Decellularized biological scaffolds with physical stabilization

Abstract

The disclosure, developed using decellularization and physical crosslinking of native tissues, removes the principal sites of calcification from the xenogeneic tissues, protects the extracellular matrix components (elastin and collagen) from rapid degradation after implantation, provides mechanical integrity and support after implantation, as well as allows for cellular infiltration, and remodeling over time, without the development of calcification or thrombosis.

Description

Decellularized Biological Scaffolds with Physical StabilizationGovernment Support

[0001] This invention was made with government support under Grant No R44HL147771-03, awarded by the National Institutes of Health. The government has certain rights in the invention.Cross-Reference to Related Application(s)

[0002] This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application 63 / 519,585, filed August 15th, 2023 and entitled “Decellularized Biological Scaffolds with Physical Stabilization for in situ Tissue Engineering Applications,” which are hereby incorporated herein by reference in their entireties.Field

[0003] The various embodiments herein relate to manufacture and use of decellularized biological scaffolds with physical stabilization for in situ tissue engineering applications.Background

[0004] In situ tissue engineering applications require scaffolds to replace tissues that are absent or deformed. Due to the various limitations of currently available scaffolds for tissue engineering, there is still a need to develop optimal tissue-based products that allow remodeling and regeneration in situ. The present disclosure, developed using decellularization and physical crosslinking of native tissues, removes the principal sites of calcification from the xenogeneic tissues, protects the extracellular matrix components (elastin and collagen) from rapid degradation after implantation, provides mechanical integrity and support after implantation, as well as allows for cellular infiltration, and remodeling over time, without the development of calcification or thrombosis.

[0005] Pediatric CHD patients often require pulmonary conduit replacements to correct critical congenital conditions like pulmonary stenosis, tetralogy of Fallot, pulmonary atresia, truncus arteriosus, and transposition of the great arteries along with ventricular septal defects. Currently used homografts, glutaraldehyde crosslinked xenografts, or synthetic grafts are suboptimal Calcification, thrombosis, and the inability to remodel require multiple replacement surgeries Data reviewed from -300 pediatric patients who underwent placement of the right ventricle-to-pulmonary artery valved conduit using glutaraldehyde-treated tissues concluded that stenosis leading to the xenograft conduit failure was present in all types of implants. A CDRH (Center for Devices and Radiological Health, FDA) report for crosslinked xenografts showed 44% stenosis, 42% device replacement, and a subsequent valved conduit or valve replacement required in 79 out of 84 pediatric patients undergoing implantation. One of the main reasons for calcification, stenosis, and immune response in glutaraldehyde-fixed xenografts may be related to the remnant bovine cellular material, such as membrane phospholipids since these conduits are not decellularized before glutaraldehyde treatment Efforts have been made to develop pulmonary valve conduits from decellularized xenograft tissues, but results are mixed. The initial iteration of a porcine xenograft showed promising preliminary results in adults but elicited a severeimmune response and catastrophic failures in children, primarily due to incomplete decellularization. Pulmonary conduits were also developed with decellularized porcine small intestinal submucosa

[0006] Currently available products for tissue engineering applications encounter various limitations with flexibility, non-degradation, calcification, thrombosis, inflammation, infection, and an inability to repopulate with cells in-vivo. There is a need to develop an optimal replacement conduit for patients that can last for several decades. Such a conduit should provide optimum mechanical strength on implantation, allow cellular infiltration, and be able to remodel over time, thereby displaying a potential to grow along with the patient. There is a need for treatments and products aimed at overcoming these limitations, and providing the potential of producing off-the-shelf tissue products, that can be derived from a wide range of tissues depending upon the application and need, as well as improved methods to reduce manufacturing production times.Brief Summary

[0007] Discussed herein are various methods for the manufacture and use of decellularized and PGG treatment of xenogeneic tissues, and providing the potential of producing off-the-shelf tissue products that can be derived from a wide range of tissues depending upon the application and need. Temporarily stabilized decellularized scaffolds also have the potential to degrade slowly after implantation, allowing cellular infiltration, and remodeling in situ.

[0008] In Example 1 , was designed to optimize the water rinses that follow tissue trimming

[0009] Example 2 follows the work performed in Example 1, wherein water rinses / incubations and decellularizations solutions conditions are optimized

[0010] Example 3 follows the work performed in Example 2 wherein the process for alcohol rinses of samples is optimized.

