases, after decellularization, grinding, digestion, freeze-drying, and / or washing, the tendon matrix can be present in an amount of about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 50% to about 55%, 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 60% to about 65%, 70% to about 90%, about 70% to about 80%, about 70% to about 75%, 80% to about 90%, about 80% to about 85%, or about 85% to about 90% by weight of the decellularized, grinded, digested, freeze-dried, and / or washed tendon tissue.

[0068] In some cases, after decellularization, grinding, digestion, freeze-drying, and / or washing, the tendon matrix can be present in an amount of greater than about 99% by weight, greater than about 95% by weight, greater than about 90% by weight, greater than about 85% by weight, greater than about 80% by weight, greater than about 75% by weight, greater than about 70% by weight, greater than about 65% by weight, greater than about 60% by weight, greater than about 55% by weight, greater than about 50% by weight, greater than about 45% by weight, greater than about 40% by weight, greater than about 35% by weight, greater than about 30% by weight, greater than about 25% by weight, greater than about 20% by weight, greater than about 15% by weight, or greater than about 10% by weight of the decellularized, grinding, digestion, freeze-drying, and / or washed tendon tissue.

[0069] In some cases, after decellularization, grinding, digestion, freeze-drying, and / or washing, the tendon matrix can be present in an amount of about 40% by volume, about 45% by volume, about 50% by volume, about 55% by volume, about 60% by volume, about 65% by volume, about 70% by volume, about 75% by volume, about 80% by volume, about 85% by volume, about 90% by volume, about 95% by volume, or about 99% by volume of the decellularized, grinded, digested, freeze-dried, and / or washed tendon tissue.

[0070] In some cases, after decellularization, grinding, digestion, freeze-drying, and / or washing, the tendon matrix can be present in an amount of about 50% to about 90% by volume, about 50% to about 80% by volume, about 50% to about 70% by volume, about 50% to about 60% by volume, about 50% to about 55% by volume, 60% to about 90% by volume, about 60% to about 80% by volume, about 60% to about 70% by volume, about 60% to about 65% by volume, 70% to about 90% by volume, about 70% to about 80% by volume, about 70% to about 75% by volume, 80% to about 90% by volume, about 80% to about 85% by volume, or about 85% to about 90% by volume of the decellularized, grinding, digestion, freeze-drying, and / or washed tendon tissue.

[0071] In some cases, after decellularization, grinding, digestion, freeze-drying, and / or washing, the tendon matrix can be present in an amount of greater than about 99% by volume, greater than about 95% by volume, greater than about 90% by volume, greater than about 85% by volume, greater than about 80% by volume, greater than about 75% by volume, greater than about 70% by volume, greater than about 65% by volume, greater than about 60% by volume, greater than about 55% by volume, greater than about 50% by volume, greater than about 45% by volume, greater than about 40% by volume, greater than about 35% by volume, greater than about 30% by volume, greater than about 25% by volume, greater than about 20% by volume, greater than about 15% by volume, or greater than about 10% by volume of the decellularized, grinding, digestion, freeze-drying, and / or washed tendon tissue.

[0072] In one aspect, the decellularization process comprises exposing the shredded tendon tissue sample to a solution comprising one or more components selected from a chaotropic salt, a non-ionic detergent, a zwitterionic detergent, a cationic detergent, an anionic detergent, or a combination thereof. In some embodiments, the decellularization process comprises one or more freeze / thaw cycles. In some aspects, the decellularization process further comprises treatment with DNase and / or RNase. In some aspects, the decellularization process further comprises one or more washes in a balanced salt solution, e.g., Hank's balanced salt solution, phosphate buffered saline.

[0073] In some embodiments, the minced tendon tissue sample is rinsed with ultrapure water and then decellularized using a solution containing 1% w / v sodium dodecyl sulfate (SDS) under moderate agitation, in some embodiments, the moderate agitation is intermittent.

[0074] In another aspect, the minced tendon tissue sample is decellularized using a solution comprising one or more of an ionic surfactant, a non-ionic surfactant, an anionic surfactant, or a cationic surfactant. In some aspects, the decellularization solution further comprises a chaotropic salt. In some embodiments, the chaotropic salt is urea. In some embodiments, the decellularization solution comprises between 0.5 M urea and 8 M urea. In some embodiments, the decellularization solution comprises between 2 M and 5 M urea. In some embodiments, the decellularization solution comprises about 3 M urea.

[0075] In some embodiments, the decellularization solution comprises a surfactant and a chaotropic salt. In some embodiments, the decellularization solution further comprises an anti-foaming agent, for example, Antifoam 204.

[0076] In another aspect, the process further comprises precipitating cellular proteins, the process further comprising treating the minced tendon tissue sample with a concentrated kosmotropic solution. In some embodiments, the concentrated kosmotropic solution is ammonium sulfate. Kosmotropic salting out is accomplished, for example, according to the method summarized in Wingfield, Curr. Protoc. Protein Sci., APPENDIX 3:Appendix-3F (2001).

[0077] Mincing can be accomplished using methods known in the art, for example, by first removing the sheath, fat, and synovial tissue from the tendon tissue sample. The tendon tissue sample is then cut into approximately 1-4 mm pieces. 3 After chopping to a size of 100 mm, the tissue is washed with phosphate buffered saline (PBS).

[0078] In one aspect, the quenching and neutralization step comprises quenching and neutralizing with a solution comprising one or more protease inhibitors selected from the group consisting of TAPI-0, TAPI-1, TAPI-2, marimastat, phosphoramidon, luteolin, PMSF, pepstatin A, leupeptin, E-64, sodium orthovanadate, or combinations thereof.

[0079] Decellularization can be monitored by methods known in the art, such as sectioning decellularized and control samples (i.e., untreated samples of starting donor tendon tissue) and staining them with hematoxylin-eosin and Masson-Goldner trichrome stains to detect cellular components and collagen fiber structure, respectively. DNA may be extracted from decellularized and untreated starting samples. Decellularized samples typically yield at least four-fold less DNA than comparable starting weights. See, e.g., Seif-Naraghi et al., Acta Biomater. 8:3695-3703 (2012).

[0080] Decellularized tissues retain extracellular matrix (ECM) components from all or most regions of the tissue, including the ECM components of the vascular tree. ECM components may include any combination of fibronectin, fibrillin, laminin, elastin, collagen family members (e.g., collagen types I, III, and IV), ECM-associated growth proteins such as growth factors and cytokines, glycosaminoglycans, ground substance, reticular fibers, and thrombospondin, which may remain organized as defined structures such as basement membranes. Successful decellularization can be defined as the absence of detectable myofilaments, endothelial cells, smooth muscle cells, and nuclei in tissue sections using standard histological staining procedures, or the removal of 97% or more of detectable DNA (e.g., as measured by fluorescent assays). Residual cellular debris can be removed from the decellularized tissue.

[0081] The morphology and architecture of the ECM can be maintained during and after the decellularization process. As used herein, "morphology" refers to the overall shape of the ECM, and "architecture" refers to the ECM on the outer surface, inner surface, and the surfaces between them. The morphology and architecture of the ECM can be examined visually and / or histologically.

[0082] One or more compounds can be applied into or onto the decellularized tissue to, for example, preserve the decellularized tissue or prepare the decellularized tissue for recellularization or integration or implantation into a host. Such compounds include, but are not limited to, one or more growth factors (e.g., VEGF, DKK-1, FGF, BMP-1, BMP-4, SDF-1, IGF, and HGF), immunomodulators (e.g., cytokines, glucocorticoids, IL2R antagonists, leukotriene antagonists), and / or factors that modulate the coagulation cascade (e.g., aspirin, heparin-binding proteins, and heparin). Additionally, the decellularized tissue can be further treated, for example, with irradiation (e.g., UV, gamma) to reduce or eliminate the presence of any type of microorganism remaining on or in the decellularized tissue.