[0011] Example 4 follows the work performed in Example 3 wherein improvements were made to DNase and RNase treatments of the samples. This step is performed to bring tissue to isotonic physiological environment for optimal DNase / RNase activity and then subsequent removal of RNase and DNase from the tissue.

[0012] Example 5 follows the work performed in Example 4 and demonstrates the peracetic acid (PAA) treatment step used to sterilize the tissue

[0013] Example 6 follows the work performed in Example 5 and outlines the optimized Penta-Galloyl Glucose (PGG) treatment processes.

[0014] Example 7 follows the work performed in Example 6 and outlines the processes for terminal sterilization and packaging. Terminal sterilization was added to enable production of a sterilized product in its final packaging that is free of contamination from living microorganisms, including bacteria, yeasts, and viruses while maintaining biomechanical and structural properties of the scaffold.

[0015] Example 8 describes terminal sterilization methods and compositions utilizing Supercritical carbon-dioxide (SCCO2) for PGG stabilized decellularized tissues with low concentrations of Peracetic acid (PAA) (75-100 ppm) as the additive for 60-120 minutes

[0016] Example 9 tested decellularized PGG treated bovine jugular vein valved conduits (BJV) implantation in sheep.

[0017] Example 10 details the manufacture and use of decellularized xenograft valved conduits for pediatric patients.

[0018] While multiple embodiments are disclosed, still other embodiments will become apparent to those skilled in the art from the following detailed description, which shows and describes various illustrative implementations. As will be realized, the various embodiments herein are capable of modifications in various obvious aspects, all without departing from the spirit and scope thereof. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not restrictiveBrief Description of the Drawings

[0019] FIG. 1 shows the DNA levels measured during the control process. DNA measured in samples taken after each step of the process are presented alongside DNA levels of native tissue (unprocessed bovine jugular vein valves).

[0020] FIG. 2 is a representative image of trimmed, rinsed, and mounted veins

[0021] FIG. 3 illustrates the experimental design for decellularization.

[0022] FIG. 4 illustrates incubation steps for the decellularization process.

[0023] FIG. 5 illustrates the experimental design to determine the level of DNA removal

[0024] FIG. 6 illustrates the protocol differences with the present protocol using reduced rinse steps.

[0025] FIG. 7 details the alcohol rinse optimization protocol.

[0026] FIG. 8 details SDS levels measured in various tissue samples. For control and optimized process “Decel.” tissue was collected and assessed post decellularization; for the rest of the sample groups tissue was collected after the alcohol incubations.

[0027] FIG. 9 details additional residual SDS at the steps listed in Example 5. Levels of SDS were measured in native non-processed tissue and on tissue that was collected after PBS rinses post- DNase / RNase incubation.

[0028] FIG. 10 is a table detailing the specific testing parameters for 5 tested groups.

[0029] FIG. 11 details the results of residual DNA measured using PicoGreen assay. Percent reduction is measured against Native Tissue DNA results.

[0030] FIG. 12 shows residual DNA measured via PicoGreen assay on the left. DNA levels ofNative tissue are contrasted with that of DNA levels in decellularized tissue. This testing was done on group 7 of Example 6 (post DNase / RNase incubation and rinsing). Right) DAPI and H&E staining on the right. Image is representative. H&E staining was done on groups 1 and 2 of experiment 6. Samples were collected post PGG rinsing

[0031] FIG. 13 details the testing conditions for each group for PGG treatment.

[0032] FIG. 14 shows the pH measurements of final 1xPBS rinses of various groups (1-4). pH of the final 1xPBS rinse were compared to pH of the PGG solution. Target was set at pH 7.4.

[0033] FIG. 15 shows the residual PGG in tissue post PBS rinsing. Group 3 was the control as it used a protocol in the art for post PGG rinsing. Groups 4, 5, and 6 were experimental groups with different rinsing strategies None of these groups underwent PAA treatment

[0034] FIG 16 shows the residual PGG in samples that underwent PAA treatment (ControlProcess, Group 1) vs samples that were not PAA- treated (Groups 2 and 3).

[0035] FIG. 17 shows the total amount of PGG (mg) in various solutions. Data was collected as part of Example 7. Start PGG Solution shows PGG amount in PGG solution at the beginning of incubation. End PGG Solution shows PGG levels in the same solution at the end of PGG incubation. 1st, 3rd, 6th rinses show amounts of PGG at the end of each respective 1xPBS rinse.