[0083] In some embodiments, the present invention provides methods of producing a decellularized tendon matrix (DTM) composition using a method further comprising maintaining at least 100, at least 99, at least 98, at least 97, at least 96, at least 95, at least 94, at least 93, at least 92, at least 91, or at least 90% of the growth factors present in the shredded tendon tissue. In some embodiments, the present invention provides compositions produced using a method of producing a decellularized tendon matrix (DTM) composition using a method further comprising maintaining at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, or at least 40% of the growth factors present in the shredded tendon tissue. In some embodiments, the present invention provides a method for producing a decellularized tendon matrix (DTM) composition, wherein the composition contains at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85%, at least 84%, at least 83%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 9 ... 2%, at least 81%, at least 80%, at least 79%, at least 78%, at least 77%, at least 76%, at least 75%, at least 74%, at least 73%, at least 72%, at least 71%, at least 70%, at least 69%, at least 68%, at least 67%, at least 66%, at least 65%, at least 64%, at least 63%, at least 62%, at least 61%, at least 60%, at least 59%, at least 58%, at least 57%, at least at least 56%, at least 55%, at least 54%, at least 53%, at least 52%, at least 51%, at least 50%, at least 49%, at least 48%, at least 47%, at least 46%, at least 45%, at least 44%, at least 43%, at least 42%, at least 41%, at least 40%, at least 39%, at least 38%, at least 37%, at least 36%, at least 35%, at least 34%, at least 33%, at least 32%, at least 31% , at least 31%, at least 30%, at least 29%, at least 28%, at least 27%, at least 26%, at least 25%, at least 24%, at least 23%, at least 22%, at least 21%, at least 20%, at least 19%, at least 18%, at least 17%, at least 16%, at least 15%, at least 14%, at least 13%, at least 12%, at least 11%, or at least 10%.In some embodiments, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, wherein the composition produced using the method maintains about 70% to about 100% of the growth factors present in the shredded tendon tissue prior to decellularization. In some embodiments, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, wherein the composition produced using the method maintains about 70% to about 75% of the growth factors present in the shredded tendon tissue prior to decellularization. In some embodiments, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, wherein the composition produced using the method maintains about 75% to about 80% of the growth factors present in the shredded tendon tissue prior to decellularization. In some embodiments, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, wherein the composition produced using the method maintains about 80% to about 85% of the growth factors present in the shredded tendon tissue prior to decellularization. In some embodiments, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, wherein the composition produced using the method maintains about 85% to about 90% of the growth factors present in the shredded tendon tissue prior to decellularization. In some embodiments, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, wherein the composition produced using the method maintains about 90% to about 95% of the growth factors present in the shredded tendon tissue prior to decellularization. In some embodiments, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, wherein the composition produced using the method maintains about 95% to about 100% of the growth factors present in the shredded tendon tissue prior to decellularization. In some embodiments, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, wherein the composition produced using the method maintains about 75% to about 95% of the growth factors present in the shredded tendon tissue prior to decellularization. In some aspects, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, wherein the composition produced using said method retains about 70% to about 80% of the growth factors present in the minced tendon tissue prior to decellularization.In some aspects, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, wherein the composition produced using the method retains about 80% to about 90% of the growth factors present in the shredded tendon tissue prior to decellularization. In some embodiments, the growth factors are selected from the group consisting of IGF-1, TGF-β, PDGF, VEGF, bFGF, GDF-5, GDF-6, GDF-7, HGF, and combinations thereof. In some embodiments, the growth factors include at least TGF-β.

[0084] In some aspects, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, further comprising maintaining at least 90% of the cytokines present in the shredded tendon tissue, wherein the growth factors are selected from the group consisting of IGF-1, TGF-β, PDGF, VEGF, bFGF, GDF-5, GDF-6, GDF-7, HGF, and combinations thereof.In some embodiments, there is provided a method of producing a decellularized tendon matrix (DTM) composition, comprising: Removing at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85%, at least 84%, at least 83%, at least 82%, at least 81%, at least 80%, at least 91%, at least 9 ... 79%, at least 78%, at least 77%, at least 76%, at least 75%, at least 74%, at least 73%, at least 72%, at least 71%, at least 70%, at least 69%, at least 68%, at least 67%, at least 66%, at least 65%, at least 64%, at least 63%, at least 62%, at least 61%, at least 60%, at least 59%, at least 58%, at least 57%, at least 56%, at least 55%, at least 54%, at least 53%, at least 5 2%, at least 51%, at least 50%, at least 49%, at least 48%, at least 47%, at least 46%, at least 45%, at least 44%, at least 43%, at least 42%, at least 41%, at least 40%, at least 39%, at least 38%, at least 37%, at least 36%, at least 35%, at least 34%, at least 33%, at least 32%, at least 31%, at least 31%, at least 30%, at least 29%, at least 28%, at least 27%, at least 26% %, at least 25%, at least 24%, at least 23%, at least 22%, at least 21%, at least 20%, at least 19%, at least 18%, at least 17%, at least 16%, at least 15%, at least 14%, at least 13%, at least 12%, at least 11%, or at least 10%, and wherein the growth factor is selected from the group consisting of IGF-1, TGF-β, PDGF, VEGF, bFGF, GDF-5, GDF-6, GDF-7, HGF, and combinations thereof.

[0085] In some embodiments, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, the method further comprising maintaining at least 90% of the TGF-β present in the shredded tendon tissue. In some embodiments, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, the method further comprising maintaining at least 95% of the TGF-β present in the shredded tendon tissue. In some embodiments, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, the method further comprising maintaining at least 99% of the TGF-β present in the shredded tendon tissue. In some embodiments, there is provided a method of producing a decellularized tendon matrix (DTM) composition, comprising: decellularizing at least 99% by weight, at least 98% by weight, at least 97% by weight, at least 96% by weight, at least 95% by weight, at least 94% by weight, at least 93% by weight, at least 92% by weight, at least 91% by weight, at least 90% by weight, at least 89% by weight, at least 88% by weight, at least 87% by weight, at least 86% by weight, at least 85% by weight, at least 84% by weight, at least 83% by weight, at least 82% by weight, at least 81% by weight, at least 80% by weight, at least 79% by weight, at least 78% by weight, at least 77% by weight, at least 76% by weight, at least 75% by weight, at least 74% by weight, at least 73 ...97% by weight, at least 96% by weight, at least 95% by weight, at least 94% by weight, at least 93% by weight, at least 92% by weight, at least 91% by weight, at least 90 72% by weight, at least 71% by weight, at least 70% by weight, at least 69% by weight, at least 68% by weight, at least 67% by weight, at least 66% by weight, at least 65% by weight, at least 64% by weight, at least 63% by weight, at least 62% by weight, at least 61% by weight, at least 60% by weight, at least 59% by weight, at least 58% by weight, at least 57% by weight, at least 56% by weight, at least 55% by weight, at least 54% by weight, at least 53% by weight, at least 52% by weight, at least 51% by weight, at least 50% by weight, at least 49% by weight, at least 48% by weight, at least 47% by weight, at least 46% by weight, at least 45% by weight, at least 44% by weight, at least 43% by weight, at least 42% by weight, at least 41% by weight, at least 40% by weight,maintaining at least 39%, at least 38%, at least 37%, at least 36%, at least 35%, at least 34%, at least 33%, at least 32%, at least 31%, at least 31%, at least 30%, at least 29%, at least 28%, at least 27%, at least 26%, at least 25%, at least 24%, at least 23%, at least 22%, at least 21%, at least 20%, at least 19%, at least 18%, at least 17%, at least 16%, at least 15%, at least 14%, at least 13%, at least 12%, at least 11%, or at least 10% by weight.

[0086] In some aspects, methods of producing a decellularized tendon matrix (DTM) composition are provided, further comprising maintaining at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, or at least 40% of the growth factors present in the shredded tendon tissue, wherein the growth factors are selected from the group consisting of IGF-1, TGF-β, PDGF, VEGF, bFGF, GDF-5, GDF-6, GDF-7, HGF, and combinations thereof.

[0087] In some embodiments, methods of producing a decellularized tendon matrix (DTM) composition are provided, further comprising increasing the concentration of a growth factor present in the decellularized tissue or DTM by at least 500%, at least 250%, at least 200%, at least 150%, at least 100%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, or at least 5%, wherein the growth factor is selected from the group consisting of IGF-1, TGF-β, PDGF, VEGF, bFGF, GDF-5, GDF-6, GDF-7, HGF, and combinations thereof.

[0088] In one embodiment, the composition maintains two or more of the growth factors, three or more of the growth factors, four or more of the growth factors, five or more of the growth factors, six or more of the growth factors, or seven or more of the growth factors. In one embodiment, the composition maintains one or more growth factors selected from the group consisting of IGF-1, TGF-β, PDGF, VEGF, bFGF, GDF-5, GDF-6, and GDF-7. In one embodiment, the composition maintains IGF-1 and HGF.

[0089] In one embodiment, the DTM composition further comprises at least 85%, at least 84%, at least 83%, at least 82%, at least 81%, at least 80%, at least 79%, at least 78%, at least 77%, at least 76%, at least 75%, at least 74%, at least 73%, at least 72%, at least 71%, at least 70%, at least 69%, at least 68%, at least 67%, at least 66%, at least 65%, at least 64%, at least 63%, at least 62%, at least 61%, at least 60%, at least 59%, at least 58%, at least 57%, at least 56%, at least 55%, at least 54%, at least 53%, at least 52%, at least 51%, at least 50%, at least 49%, at least 5 ... %, at least 48%, at least 47%, at least 46%, at least 45%, at least 44%, at least 43%, at least 42%, at least 41%, at least 40%, at least 39%, at least 38%, at least 37%, at least 36%, at least 35%, at least 34%, at least 33%, at least 32%, at least 31%, at least 31%, at least 30%, at least 29%, at least 28%, at least 27%, at least 26%, at least 25%, at least 24%, at least 23%, at least 22%, at least 21%, at least 20%, at least 19%, at least 18%, at least 17%, at least 16%, at least 15%, at least 14%, at least 13%, at least 12%, at least 11%, at least 10%.