[0036] FIG. 18 shows weight loss during elastase enzyme challenge. Degree of mass loss is inversely proportional to the degree of elastase resistance.

[0037] FIG. 19 shows weight loss during collagenase enzyme challenge. Degree of mass loss is inversely proportional to the degree of collagenase resistance

[0038] FIG. 20 shows the biaxial stretching of samples with PAA vs no PAA before PGG treatment. Control process samples were collected at the same phase as Group 1 and 2, (post-PGG incubation and subsequent rinses). All groups were compared to the stress strain curve of the native tissue.

[0039] FIG. 21 shows the suture pullout data of samples with PAA vs no PAA before PGG treatment. Control process samples were collected at the same phase as Group 1 and 2 (post-PGG incubation and subsequent rinses). All groups were compared to stress / strain suture pullout curve of the native tissue.

[0040] FIG 22 shows the PGG Levels in Storage Solution Group 3 - pre-sterilization levelsGroups 4 and 5 - storage solution was not changed during sterilization, measured post E-beam sterilization. Groups 6 and 7 - storage solution changed during sterilization, measured post SCCO2 sterilization.

[0041] FIG. 23 shows the levels of PGG in dry tissue. Post PGG Treatment results were calculated by subtracting the PGG levels in solution from pre- and post-PGG treatment results; this data set indicates how much PGG was present in the tissue immediately after the PGG treatment. Group 3 samples underwent processing but were not sterilized. Group 4 - Low E-beam Group 5 - High E- beam. Group 6 - Low SCCO2. Group 7 - High SCCO2.

[0042] FIG. 24 shows the Weight loss during elastase enzyme challenge. Degree of mass loss is indirectly proportional to the degree of elastase resistance “Fresh” refers to native tissue samples.

[0043] FIG. 25 shows the weight loss during collagenase enzyme challenge. Degree of mass loss is indirectly proportional to the degree of collagenase resistance.

[0044] FIG. 26 shows one cycle of biaxial stretching of treated groups vs native tissue stress strain curves.

[0045] FIG. 27 shows suture pullout testing of treated groups vs native tissue stress strain curves.

[0046] FIG. 28 shows suture pullout / biaxial graphs that include Max Stress, Max Stress at150% Extension, Young’s Modulus for Suture Pullout, and Young’s Modulus for Biaxial testingcomparing treated tissue groups to native tissue. Left-side columns represent longitudinal stress and right-side columns represent circumferential.

[0047] FIG 29 illustrates the decellularization of native tissues: DNA quantification in (A) bovine jugular vein valved conduit; (B) Porcine Fascia; (C) Bovine Vena Cava (N=5); *** p<0.001.

[0048] FIG. 30 shows VVG staining for elastin: TxGuardTM conduit walls (bottom) retain elastin (black) after treatment with elastase enzyme, glutaraldehyde walls (top) lose elastin after enzyme treatment.

[0049] FIG. 31 shows (A) &(B) Biaxial Testing of Bovine Jugular Vein conduit walls showing comparable stress at maximum strain, and young’s modulus for native, Glut and PGG treated tissues; (C) Elastase Challenge with 0.15% PGG treated Bovine Jugular Vein tissues stored for up-to 24-months (Statistically significant difference compared to decellularized BJV), (N=8); (D) Degradation of native, glutaraldehyde treated tissues, and TxGuard tissues exposed to 100 units / mL Collagenase enzyme.

[0050] FIG. 32 are pictures of rat subdermal implant calcification of decellularized bovine valved jugular vein and prevention with TxGuard (PGG) treatment. Alizarin red stain for calcium.

[0051] FIG. 33 shows BJV conduits after 3 months of implant in sheep showing (A) no tissue overgrowth or thrombosis; (B) cellular infiltration (H&E); (C) Luminal reendothelialization shown by VWF staining; (D) No calcification shown by Von Kossa staining; (E) HSP-47 staining for collagen remodeling; (F) a-SMA and (G) Vimentin staining showing infiltration of myofibroblast-like cells in the walls; (H) Leaflet showing cellular infiltration and no thickening.

[0052] FIG. 34 shows Phase I original and Phase II optimized decellularization and PGG stabilization process. The optimization reduced active production days from 19 to 8 days, without change in the chemical and mechanical properties of the scaffolds. (C) <50 ng residual DNA / mg dry weight of decellularized tissue using optimized decellularization procedure.

[0053] FIG. 35 shows (A) Elastase and (B) Collagenase enzyme challenge with native, andPGG treated decellularized Bovine Jugular Vein before and after sterilization processing. (C) Suture retention and biaxial mechanical characterization of native, and PGG treated decellularized bovine jugular vein before and after sterilization. No significant difference observed in tissues before and after SCCO2 sterilization

[0054] FIG 36 shows three photos of sterile packaging: (A) Final packaging container with glass bottle and semi porous cap. (B) PTFE filter fitted semi-porous cap; (C) Sterile cap in secondary packaged pouched after sterilization.

[0055] FIG 37A-D outlines an embodiment of an optimized Process Flow Chart for a tri-leaflet valve for use in biological scaffolds.Detailed Description

[0056] The various tissue embodiments disclosed or contemplated herein include compositions for biological scaffolding, as well as novel methods of manufacture. The present disclosure, developed using decellularization and physical crosslinking of native tissues, removes the principal sites of calcification from the xenogeneic tissues, protects the extracellular matrix components (elastin and collagen) from rapid degradation after implantation, provides mechanical integrity and support afterim pla ntation , as well as allows for cellular infiltration, and remodeling over time, without the development of calcification or thrombosis.

[0057] Prior studies (Phase I) with the developed decellularization and PGG stabilization process had shown the ability of the process to optimally remove nuclear and cellular components from native tissues (< 50 ng residual DNA / mg decellularized tissue), while maintaining the integrity of the extracellular matrix. Treated tissues were shown to be more resistant to elastase and collagenase enzyme degradation, while exhibiting comparable mechanical strength as native tissues. Decellularized stabilized tissues implanted subcutaneously in rats for up to 90 days have been shown to resist calcification and allow infiltration of fibroblast and myofibroblast cells. GLP compliant biocompatibility studies performed with the decellularized crosslinked tissues have shown that the developed scaffolds do not cause sensitization, irritation, and are non-cytotoxic as per ISO-10993 standards. Decellularized and PGG treated valved bovine jugular vein conduits implanted in sheep as a pulmonary conduit replacement have also shown adequate functionality in a circulatory environment. The animals, after 3 months of implantation, did not show any thrombosis or calcification. The replacement conduits did not demonstrate any thickening of the implanted tissues and allowing cellular infiltration. Explants observed after 3 months showed the infiltration of fibroblasts, myofibroblasts, and endothelial cells along the luminal side Explants also demonstrated a 10% increase in the diameter before and after implantation

[0058] The current design phase has been aimed to scale-up, and develop an optimized processing, generating uniform and repeatable results. The decellularization and PGG stabilization process has been optimized to reduce the active production days (19 to 8 days), without affecting the chemical, and mechanical properties of the final scaffolds, and enable production on a manufacturing scale. A terminal sterilization process, utilizing SCCO2 sterilization with peracetic acid (75-150 ppm) has been established. This novel approach has shown to reduce bioburden levels in controlled contamination up-to 95%. A prototype packaging for the final sterilized product has been developed with a semi-porous cap allowing gas permeation during sterilization, while maintaining sterility of the contents post-processing. Additional secondary packaging has also been explored, and the prototype has been confirmed to be able to maintain sterility through the delivery process, until clinical use. Further optimizations are being carried out to eliminate the secondary packaging, add an additional lid to the semi-permeable cap, and thereby increase ease of handling the product in a clinical setting.

[0059] Current technologies in tissue engineered products use allografts, homografts, or glutaraldehyde fixed tissues that are not degradable and will not remodel. The present disclosure provides slowly degrading scaffolds that allow remodeling, growth and assimilation. Tissue engineering scaffolds used today are either made from synthetic polymers or decellularized tissues, but these require in vitro cell seeding and their controlled degradation is difficult. The present disclosure provides decellularized and physically stabilized tissue scaffolds that slowly degrade and allow in situ tissue repair. The unique approach of the present disclosure is to effectively decellularize and physically crosslink soft tissues, sterilize them to reduce bioburden, and maintain their sterility until being used. Tissue engineered products currently available in the marketplace include homografts, allografts, synthetic, or tissue-based products. While homografts and allografts are not always easily available, synthetic scaffolds are incapable of growth and regeneration. Most of the available tissue-basedproducts are crosslinked with glutaraldehyde, which makes the scaffolds stiff, non-degradable, and prone to calcification.