[0090] In some embodiments, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, the method further comprising removing at least 90% of the cellular material present in the shredded tendon tissue. In some embodiments, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, the method further comprising removing at least 95% of the cellular material present in the shredded tendon tissue. In some embodiments, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, the method further comprising removing at least 99% of the cellular material present in the shredded tendon tissue. In some embodiments, there is provided a method of making a decellularized tendon matrix (DTM) composition, comprising removing at least 99% by weight, at least 98% by weight, at least 97% by weight, at least 96% by weight, at least 95% by weight, at least 94% by weight, at least 93% by weight, at least 92% by weight, at least 91% by weight, at least 90% by weight, at least 89% by weight, at least 88% by weight, at least 87% by weight, at least 86% by weight, at least 85% by weight, at least 84% by weight, at least 83% by weight, at least 82% by weight, at least 81% by weight, at least 80% by weight, at least 79% by weight, at least 78% by weight, at least 77% by weight, at least 76% by weight, at least 75% by weight, at least 74% by weight, at least 73% by weight, at least 89% by weight, at least 88% by weight, at least 97% by weight, at least 96% by weight, at least 95% by weight, at least 94% by weight, at least 93% by weight, at least 92% by weight, at least 91% by weight, at least 90% by weight, at least 89% by weight, at least 88% by weight, at least 87% by weight, at least 86% by weight, at least 85% by weight, at least 84% by weight, at least 83% by weight, at least 82% by weight, at least 81% by weight, at least 80% by weight, at least 79% by weight, at least 78% by weight, at least 77% by weight, at least 76% by weight, at least 75% by weight, at least 74% by weight, at least 73% by weight 72% by weight, at least 71% by weight, at least 70% by weight, at least 69% by weight, at least 68% by weight, at least 67% by weight, at least 66% by weight, at least 65% by weight, at least 64% by weight, at least 63% by weight, at least 62% by weight, at least 61% by weight, at least 60% by weight, at least 59% by weight, at least 58% by weight, at least 57% by weight, at least 56% by weight, at least 55% by weight, at least 54% by weight, at least 53% by weight, at least 52% by weight, at least 51% by weight, at least 50% by weight, at least 49% by weight, at least 48% by weight, at least 47% by weight, at least 46% by weight, at least 45% by weight, at least 44% by weight, at least 43% by weight, at least 42% by weight, at least 41% by weight, at least 40% by weight,Methods further include removing at least 39%, at least 38%, at least 37%, at least 36%, at least 35%, at least 34%, at least 33%, at least 32%, at least 31%, at least 31%, at least 30%, at least 29%, at least 28%, at least 27%, at least 26%, at least 25%, at least 24%, at least 23%, at least 22%, at least 21%, at least 20%, at least 19%, at least 18%, at least 17%, at least 16%, at least 15%, at least 14%, at least 13%, at least 12%, at least 11%, or at least 10% by weight. In certain embodiments, the invention provides methods of producing a decellularized tendon matrix (DTM) composition, wherein the DTM is substantially free of cellular material. In certain embodiments, the present invention provides a method for producing a decellularized tendon matrix (DTM) composition, wherein the DTM is substantially free of TGF-β-producing cells.

[0091] In some embodiments, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, the method further comprising removing at least 90% of the nucleic acid (e.g., DNA or RNA) present in the shredded tendon tissue. In some embodiments, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, the method further comprising removing at least 95% of the nucleic acid (e.g., DNA or RNA) present in the shredded tendon tissue. In some embodiments, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, the method further comprising removing at least 99% of the nucleic acid (e.g., DNA or RNA) present in the shredded tendon tissue. In some embodiments, there is provided a method of making a decellularized tendon matrix (DTM) composition, comprising: decellularizing at least 99% by weight, at least 98% by weight, at least 97% by weight, at least 96% by weight, at least 95% by weight, at least 94% by weight, at least 93% by weight, at least 92% by weight, at least 91% by weight, at least 90% by weight, at least 89% by weight, at least 88% by weight, at least 87% by weight, at least 86% by weight, at least 85% by weight, at least 84% by weight, at least 83% by weight, at least 82% by weight, at least 81% by weight, at least 80% by weight, at least 79% by weight, at least 78% by weight, at least 77% by weight, at least 76% by weight, at least 75% by weight, at least 74% by weight, at least 73% by weight, at least 72% by weight, at least 71% by weight, at least 70% by weight, at least 69% by weight, at least 68% by weight, at least 67% by weight, at least 66% by weight, at least 65% by weight, at least 64% by weight, at least 63% by weight, at least 62% by weight, at least 61% by weight, at least 60% by weight, at least 59% by weight, at least 58% by weight, at least 57% by weight, at least 56% by weight, at least 55% by weight, at least 54% by weight, at least 53% by weight, at least 52% by weight, at least 51% by weight, at least 50% by weight, at least 49% by weight, at least 48% by weight, at least 47% by weight, at least 46% by weight, at least 45% by weight, at least 44% by weight,removing at least 43%, at least 42%, at least 41%, at least 40%, at least 39%, at least 38%, at least 37%, at least 36%, at least 35%, at least 34%, at least 33%, at least 32%, at least 31%, at least 31%, at least 30%, at least 29%, at least 28%, at least 27%, at least 26%, at least 25%, at least 24%, at least 23%, at least 22%, at least 21%, at least 20%, at least 19%, at least 18%, at least 17%, at least 16%, at least 15%, at least 14%, at least 13%, at least 12%, at least 11%, or at least 10% by weight. In certain embodiments, the present invention provides methods for producing a decellularized tendon matrix (DTM) composition, wherein the DTM is substantially free of nucleic acids (e.g., DNA or RNA).

[0092] Various methods are known in the art, including those summarized in Gilpin and Yang, Biomed. Res. Int. 2017:9831534 (2017). Many methods involve aggressive detergent extraction and prolonged treatment with promiscuous proteases, such as pepsin, at extreme, non-physiological pH. The methods and processes of the present invention differ from those known in the art by using lower amounts of promiscuous proteases that are active at physiological pH. Without being bound by theory, the methods and processes of the present invention result in less protein denaturation and preserve more functional growth factors in the decellularized tendon matrix. In some embodiments, MMP2, MMP9, MMP14, or a combination thereof, are used to prepare the decellularized tendon matrix composition of the present invention. The target cleavage sites of the MMP family, including MMP2, MMP9, and MMP14, have been mapped using a whole-proteome approach by Eckhard et al., Data Brief, 7:299-310 (2017).

[0093] DTM Hydrogel In another aspect, the present disclosure provides decellularized tendon matrix hydrogels. Hydrogels can be generated using the inherent polymerization ability of pepsin-treated monomeric collagen by adjusting temperature or pH. These approaches are well-known, albeit somewhat unexpected; for example, Drake et al., Biochemistry 5:301-312 (1966) details the generation of polymerizable proteolytic fragments of collagen. Other well-known methods are also known, such as those taught by Bahney et al., FASEB J, 25:1486-1496 (2011) and Ungerleider et al., Methods, 84:53-59 (2015). These well-known methods are particularly unexpected when applied to protein-rich extracellular matrix tissues.

[0094] The use of carbodiimide crosslinker chemistry results in more reliable and controlled crosslinking. In some embodiments, hydrogels are produced by mixing the DTM composition and reacting it with a carboxyl-reactive crosslinker, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, "EDC." EDC crosslinking is most efficient under acidic conditions (e.g., about pH 4.5) and is best performed in buffer without exogenous carboxyls and amines. MES buffer (4-morpholinoethanesulfonic acid) is a suitable carbodiimide reaction buffer. Phosphate buffer and neutral pH (up to 7.2) conditions are compatible with the reaction chemistry but are less efficient; increasing the amount of EDC in the reaction solution can easily compensate for the reduced efficiency. EDC is mixed 1:1 with N-hydroxysuccinimide (NHS) or its water-soluble analog (sulfo-NHS) to further improve crosslinking. EDC attaches NHS to carboxyl groups, forming an NHS ester that is much more stable than the O-acylisourea intermediate, while allowing efficient conjugation to primary amines at physiological pH.

[0095] In another embodiment, the DTM hydrogel is formed by reconstituting the DTM in a sterile, pharmaceutically acceptable solution for injection.