[0060] The applicants used decellularized bovine jugular vein that was stabilized with pentagalloyl glucose (PGG) (PGG-DBJVC). Bovine jugular veins have a tri-leaflet valvular apparatus, making it an attractive option as a pulmonary conduit replacement. Decellularization removes the scaffold's immunogenic cellular components while preserving the extracellular matrix structure. Decellularization might lead to the loss of extracellular tissue matrix (ECM) integrity, increased degenerative structural failure, and calcification after implantation. To optimize the biodegradation process and maintain ECM integrity, crosslinking or stabilization of decellularized ECM is utilized. PGG is a non-cytotoxic polyphenolic tannin composed of a hydrophobic inner core and numerous external hydroxyl groups, which specifically binds to hydrophobic regions of proteins and form several hydrogen bonds. NMR studies have shown that PGG has a strong affinity for proline-rich proteins like elastin and collagen, binding through the hydrophobic stacking of the polyphenol ring against the pro-S surface of proline. The addition of PGG in vitro has been shown to increase the rate of coacervation and selfassembly of tropoelastin, a precursor to elastin fibers within the ECM. While glutaraldehyde fixation crosslinks only the collagen fibers, PGG binds to both collagen and elastin. Since -50% of the dry weight of vascular tissues is composed of elastin, stabilization of elastin is essential in maintaining the structure and mechanical behavior of native vascular tissues.

[0061] Furthermore, PGG is one of the most potent antioxidant within the tannin group and is also known for its antimicrobial, antiviral, anti-diabetic, anti-inflammatory, and anti-tumor properties. PGG-treated elastin-rich tubular vascular grafts (ETVGs) have shown good mechanical and biological properties in multiple in vivo subdermal implantation models, reducing rapid biodegradation and calcification of the implants.

[0062] The decellularization process involves treating native tissues obtained from animal sources with a combination of detergents, chelating agents (EDTA), DNase and RNase enzymes. A successful decellularization involves removal of all cellular and nuclear material from the native tissues, with a final DNA concentration < 50ng / mg dry weight of the tissue, and no visible nuclear material observed from histology. The decellularization process has demonstrated successful removal of cells in tissues obtained from different animal sources (bovine, porcine, murine), as well as different tissue types (jugular vein, pulmonary arteries, aortic and pulmonary valves, pericardium, vena cava, urinary bladder tissue, peritoneum, fascia), with final DNA concentration of <50ng / mg dry weight of decellularized tissue (See FIG. 29). The process is therefore capable of removing native cells consistently from different tissues, allowing for the development of customizable tissue engineering scaffolds depending on the specific application.

[0063] The temporary crosslinking of the extracellular matrix is achieved with polyphenol pentagalloyl glucose (PGG). Unlike glutaraldehyde, which covalently crosslinks collagen fibers, PGG hydrophobically binds to both elastin and collagen within the ECM (FIG. 30). This property can be targeted towards elastin rich tissues, majorly found in the cardiovascular system, or where tissue elasticity is essential to be maintained after implantation. Moreover, temporary crosslinking, allows for gradual degradation over time, enabling remodeling of graft (FIG 31 ) Temporarily crosslinked tissuesalso maintain mechanical integrity, allowing for sufficient mechanical support at the point of implantation. The present disclosure shows the mechanical properties of PGG crosslinked decellularized tissues comparable to those of native glutaraldehyde fixed tissues, which are currently used commercially (FIG. 31 ) Stabilized decellularized tissues have been shown to resist degradation by elastase and collagenase enzymes after extended storage, demonstrating the ability of the prepared scaffolds to be stored for an extended period until use, and therefore be used as off-the-shelf scaffolds (FIG. 31).

[0064] Stabilized crosslinked tissues have shown resistance to calcification when implanted subcutaneously in rats in an accelerated calcification model, while glutaraldehyde crosslinked tissues calcify heavily and consistently in different tissue types Subcutaneously implanted stabilized treated tissues have also shown cellular infiltration over the period of 90 days. Analysis of these infiltrated cells have indicated that most of the infiltrated cells are fibroblasts, or myofibroblasts, with very little infiltration of immune cells, indicating no major immune response to the decellularized stabilized scaffolds. GLP compliant biocompatibility studies performed with the decellularized crosslinked tissues have shown that the developed scaffolds do not cause sensitization, irritation, and are non-cytotoxic as per ISO- 10993 standards.

[0065] The following examples demonstrate processing techniques designed to decellularize tri-leaflet bovine jugular vein valves for the surgical treatment of congenital heart malformations The designs are aimed to develop a pathway to an optimized manufacturing process that yields a commercially viable product, while increasing regulatory compliance and minimizing process changes.

[0066] Decellularization of tissue to create an extracellular tissue matrix (ECM) has been long established as an excellent technique to preserve a tissue's native composition and ultrastructure, while ridding the tissue of native cells and genetic material. This removal of native cellular and genetic materials is extremely important to prevent an immune rejection when the tissue is implanted in a patient.

[0067] In the present disclosure, effective removal of cells (decellularization) from bovine jugular vein segment containing a tri-leaflet valve will allow it to be utilized in the treatment of congenital heart defects in a neonatal population. The decellularized jugular vein segment will be primarily composed of insoluble collagen and elastin that has been chemically stabilized with penta-galloyl glucose (PGG). A key feature of this conduit is that it maintains function of the tri-leaflet valves in its structure.

[0068] The following is the decellularization acceptance criteria for the product:- DNA content in decellularized tissue should be < 50 ng DNA per mg dry tissue. When compared to fresh tissue, each segment should have reduction of DNA content by >95%.- Lack of cell nuclei on H&E and DAPI stained sections- Confirmed matrix integrity by H&E staining

[0069] A series of experiments was designed to optimize the decellularization process which achieved the above acceptance criteria, maintained the spirit of the key processing steps, and implemented terminal sterilization. The new optimized production protocol increased the efficiency of the process through the reduction of chemical & labor requirements as well as a reduction in step times. Overall, the production process has been reduced from 19 to 8 active production days not including terminalsterilization which is anticipated to require 1-2 additional processing days. These changes also have a positive environmental effect through the reduction in the amount of hazardous waste generated. Additionally, a supplemental terminal sterilization step was added to allow for a final product free of contamination from living microorganisms, including bacteria, yeasts, and viruses while maintaining biomechanical and structural properties of the scaffold. The improved process will allowfor achievement a SAL of 106per the appropriate standard contained in ISO section: 11.080.01.

[0070] Optimization of Decellularization Process of Bovine Jugular Veins for the T reatment of Human Congenital Defects was performed. This report discloses the optimization parameters, and the rationale for the final process conditions that were selected for the optimized decellularization process.

[0071] Control Process: Before any optimization experiments were conducted, a control process run was executed This run followed provided protocol exactly with two changes - all sodium azide was removed from any steps in which it was previously included due to its known toxicity and the 4th DNase / RNase treatment step was also removed due to previous data suggesting that it added no further benefit compared to 3 treatment steps. The purpose of this control process run was for Collagen Solutions operators to learn the process and to collect samples to be referenced as standards for comparison with collected experimental samples. After the control process was conducted and data was analyzed on the control process samples, Experiments 1-7 were conducted. Any references made in the experimental data labeled “Control Process” is referring to data collected during this control run

[0072] To improve experimental rigor and confirm reproducibility of results, the conventional protocol was sometimes repeated during experiments for a given step and the results were compared to those of the optimized groups. FIG. 1 is a graph displaying measured DNA levels in samples collected during the control process as a baseline.

[0073] a table describing the test methods performed for this series of experiments.EXAMPLE 1

[0074] The following example was designed to optimize the water rinses that follow tissue trimming. Required specifications for the bovine jugular vein valves:- Valve must be functional (conduit must retain solution at the valve site)- Valve must have 3 leaflets- Conduit must be 8-10 cm in length with >3 cm per side from the end of each leaflet

[0075] Phase 1 - Identification of Appropriate Candidates1. Transfer veins to a colander. Rinse in PBS by passing 4-5L of 1xPBS over the colander with the veins.2. Place all veins in a beaker with 1xPBS. Veins must be submerged.3. Invert veins & check for the number of leaflets. Cut veins to size (8-10 cm, at least 3 cm away from the valve on each side), if there is more than one valve, select most appropriate way to cut as to preserve as many valves as possible. Without re-inverting, place into beakers as described below. i. If 2 leaflets, place into beaker “Inverted, 2-leaflet” ii. If 3 leaflets, place into beaker “Inverted, 3-leaflet”4. Take Inverted, 3-leaflet veins. i. Restore vein to original conformation (invert again)ii. Check for function - add PBS to distal end of vein. Test both sides. a. If functional, place in a beaker with fresh 1xPBS and name it “Functional, 3- leaflet” veins. b. If non-functional, place in a beaker with fresh 1xPBS and name it “Nonfunctional, 3-leaflet” veins.5. Discard any veins that feel to meet the specifications listed above.