[0096] Injectable pharmaceutical composition In one aspect, the present invention provides a pharmaceutical composition for use in repairing or treating a tendon rupture. In a preferred embodiment, the present invention provides a pharmaceutical composition comprising a DTM hydrogel that is applied directly to the site of tendon injury. In one aspect, the site of tendon injury is a first-degree tear. In another aspect, the site of tendon injury is a second-degree tear. In one aspect, the site of tendon injury is a third-degree tear.

[0097] Pharmaceutical compositions are typically formulated to provide a therapeutically effective amount of the DTM hydrogel, and may further include one or more pharmaceutically acceptable excipients, carriers such as inert solid diluents and fillers, diluents such as sterile aqueous solutions and various organic solvents, penetration enhancers, solubilizers, and adjuvants.

[0098] When preparing the compositions of the present disclosure, compositions containing decellularized tendon matrix can also contain excipients. Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, PEG, polyvinylpyrrolidone, cellulose, water, sterile saline, syrup, and methylcellulose. The formulation can further include lubricants such as talc, magnesium stearate, and mineral oil, wetting agents, emulsifying and suspending agents, preservatives such as methyl- and propylhydroxybenzoates, sweeteners, and flavoring agents. The compositions of the present disclosure can be formulated to provide rapid or sustained delayed release of the active ingredient (e.g., growth factor) after implantation in a patient using procedures known in the art.

[0099] In some cases, the pharmaceutical compositions described herein can include excipients that can provide long-term storage, bulk formulations containing potent active ingredients, promote drug absorption, reduce viscosity, add taste, or increase the solubility of the pharmaceutical composition. Non-limiting examples of excipients include anti-adherents, binders (e.g., sucrose, lactose, starch, cellulose, gelatin, or polyethylene glycol), coatings (e.g., hydroxypropyl methylcellulose or gelatin), disintegrants, glidants, lubricants, or preservatives (e.g., acids, esters, phenols, mercury compounds, or ammonium compounds). The pharmaceutical compositions of the present disclosure can contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more than about 50% excipients by weight or volume. For example, the pharmaceutical composition can contain 5% by volume of excipients. In another example, the pharmaceutical composition may contain 8% by weight of excipients. It is contemplated that one or more vehicles may be selected based on the active ingredients in the pharmaceutical composition.

[0100] In certain embodiments, pharmaceutical compositions of the present disclosure can include one or more solubilizing agents. As used herein, "solubilizing agent" refers to compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium docusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide. Pharmaceutical compositions of the present disclosure can include about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more than about 50% solubilizing agent by weight or volume. For example, the pharmaceutical composition may include 10% solubilizer by volume. In another example, the pharmaceutical composition may include 5% solubilizer by weight.

[0101] In some embodiments, the composition includes a stabilizer. In some embodiments, the stabilizer is selected from, for example, fatty acids, fatty acid alcohols, alcohols, long-chain fatty acid esters, long-chain ethers, hydrophilic derivatives of fatty acids, polyvinylpyrrolidone, polyvinyl ethers, polyvinyl alcohols, hydrocarbons, hydrophobic polymers, hygroscopic polymers, and combinations thereof. In some embodiments, amide analogs of stabilizers are also used. Other useful compositions optionally include one or more antioxidants to enhance chemical stability. Suitable antioxidants include, by way of example only, ascorbic acid and sodium metabisulfate. In one embodiment, the antioxidant is selected from metal chelators, thiol-containing compounds, and other common stabilizers.

[0102] Still other useful compositions include one or more surfactants to enhance physical stability or for other purposes. Suitable nonionic surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, polyoxyethylene, hydrogenated castor oil, polyoxyethylene alkyl ethers, alkylphenyl ethers, Octoxynol 10, and Octoxynol 40.

[0103] In some embodiments, the compositions disclosed herein include a preservative. Preservatives suitable for use in the compositions described herein include, but are not limited to, benzoic acid, boric acid, p-hydroxybenzoates, phenols, chlorinated phenolic compounds, alcohols, quaternary compounds, quaternary ammonium compounds (e.g., benzalkonium chloride, cetyltrimethylammonium bromide, or cetylpyridinium chloride), stabilized chlorine dioxide, mercury (e.g., merfen or thiomersal), or mixtures thereof. In some embodiments, the preservative is methylparaben. In some embodiments, the methylparaben is present in an amount of about 0.05% to about 1.0%, or about 0.1% to about 0.2%, by weight or volume.

[0104] In some embodiments, the compositions of the present disclosure can include a base, such as sodium stearyl fumarate, diethanolamine cetyl sulfate, isostearate, polyethoxylated castor oil, benzalkonium chloride, nonoxyl 10, octoxynol 9, sodium lauryl sulfate, sorbitan esters (sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, sorbitan sesquioleate, sorbitan trioleate, sorbitan tristearate, sorbitan laurate, sorbitan oleate, sorbitan palmitate, sorbitan stearate, sorbitan diolate, sorbitan sesquiisostearate, sorbitan sesquistearate, sorbitan triisostearate), lecithin, pharmaceutically acceptable salts thereof, combinations thereof, or derivatives thereof.

[0105] In embodiments, the concentration of decellularized tendon matrix (DTM) in the DTM hydrogel pharmaceutical composition is about 0.2 mg / mL to 20 mg / mL; 0.2 mg / mL to 19 mg / mL; 0.2 mg / mL to 18 mg / mL; 0.2 mg / mL to 17 mg / mL; 0.2 mg / mL to 16 mg / mL; 0.2 mg / mL to 15 mg / mL; 0.2 mg / mL to 14 mg / mL; 0.2 mg / mL to 13 mg / mL; 0.2 mg / mL to 12 mg / mL; 0.2 mg / mL to 11 mg / mL; 0.2 mg / mL to 10 mg / mL; 0.2 mg / mL to 9 mg / mL; 0.2mg / mL~8mg / mL;0.2mg / mL~7mg / mL;0.2mg / mL~6mg / mL;0.3mg / mL~6mg / mL;0.4mg / mL~6mg / mL;0.5mg / mL~6mg / mL;0.6mg / mL~6mg / mL;0.7mg / mL~6mg / mL; 0.8 mg / mL to 6 mg / mL; 0.9 mg / mL to 6 mg / mL; 1 mg / mL to 6 mg / mL; 2 mg / mL to 6 mg / mL; 3 mg / mL to 6 mg / mL; about 3 mg / mL; about 4 mg / mL; about 5 mg / mL; and about 6 mg / mL.

[0106] In the embodiment, the concentration of the decellularized tendon matrix (DTM) in the DTM hydrogel pharmaceutical composition is selected from the group consisting of about 1.0 mg / mL to 6 mg / mL; 1.1 mg / mL to 6 mg / mL; 1.2 mg / mL to 6 mg / mL; 1.3 mg / mL to 6 mg / mL; 1.4 mg / mL to 6 mg / mL; 1.5 mg / mL to 6 mg / mL; 1.6 mg / mL to 6 mg / mL; 1.7 mg / mL to 6 mg / mL; 1.8 mg / mL to 6 mg / mL; 1.9 mg / mL to 6 mg / mL; 2.0 mg / mL to 6 mg / mL; 2.1 mg / mL to 6 mg / mL; 2.2 mg / mL to 6 mg / mL; 2.3 mg / mL to 6 mg / mL; 2.4 mg / mL to 6 mg / mL; 2.5 mg / mL to 6 mg / mL; 2.6 mg / mL to 6 mg / mL; 2.7 mg / mL to 6 mg / mL; 2.8 mg / mL to 6 mg / mL; 2.9 mg / mL to 6 mg / mL; 3.0 mg / mL to 6 mg / mL; 3.1 mg / mL to 6 mg / mL; 3.2 mg / mL to 6 mg / mL; 3.3 mg / mL to 6 mg / mL; 3.4 mg / mL to 6 mg / mL; 3.5 mg / mL to 6 mg / mL; 3.6 mg / mL to 6 mg / mL; 3.7 mg / mL to 6 mg / mL; 3.8 mg / mL to 6 mg / mL; 3.9 mg / mL to 6 mg / mL; 4.0 mg / mL to 6 mg / mL; 4.1 mg / mL to 6 mg / mL; 4.2 mg / mL to 6 mg / mL; 4.3 mg / mL to 6 mg / mL; 4.4 mg / mL to 6 mg / mL; 4.5 mg / mL to 6 mg / mL; 4.6 mg / mL to 6 mg / mL; 4.7 mg / mL to 6 mg / mL; 4.8 mg / mL to 6 mg / mL; 4.9 mg / mL to 6 mg / mL; 5.0 mg / mL to 6 mg / mL; 5.1 mg / mL to 6 mg / mL; 5.2 mg / mL to 6 mg / mL; 5.3 mg / mL to 6 mg / mL; 5.4 mg / mL to 6 mg / mL; 5.5 mg / mL to 6 mg / mL; 5.6 mg / mL to 6 mg / mL; 5.7 mg / mL to 6 mg / mL; 5.8 mg / mL to 6 mg / mL; and 6 mg / mL.