[0076] Phase 2 - T rimming, Mounting, & Rinsing1. Remove as much adventitious tissue as possible from veins.2. Once all tissue is cleaned, thoroughly rinse (move the tissue around mechanically) with Dl- H2O by filling the beaker containing tissue with water and subsequently draining the solution using a colander. Repeat 2 additional times for a total of 3 washes Use 50-100 ml per vein.3. Mount onto clamps and zip-tie closed. Place mounted veins onto fixture and place in container.4. Pour 800-1000 mL of DI-H2O and store overnight at 2-8 °C without agitation.

[0077] Final Water Rinse Optimization: The purpose of this step is to clean and prepare tissue for further processing by removing unwanted elements which may include fat, flesh, blood, fascia, etc. and to retain the tissue of interest, in this case jugular veins containing tri-leaflet valves. This step is also necessary to confirm the appropriate number of leaflets and function of the valves Optimization of this step will allow production technicians to use their time more efficiently. Shortening the water rinses to three cycles of filling the container containing tissue with water and then draining the solution using a colander will allow the technician to briefly rinse out all the undesirable material without having to set timers and use extra equipment. Optimization is measured by duration and number of water washes. The results were visually inspected

[0078] Results: Unwanted elements which include fat, flesh, blood, fascia, etc., were not visually detected in the final solution or on the processed jugular veins. See FIG. 2 for representative images of trimmed, rinsed and mounted veins. It has been shown visually that after cleaning and brief rinsing (three cycles of filling the container containing tissue with water and then draining the solution using a colander) there is no visible excess tissue or blood remaining in the final water solution. Also, based on further experiments, this water rinse and subsequent processing steps were collectively sufficient to remove all necessary tissue residuals to achieve the desired physical properties and DNA content specifications.EXAMPLE 2

[0079] The following example was designed to optimize the water rinses / incubations taking place the day of decellularization. Decellularization duration and number of decellularizations solutions were also optimized. The general optimized decellularization process is to first incubate in NaOH for one hour; rinse with DI-H2O twice for 15-30 minutes; incubate in 1xPBS one time for 15-30 minutes; and incubate in decellularization solution (50 mM Tris; 0.25% SDS; 0.5% sodium deoxycholate; 0.5% Triton-X 100; 0.2% EDTA) for 24-48 hours.

[0080] Prior processes called for multiple water rinses prior to NaOH incubation, but unwanted elements which include fat, flesh, blood, fascia, etc., were not visually detected in the final solutiondespite no water rinses before NaOH incubation. Therefore, water rinses before NaOH treatment were eliminated as it was determined that water rinses from the previous day followed by an overnight water incubation was sufficient in removing excess blood and fat from tissue trimming thereby preparing the tissue for NaOH incubation and further treatments; further experimentation revealed desired physical and DNA specifications were achieved using this rinsing method in addition to subsequent processing steps.

[0081] Rinse #2, post NaOH incubation: Rinse out residual NaOH and bring tissue to physiological pH of 7.4. The purpose is to reduce chemical usage, labor usage, and hold times. The design of experiments (DOE) is illustrated in FIG. 3. The incubation steps are detailed in FIG. 4 It was determined that 2 x 15-30 min water washes followed by 1 x 15-30 min 1xPBS wash was sufficient to rinse out residual NaOH and bring tissue to physiological pH of 7.4.