[0107] In embodiments, the DTM hydrogel percentage (%) in the pharmaceutical composition may be, for example, independently, about 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.1% or 0.2% of the pharmaceutical composition. 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w / w, w / v, or v / v.

[0108] The composition may further comprise one or more pharmaceutically acceptable additives and excipients, including, but not limited to, anti-adherents, anti-foaming agents, buffers, polymers, antioxidants, preservatives, chelating agents, viscosity modifiers, tonics, suspending agents, binders, fillers, plasticizers, lubricants, and mixtures thereof.

[0109] Ethanol, glycerol, propylene glycol, and liquid polyethylene glycol (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils can also be used. Suitable fluidity can be maintained, for example, by the use of a coating such as lecithin to maintain the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.

[0110] The composition may further comprise a peptide. The composition may further comprise a protein. The composition may further comprise an amino acid. The composition may further comprise water.

[0111] The composition can further include at least one growth factor. In some cases, the at least one growth factor can include insulin-like growth factor-1, insulin-like growth factor binding protein-3, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), placental growth factor (PLGF), or any combination thereof. The at least one growth factor can enhance viability, product stability, cell differentiation, maintenance of sternness, reduce anti-inflammatory properties, or any combination thereof. The at least one growth factor can be added to the composition. The at least one growth factor can be added to a subcomponent of the composition. The at least one growth factor can be added to the viscosity-modifying component, the plurality of isolated stem cells, the isolated induction component, the isolated scaffold component, or any combination thereof. For example, at least one growth factor can be added to a composition of the present disclosure comprising a decellularized tendon matrix to improve integration of the composition with host tissue upon implantation into a host. The at least one growth factor can be added prior to formation of the composition. After the composition is formed, at least one growth factor can be added.

[0112] The composition may further comprise chemokine ligand 2, macrophage inflammatory protein-1 (MIP-1) alpha, MIP-1 beta, MIP-2, beta chemokine ligand-5, beta chemokine ligand-20, alpha-chemokine ligand-14, lipopolysaccharide-induced alpha-chemokine, granulocyte-macrophage colony-stimulating factor, interleukin IL-1 beta, phorbol myristate acetate, epidermal growth factor, fibroblast growth factor, vascular endothelial growth factor, connective tissue growth factor, platelet-derived growth factor, erythrocyte-associated protein-1 (YAG), ... The composition may further comprise at least one of the following: growth factor, insulin-like growth factor, nerve growth factor, hepatocyte growth factor, colony-stimulating factor, stem cell factor, keratinocyte growth factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, glial-derived neurotrophic factor, ciliary neurotrophic factor, endothelial-monocyte-activating polypeptide, epithelial neutrophil-activating peptide, erythropoietin, bone morphogenetic protein, brain-derived neurotrophic factor, transforming growth factor beta, tumor necrosis factor, or any combination thereof. The composition may further comprise at least one hormone. In some cases, the at least one hormone may be prolactin or leptin.

[0113] In some cases, there may be six major families of growth factors associated with healing (EGF, FGF, IGF, PDGF, TGF, and VEGF). Examples of such growth factors include, but are not limited to, platelet-derived growth factors (PDGF-A, PDGF-B, PDGF-C, and PDGF-D), insulin-like growth factors I and II (IGF-I and IGF-II), acidic and basic fibroblast growth factors (aFGF and bFGF), alpha and beta transforming growth factors (TGF-a and TGF-β (e.g., TGF-beta1, TGF-beta2, TGF-beta3)), epidermal growth factor (EGF), and others. These growth factors can stimulate mitosis of one or more cells involved in healing and can be combined.

[0114] Other positive angiogenic agents that may be co-administered with the compositions disclosed herein include, but are not limited to, HGF, TNF-α, angiogenin, IL-8, and the like. Further examples of additional agents include platelet-derived growth factor (PDGF) (e.g., becaplermin (rhPDGF-BB) such as REGRANEX®), adenosine-A2A receptor agonists; keratinocyte growth factor (KGF-2, repifermin; lactoferrin (LF); thymosin beta-4 (Τβ4); thrombin-derived activating receptor peptide (TP508; CHRYSALIN®); adenoviral vectors encoding platelet-derived growth factor (PDGF-B); autologous bone marrow stem cells (BMSC); engineered tissue transplants (e.g., Apligraf, etc.). Antibiotic and antiseptic ulcer agents can also be combined. Immunosuppressive treatments (e.g., corticosteroids, radiation therapy, chemotherapy) can be combined with the compositions disclosed herein.

[0115] One of ordinary skill in the art will appreciate that the additional agents can be co-administered with the compositions disclosed herein or administered separately.

[0116] The compositions of the present invention can contain various other ingredients as enumerated above in the required amounts in an appropriate solvent, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.

[0117] Other pharmaceutical compositions Pharmaceutical compositions can also be prepared from the compositions described herein and one or more pharmaceutically acceptable excipients suitable for sublingual, buccal, rectal, intraosseous, intraocular, intranasal, epidural, or intraspinal administration. The preparation of such pharmaceutical compositions is well known in the art. See, for example, Anderson, et al., eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill, 2002; and Pratt and Taylor, eds., Principles of Drug Action, Third Edition, Churchill Livingston, NY, 1990, each of which is incorporated by reference in its entirety.

[0118] The compositions of the present invention can also be delivered via impregnated or coated devices such as sutures, e.g., suture anchors. Such administration methods can be useful, for example, for preventing or ameliorating tendon damage or injury. The compositions of the present invention can be administered, for example, by local delivery from sutures or suture anchors. In some embodiments, the compounds of the present invention are mixed with a matrix. Such a matrix can be a polymer matrix and can be useful for binding the compound to the stent. Suitable polymeric matrices for such use include, for example, lactone-based polyesters or copolyesters, such as polylactides, polycaprolactone glycolides, polyorthoesters, polyanhydrides, polyamino acids, polysaccharides, polyphosphazenes, poly(ether-ester) copolymers (e.g., PEO-PLLA); polydimethylsiloxane, poly(ethylene-vinyl acetate), acrylate-based polymers or copolymers (e.g., polyhydroxyethylmethylmethacrylate, polyvinylpyrrolidinone), fluorinated polymers such as polytetrafluoroethylene and cellulose esters; and polyetheretherketone (PEEK). Metallic or biocomposite materials, such as poly(lactic acid) (PLA) and beta-tricalcium phosphate (β-TCP), are also suitable. PLA / hydroxyapatite can also be used. See, e.g., Dorozhkin, Biomatter, 1:3-56 (2011). Suitable matrices are non-degradable or can degrade over time to release the compound. The compositions of the present invention can be applied directly to the tendon injury site and / or directly to the tendon injury site. In some embodiments, the compositions of the present invention are applied to a site adjacent to the tendon injury site and / or adjacent to the tendon injury site. In another embodiment, the compositions of the present invention are applied to the tendon in need of regeneration.

[0119] DTM hydrogels can be applied to the surface of sutures, suture anchors, or medical devices by various methods, such as dip / spin coating, spray coating, dip coating, and / or brush coating. The compound can be applied in a solvent, and evaporation of the solvent can form a hydrogel layer on the suture, suture anchor, or medical device. Alternatively, the compound can be located in the body of the suture, suture anchor, or medical device, e.g., in microchannels or micropores. Upon implantation, the compound diffuses from the body of the suture, suture anchor, or medical device to contact the tendon. Such sutures, suture anchors, or medical devices can be prepared by immersing a suture, suture anchor, or medical device fabricated to contain such micropores or microchannels in a solution of the composition of the present invention in an appropriate solvent, followed by evaporation of the solvent. Excess hydrogel on the surface of the suture, suture anchor, or medical device can be removed by a further brief solvent wash. In yet another embodiment, the compounds of the present invention can be covalently attached to a suture, suture anchor, or medical device. Covalent linkers that degrade in vivo to release the compounds of the present invention can be used. Biolabile bonds, such as ester, amide, or anhydride bonds, can be used for this purpose.

[0120] In some embodiments, the DTM hydrogels of the present invention are applied directly to the tendon. In some embodiments, the DTM hydrogels of the present invention are applied directly to the tendon using a surgical or medical needle ranging from 10 gauge to 25 gauge. The needle can be 10 gauge, 11 gauge, 12 gauge, 13 gauge, 14 gauge, 15 gauge, 16 gauge, 18 gauge, 20 gauge, 22 gauge, 23 gauge, 24 gauge, or 25 gauge. In some embodiments, the needle is 16 gauge to 20 gauge. The viscosity of the DTM hydrogel can be adjusted to optimize the composition for delivery through a particular gauge needle, for example, a 16 gauge or 20 gauge needle.

[0121] The rheological properties of the DTM hydrogels of the present invention can be tailored to the gauge of a particular medical or surgical needle for optimal injection. For example, the dynamic viscosity of the DTM hydrogels of the present invention is about 0.05 Pa. * s ~ approx. 1.0 Pa * It is s.

[0122] The present invention also provides kits. The kits contain a lyophilized DTM composition and a carbodiimide crosslinking reagent, alone or in combination, in suitable packaging, and include documentation that may include instructions for use, a description of clinical trials, and a list of side effects. In some embodiments, the kits further include an applicator for applying the composition to a tendon in need thereof. In some embodiments, the kits further include a removable attachment that allows for mixing. In one aspect, the kit includes a syringe containing the lyophilized DTM, a second syringe containing an aqueous resuspension buffer, and a mixing connector that connects the syringes and allows mixing between the two syringes. Such kits can also include information such as scientific literature references, package inserts, clinical trial results, and / or summaries thereof, which demonstrate or establish the activity and / or benefits of the composition and / or describe dosage, administration, side effects, drug interactions, or other information useful to healthcare providers. Such information may be based on the results of various studies, such as studies using laboratory animals, including in vivo models, and studies based on human clinical trials.

[0123] Tendon Treatment Methods In one embodiment, the compositions of the present invention are used to stimulate tendon regeneration, and the method includes (i) resuspending a DTM composition of the present invention in a pharmaceutically acceptable carrier and (ii) applying the resuspended DTM composition to a tendon site requiring stimulated tendon regeneration.

[0124] In another embodiment, the DTM hydrogel is prepared immediately prior to treating a subject in need thereof, the method comprising: (i) resuspending a DTM composition of the present invention in a pharmaceutically acceptable carrier; (ii) preparing a DTM hydrogel; and (iii) applying the DTM hydrogel to a tendon site requiring stimulated tendon regeneration. In some embodiments, the tendon site requiring stimulated tendon regeneration is a first-degree tear. In some embodiments, the tendon site requiring stimulated tendon regeneration is a second-degree tear. In another embodiment, the tendon site requiring stimulated tendon regeneration is a third-degree tear. In one embodiment, the site is a complete tear.

[0125] In some aspects, the tendon site in need of stimulated tendon regeneration is a site with an acute injury, hi some aspects, the tendon site in need of stimulated tendon regeneration is selected from the group consisting of lateral epicondylitis, Achilles tendonitis, peroneal tendonitis, patellar tendon, quadriceps tendonitis, and combinations thereof.

[0126] In some embodiments, the DTM hydrogel is prepared using carbodiimide chemistry. In some embodiments, the DTM hydrogel is prepared by reconstituting the DTM in a pharmaceutically acceptable sterile solution for injection.

[0127] In one embodiment, the DTM compositions of the present invention are applied to the tendon site in need of repair via a single needle injection. In one embodiment, application of the DTM compositions of the present invention is image-guided. In some embodiments, the DTM compositions of the present invention are applied to the tendon site in need of repair using arthroscopy. In another embodiment, the DTM compositions of the present invention are applied directly to the tendon site in need of repair during a surgical procedure to open the tendon.

[0128] In some embodiments, the compositions of the present invention are administered to one or more joints via image-guided injection. X-ray, computed tomography (CT), or ultrasound are useful imaging modalities for guiding joint injection.

[0129] Although the present invention has been described in considerable detail with reference to various examples thereof, other examples are possible, and therefore, the spirit and scope of the appended claims should not be limited to the description of the preferred examples contained herein.

[0130] The reader's attention is directed to any articles and documents filed concurrently with this specification and publicly accessible herewith, the contents of which or any such articles and documents are incorporated herein by reference. Any feature disclosed in this specification (including the accompanying claims, abstract, and drawings) may, unless otherwise specified, be replaced by alternative features serving the same, equivalent, or similar purpose. Thus, unless otherwise specified, each feature disclosed is only one example of a generic series of equivalent or similar features. [Example]

[0131] The embodiments contained herein are described with reference to the following examples, which are provided for illustrative purposes only, and the disclosure contained herein is not to be construed as being limited to these examples in any way, but rather as encompassing any variations that become evident as a result of the teachings provided herein.

[0132] Example 1 Preparation of decellularized tendon matrix Human cadaver Achilles tendons were washed with phosphate-buffered saline (PBS) (pH 7.4), and the sheath, fat, and synovial tissue were then removed from the tendon tissue samples. The tendon tissue samples were then cut into approximately 1-4 mm pieces. 3 The tissue is then chopped into pieces of approximately 1 / 4 size and washed with phosphate buffered saline (PBS).

[0133] The shredded tendon pieces were immersed in a decellularization solution containing 1% w / v sodium dodecyl sulfate (SDS) with moderate agitation. The shredded material was carefully washed with multiple changes of ultrapure water to remove residual SDS and cellular components.

[0134] The material is then flash frozen and pulverized to produce a heterogeneous material of varying particle sizes. The resulting material is then resuspended in MMP digestion buffer. This suspension is then incubated.

[0135] A stop solution is then added to stop the MMP digestion. The buffer is exchanged and a neutralizing solution is added. The material is then washed with multiple changes of wash buffer before being lyophilized.

[0136] Decellularization is assayed by comparing SYTO Green 11 (nuclear) staining of the native tendon starting material with the final DTM product. Decellularization is further confirmed using hematoxylin and eosin, 4',6-diamidino-2-phenylindole (DAPI) staining, agarose gel electrophoresis, and quantification of residual DNA. The DTM product is essentially free of nuclear staining. Residual DNA is present at approximately 2 ng / mL or less.

[0137] MALDI-TOF mass spectrometry is used to demonstrate the presence of TGF-β in the DTM product.

[0138] Example 2 Characterization of DTM Hydrogel DTM hydrogels are prepared by resuspending the DTM of the present invention in a pharmaceutically acceptable sterile solution for injection. The resulting DTM hydrogels are then characterized using the following methods according to Zuidema et al., J. Biomed. Mater. Res. B Appl. Biomater., 102:1063-73 (2014): (1) a time sweep to determine the gelation time of the hydrogel; (2) a strain sweep to determine the linear viscoelastic region of the hydrogel versus strain; (3) a frequency sweep to determine the linear equilibrium modulus plateau of the hydrogel; and (4) a time sweep with values ​​obtained from the strain and frequency sweeps to accurately report the equilibrium modulus and gelation time.

[0139] Example 3 DTM Treatment to Maintain Native Growth Factor Profile We developed a decellularization and enzyme treatment technique to produce a decellularized tendon matrix putty that maintains TGF-β bioactivity to promote tissue regeneration.

[0140] Tendons have a poor regenerative capacity and typically heal through scarring rather than a native-like tissue structure, resulting in reduced mechanical strength. As a result, tendon repairs, such as rotator cuff repairs, have failure rates ranging from 20% to 90%, depending on the patient's age, tear size, and other biological factors. There is an unmet clinical need to stimulate tendon healing, resulting in stronger regeneration, to improve patient outcomes.

[0141] Decellularized extracellular matrix (ECM) is frequently used as a regenerative material for tissue engineering because it maintains tissue-specific proteins and growth factors and can also provide structural support. Several growth factors, particularly transforming growth factor beta (TGF-β), which promote tendon remodeling, have been studied for their role in regenerative healing. TGF-β signaling has been shown to be important for tendon formation during development. After injury, TGF-β is temporally regulated to promote healing by stimulating collagen production and angiogenesis. Furthermore, exogenous TGF-β1 injection has been reported to increase the mRNA levels of type I and type III collagen, and improved biomechanical function of repaired tendons was also observed in this group.

[0142] the purpose (i) to develop decellularization techniques, (ii) to develop methods for enzymatically digesting decellularized tendons, and (iii) to characterize the protein profile of decellularized tendon matrix (DTM).

[0143] Characterization of Native Tendons—The goal was to determine the optimal tendon for developing an allograft product. Patellar and Achilles tendons were characterized for DNA content and native protein concentration. Differences in protein profile by location within each source (i.e., proximal vs. distal) were also determined. As shown in Figures 1A and 1B and 2A and 2B, no significant differences were observed between patellar and Achilles tendons. DNA content was measured using a DNEasy kit (Qiagen). Total protein content was measured using a BCA kit (Thermo Scientific). Because TGF-β is a crucial growth factor in tendon healing, it is important to determine the pre-treatment (native) TGF-β concentration and its location (proximal, central, distal) in each tendon (patellar and Achilles tendons) (e.g., see Figures 2A and 2B).

[0144] Detergent-free decellularization The goal of this study was to develop a gentler and faster decellularization method compared to conventional detergent-based methods. DNase was compared with detergents such as SDS and EDTA, which often require long processing times (1-2 weeks). DNase was tested at various times and concentrations. As shown in Figure 3, 1 hour of decellularization using 50 U of DNase resulted in significantly different native DNA content and was comparable to conventional methods.

[0145] Collagenase digestion maximizes protein content Enzymatic digestion allows decellularized tendons to be processed into surgically friendly forms such as injectable systems or putties. Enzymatic digestion was adjusted to maximize growth factor function. Collagenase I, III, and a combination of the two were compared with pepsin digestion, as shown in Figure 4. All tendon samples were measured in μg of total protein per mg of tissue (μg protein / mg tissue). To prepare the enzyme solutions, collagenase I (Life Technologies) was used at 2 mg / 1 mL in PBS, collagenase III (MP Biomedicals) at 1 mg / 1 mL in PBS, and pepsin (Sigma) at 1 mg / 1 mL in 0.1 M HCl. All samples were incubated for 24 hours.

[0146] Decellularized tendon matrix (DTM) maintains TGF-β protein To confirm that our decellularized tendon matrix (DTM) maintained its bioactivity, we compared TGF-β levels with those of native tissue. TGFβI, II, and III all play important roles in tendon healing and repair. Following enzymatic digestion and a final freeze-drying step, 30 μg of total protein per sample was measured based on BCA results. A TGF-β Milliplex kit (Millipore Sigma) was used to measure all DTM samples. As shown in Figure 5, the final DTM prototype maintained TGFβI, II, and III. As shown in Figures 6A and 6B, DTM processing promotes elastic properties, including the ability to stretch without tearing (Figure 6A).

[0147] There is an unmet clinical need to stimulate tendon healing, resulting in stronger regeneration, to improve patient outcomes. The current standard of care in tendon repair leads to high failure rates, in part due to excessive scarring that leads to reduced joint biomechanical function. In this study, we developed a technique to produce a decellularized tendon matrix putty that maintains the bioactivity of TGF-β to promote tissue regeneration. In vitro assays focused on cellular responses to DTM and further characterize the efficacy of DTM in promoting tendon repair. Further testing is underway, including an in vivo rotator cuff repair model.

[0148] Example 4 Decellularization of Tendons and Enzymatic Digestion and Reconstitution of Decellularized Tendon Matrix (DTM) The goal was to develop a gentler and faster decellularization method compared to conventional detergent-based methods. DNase was compared with detergents such as SDS and EDTA, which often require long processing times (1–2 weeks). DNase was tested at various times and concentrations. One hour of decellularization using 50 U of DNase was shown to significantly reduce native DNA content and be comparable to conventional methods. DTM was prepared according to the following procedure.

[0149] Tendon decellularization methodFirst, weigh the tendon and record its weight. Then, shred the tendon into small, uniformly sized pieces. Next, for decellularization, place the shredded pieces in DNase solution (see the table below for an example: 0.5 g tendon / mL DNase solution; DNase solution: 50 U DNase I per mL 1x PBS; for 2 grams of shredded tendon, place in 4 mL of 1x PBS and add 200 U DNase). Next, incubate at 56°C for 1 hour with moderate shaking. Next, to wash the DTM, add 1x PBS at twice the initial volume (for example, if 1 mL of DNase solution was added, add 2 mL of 1x PBS). Next, place the DTM on a 70 μm cell strainer and centrifuge at 2000 G for 5-10 minutes. Finally, freeze at -80°C for at least 30 minutes and place the tube in a freeze dryer. [Table 1]

[0150] Enzymatic Digestion (Injectable DTM) - First, weigh and record the weight of the decellularized tendon. Next, to prepare the injectable solution, weigh 0.02-0.10 g of tendon and add 1 mL of collagenase solution (2 mg / mL collagenase type I and 1 mg / mL collagenase type III in 1x PBS). Incubate at 37°C for 24 hours. Next, to wash the DTM, add 1x PBS at twice the initial volume (if 1 mL of collagenase solution was added, add 2 mL of 1x PBS). Next, place the DTM on a 70 μm cell strainer and centrifuge at 2,000 G for 5-10 minutes. Next, place the DTM into a new microcentrifuge tube containing 1 mL of PBS and vortex for 30 seconds. Next, place this solution into a 100 KDa filter and spin at 12,000 G for 5 minutes. Finally, freeze at -80°C for at least 30 min and place the tubes in a freeze dryer.

[0151] Enzymatic Digestion (Putty DTM) - First, weigh and record the weight of the decellularized tendon. Next, to prepare the putty, weigh 10-20 g of tendon and add 1 mL of collagenase solution (2 mg / mL collagenase type I and 1 mg / mL collagenase type III in 1x PBS). Incubate at 37°C for 12 hours. Next, to wash the DTM, add 1x PBS at twice the initial volume (if 1 mL of collagenase solution was added, add 2 mL of 1x PBS). Next, place the DTM into a new microcentrifuge tube containing 1 mL of PBS and vortex for 30 seconds. Next, place this solution into a 100 kDa filter and spin at 12,000 g for 5 minutes. Finally, freeze at -80°C for at least 30 minutes and lyophilize.

[0152] Reconstitution - Add 2-5 uL of 1x PBS / mg of tendon, more PBS can be added until the desired concentration is reached.

[0153] Tendons were decellularized for 1 hour using various concentrations of DNAse (10 U, 50 U, and 100 U) (see, for example, Figure 7). 1x PBS was used as a no-decellularization control. DNA concentration was determined using a DNEasy kit (Qiagen). The data indicate that as little as 50 U of DNAse is effective in decellularizing tissue. As shown in Figure 8, 50 U of DNAse was compared to traditional detergents, 1% SDS, and 0.1% EDTA. 50 U of DNAse was tested for 0.5, 1, and 2 hours, whereas the standard SDS and EDTA protocol requires 24 hours of decellularization. DNA concentration was determined using a DNEasy kit (Qiagen, n = 3). All values ​​were normalized to "no decellularization." Tukey's HSD multiple comparison post-hoc test showed no significant differences between DNAse treatment or DNAse decellularization at each time point compared to SDS and EDTA. The table below also shows the percentage of DNA remaining in patellar tendons and Achilles tendons after various decellularization methods and / or various time periods. [Table 2]

[0154] Native tendons were characterized to determine which tendons would be optimal for developing allograft products. Patellar and Achilles tendons were characterized for DNA content and native protein concentration. We also aimed to identify differences between location and protein profile within each source (i.e., proximal vs. distal). No significant differences in total protein or TGFβ content were observed between different regions of the tendon. However, we found that the Achilles tendon had a higher relative protein content. Achilles and patellar tendons were divided into three sections: the proximal end, mid-center / central portion, and distal end of the tendon (Figures 9A-9D). Total protein in native tendons was measured using a BCA Protein Quantitation Kit (Thermo Scientific) (Figures 9E-9H). TGF-β was measured using a TGF-β Magnetic Bead Panel Milliplex Kit (Millipore Sigma, #TGFBMAG-64K-03). ANOVA showed no statistically significant differences between tendon regions, so the entire tendon can be used for treatment. When comparing the two different tendons, (Figure 9D) there was no difference in total protein (P = 0.93), but (Figure 9H) TGF-β was statistically higher in the Achilles tendon than in the patellar tendon (P = 0.0045). [Table 3]

[0155] As shown in Figure 10, filtration effectively removed collagenase activity. Decellularized tendons were treated with collagenase to improve the form factor of DTM. The 100 kDa filter was highly effective in removing collagenase activity from the final product. ANOVA showed a significant difference between groups (F(4,22) = 18.06, p < 0.0001). Importantly, no significant difference in collagenase activity was observed between the native and 100 kDa filtered samples. [Table 4]

[0156] As shown in Figure 11, greater bioactivity was maintained in the DTM than with the standard method for decellularizing tendons with pepsin. Tendons were digested after decellularization using a solution containing collagenase type 1 (92.5 g tendon / 1 g collagenase 1) and collagenase type 3 (185 g tendon / 1 g Collagenase 3) or a previously published method (Farnebo et al. 2014, PMID: 24341855). ANOVA showed a significant difference between groups, F(3,11) = 5.056, p = 0.0193. Tukey's HSD post-hoc analysis showed significantly less TGF-β with pepsin (P = 0.0249).

[0157] As shown in Figure 15, normalized TGFb content across four samples from four different donors across two processing steps is shown. For each donor, the first column represents the amount of TGFb in native tendon, the second column represents the amount of TGFb in decellularized tendon, and the third column represents the amount of TGFb in digested tendon. The percent change across processing steps is also listed in the table below (the percent increase is measured from native tendon to post-collagenase treatment). [Table 5]

[0158] We investigated the differential proliferation of cells plated on different surfaces (see, for example, Figures 12A-12C). Tissue culture plates were left untreated (control, "TC-treated") or coated with collagen or DTM. Primary tenocytes (ZenBio#TEN-F) were plated at 20,000 cells / well, and cell viability was quantified using Presto Blue (Thermo Fisher Scientific) at (A) 48 hours after plating or (B) 7 days after plating. Significantly different proliferation rates were observed (C) (ANOVA = F(3,26) = 10.6, p < 0.0001). Table 6 [Primary Technology Documents]

Non-licensed literature

[0159]

Non-licensed literature 1

Claims

1. a matrix metalloproteinase (MMP) digested tendon tissue, a sterile aqueous carrier solution, and an antimicrobial agent; A decellularized tendon matrix (DTM) composition characterized by maintaining at least 50% of the growth factors present in native tendon tissue.

2. 1. A method for producing a decellularized tendon matrix (DTM) composition, comprising: the decellularized tendon matrix (DTM) composition comprises matrix metalloproteinase (MMP) digested tendon tissue, a sterile aqueous carrier solution, and an antimicrobial agent; maintains at least 50% of the growth factors present in natural tendon tissue; mincing the tendon tissue sample; decellularizing the comminuted tendon tissue sample; grinding and / or digesting, stopping and neutralizing; washing; and Freeze-drying process A method for producing a decellularized tendon matrix (DTM) composition, comprising a process including one or more steps selected from the following:

3. 3. The method for producing a composition described in claim 2, wherein the decellularizing step comprises exposing the shredded tendon tissue sample to a solution containing one or more components selected from a chaotropic salt, a nonionic surfactant, a zwitterionic surfactant, a cationic surfactant, an anionic surfactant, or a combination thereof.

4. The method for producing a composition according to claim 2, wherein the matrix metalloproteinase (MMP) is selected from the group consisting of MMP-2, MMP-9, MMP-14, and combinations thereof.

5. 3. The method of claim 2, wherein the quenching and neutralizing step comprises quenching and neutralizing with a solution comprising one or more protease inhibitors selected from the group consisting of TAPI-0, TAPI-1, TAPI-2, marimastat, phosphoramidon, luteolin, PMSF, pepstatin A, leupeptin, E-64, sodium orthovanadate, or combinations thereof.

6. A decellularized tendon matrix (DTM) hydrogel comprising the resuspended decellularized tendon matrix (DTM) composition of claim 1, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC), and PEG-N-hydroxysuccinimide (NHS) ester.

7. 1. A method for producing a soft cast decellularized tendon matrix (DTM) body, comprising: resuspending a decellularized tendon matrix (DTM) composition in a physiological buffer, the DTM composition comprising matrix metalloproteinase (MMP) digested tendon tissue, a sterile aqueous carrier solution, and an antimicrobial agent, the DTM composition maintaining at least 50% of the growth factors present in native tendon tissue; mixing the decellularized tendon matrix (DTM) composition with a PEG-N-hydroxysuccinimide (NHS) ester to form a soft hydrogel; transferring the soft hydrogel into a three-dimensional mold; and Step of curing and deactivating the polymerization reaction product A method for producing a soft cast decellularized tendon matrix (DTM) body, characterized in that the DTM body is prepared by a process including one or more steps selected from the following:

8. A decellularized tendon matrix (DTM) hydrogel comprising: A decellularized tendon matrix (DTM) hydrogel comprising the resuspended decellularized tendon matrix (DTM) composition of claim 1, further comprising 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and a water-soluble coupling agent selected from N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (sulfo-NHS) together with the (EDC) coupling agent.

9. 1. A method for producing a decellularized tendon matrix (DTM) composition from a tendon, comprising: decellularizing the tendon to produce a decellularized tendon; contacting the decellularized tendon with an enzymatic solution comprising a matrix metalloproteinase (MMP) to produce a digested decellularized tendon; freeze-drying the digested decellularized tendon to produce a freeze-dried tendon; and reconstituting the freeze-dried tendon to produce a decellularized tendon matrix.

10. the decellularizing comprises contacting the tendon with a DNase solution; 10. The method of claim 9, wherein the DNase solution comprises 10-100 units of DNase per milliliter of solvent, 25-75 units of DNase per milliliter of solvent, 40-60 units of DNase per milliliter of solvent, or 50 units of DNase per milliliter of solvent.

11. 11. The method of any of claims 9 to 10, wherein the decellularization comprises contacting the tendon with a DNase solution at a concentration of 4 milliliters to 50 milliliters per gram of tendon, 5 milliliters to 10 milliliters per gram of tendon, or 10 milliliters to 50 milliliters per gram of tendon.

12. 12. The method of claim 11, wherein the contacting is carried out for 1 hour and is carried out on a shaker.

13. 11. The method of claim 9, wherein the decellularization further comprises washing the tendon with phosphate buffered saline.

14. 11. The method of any of claims 9 to 10, wherein the freeze-drying comprises freezing the digested, decellularized tendon at minus 80°C for at least 30 minutes.

15. 14. The method of claim 13, wherein the tendon is filtered through a 70 micrometer strainer using centrifugation at 1500G to 2500G for 1 minute to 15 minutes.

16. The method of any one of claims 9 to 10, wherein the MMP comprises collagenase.

17. the collagenase is selected from the group consisting of collagenase type I, collagenase type III, and combinations thereof; 17. The method of claim 16, wherein the concentration of collagenase type I in the enzyme solution is 2 milligrams per milliliter and the concentration of collagenase type III in the enzyme solution is 1 milligram per milliliter.

18. 11. The method of any of claims 9 to 10, wherein the decellularized tendon is contacted with the enzyme solution at a concentration of 10 milliliters to 50 milliliters per gram of tendon, or at a concentration of 5 milliliters to 10 milliliters per gram of tendon.

19. 11. The method of any of claims 9 to 10, wherein the decellularized tendon is contacted with the enzyme solution for 24 hours or 12 hours.

20. 11. The method of any of claims 9 to 10, wherein the decellularized tendon is contacted with the enzyme solution at 37°C.

21. 11. The method of any of claims 9 to 10, wherein the reconstitution comprises mixing 2 microliters to 5 microliters of solvent with 1 milligram of the freeze-dried tendon.

22. 11. The method of any of claims 9 to 10, wherein the decellularized tendon matrix (DTM) comprises less than 5% by weight of the cellular material in a native tendon, less than 2% by weight of the cellular material in a native tendon, less than 1% by weight of the cellular material in a native tendon, or less than 0.1% by weight of the cellular material in a native tendon.

23. 11. The method of any of claims 9 to 10, wherein the decellularized tendon matrix (DTM) comprises more than 90% by weight of the TGF-β in natural tendon, more than 95% by weight of the TGF-β in natural tendon, or more than 99% by weight of the TGF-β in natural tendon.

24. 11. The method of any of claims 9 to 10, wherein the decellularized tendon matrix (DTM) contains less than 5% by weight of DNA in native tendons, less than 2% by weight of DNA in native tendons, less than 1% by weight of DNA in native tendons, or less than 0.1% by weight of DNA in native tendons.

25. 10. The decellularized tendon matrix (DTM) composition of claim 1 for implantation into a subject.

26. 10. A tissue regeneration scaffold for implantation into a patient, comprising the decellularized tendon matrix (DTM) composition of claim 1.

27. The decellularized tendon matrix (DTM) composition of claim 1, further comprising an excipient.

28. a sterile aqueous carrier solution and an antimicrobial agent; A decellularized tendon matrix (DTM) composition characterized by maintaining at least 50% of the growth factors present in shredded native tendon tissue.

29. A decellularized tendon matrix (DTM) hydrogel comprising the resuspended decellularized tendon matrix (DTM) composition of claim 28, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC), and PEG-N-hydroxysuccinimide (NHS) ester.

30. A decellularized tendon matrix (DTM) hydrogel comprising:

29. A decellularized tendon matrix (DTM) hydrogel comprising the resuspended decellularized tendon matrix (DTM) composition of claim 28, further comprising 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and a water-soluble coupling agent selected from N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (sulfo-NHS) together with the (EDC) coupling agent.

31. 30. The decellularized tendon matrix (DTM) composition of claim 28 for implantation into a subject.

32. 30. A tissue regeneration scaffold for implantation into a patient, comprising the decellularized tendon matrix (DTM) composition of claim 28.

33. 30. The decellularized tendon matrix (DTM) composition of claim 28, further comprising an excipient.

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