[0082] DECELLULARIZATION OPTIMIZATION: This step is intended to chemically remove the bulk of the native cells and potentially immunogenic cellular material. It was hypothesized based on the gross appearance of veins after 24 hours of incubation in decellularization solution (and supporting literature) that the decellularization process is essentially complete in <24 hours. Assuming this is correct, several days could be cut from the process and chemical and labor requirements could be reduced. The potentially negative impact of prolonged detergent exposure on the tissue biochemical and mechanical properties could also be mitigated by reducing the incubation time The residual DNA content was measured by the Pico Green Assay. FIG. 5 illustrates the experiment design. Between the present protocol steps and downstream processing, the samples contained less than 50 ng / mg dry tissue weight and >95% DNA reduction compared to native tissue.EXAMPLE 3

[0083] The following example illustrates an optimized process for alcohol rinses of samples. FIG. 6 diagrams the conventional protocol and the protocol of the present disclosure. The purpose is to reduce residual chemicals introduced through the decellularization treatment as well as remove alcohol-soluble components of the ECM such as lipids. The series of residual reduction steps described in the conventional process would be cumbersome in a manufacturing setting and difficult to validate. Additionally, alternating between aqueous and alcohol solvents often enhance the solvent’s ability to remove residual material and lipids. Sodium dodecyl sulfate (SDS) levels were measured in lyophilized tissue that was papain-digested using an SDS Methylene Blue measuring kit (Chemetrics Catalog # I- 2017). FIG. 7 details the alcohol rinse steps.

[0084] Based on the data from this example (FIG. 8) that 2 hours of incubation in 70% ethanol (EtOH), followed by 1 hour of water incubation, performed twice is the most efficient and effective method of removing SDS from tissue.

[0085] The protocol was repeated as part of experiment 5 / Example 5 to ensure the SDS levels are low, and that the data collected during experiment 3 was reproducible During this example’s testing, we observed very low (0.283 ug SDS per mg dry tissue) residual SDS in processed tissue. As part of experiment 5 fresh, non-processed tissue was tested and the average SDS content was found to be 0.075 ug per mg tissue weight. While veins per Group 7 (see DOE Example 6; same protocol asselected in Example 3) showed that average SDS per mg tissue weight was 0.049 ug. Therefore following the new protocol allows for removal of nearly all residual SDS.

[0086] Day 4 PBS incubation: This step is conducted after the EtOH incubations and before DNase / RNase incubations The incubations are done to bring tissue to physiological pH and remove residual EtOH. Rather than incubate the samples in 1xPBS multiple times for 5 minutes, the samples of the present disclosure were rinsed one time for 30 minutes. The success of the new protocol with optimized PBS rinses and DNase / RNase incubations was assessed in terms of DNA removal. This protocol was repeated twice (first time as part of experiment 4 and second time as part of experiment 5) to ensure reproducibility. Therefore, it was decided that this modified PBS rinsing / incubation step was sufficient to produce the desired DNA reduction in the bovine jugular veinsEXAMPLE 4

[0087] The following example illustrates the improvements made to DNase and RNase treatments of the samples. This step is performed to bring tissue to isotonic physiological environment for optimal DNase / RNase activity and then subsequent removal of RNase and DNase from the tissue.

[0088] Groups that have been incubated for 30 min in 1xPBS pre-DNase / RNase treatment and washed for 60 min x 3 times post DNase / RNase treatment were compared to groups that were incubated as per conventional protocols. Groups 2 and 4 had additional 24-hour 1xPBS holds introduced in order to assess if that amount of incubation was necessary. See FIG. 10.

[0089] It was determined that 1 x 30 min 1xPBS incubation followed by a 3 x 60 min 1xPBS incubations following DNase / RNase treatment is optimal in the production environment to achieve the desired residual DNA specification without sacrificing unnecessary time and resources. Also, since a series of incubations were added right before this step, including a weekend hold in 1xPBS, it was hypothesized that the tissue would be in its optimal state to be incubated in DNase / RNase. This is supported by the fact that subsequent DNase / RNase treatment yielded desirable results. As for post-DNase / RNase treatment 1xPBS rinses, it was determined that they were sufficient at removing residual DNA fragments at 3x60 min incubations; a 16-24-hour hold can also be introduced as needed but does not lead to significant DNA reduction beyond that produced by the additional 3x60 min incubations.

[0090] DNase / RNase treatment to remove DNA and RNA from the ECM of bovine jugular veins: It was determined that a higher concentration of DNase allows for shorter incubation times and may improve DNA reduction without compromising the tissue’s physical properties. FIG 10 outlines the experimental parameters for five groups. FIG. 11 details the residual DNA from each group using a PicoGreen assay as described supra.

[0091] As shown by the data in FIG. 11 , the highest level of DNA reduction was achieved by Group 3 (98% DNA reduction). It was decided, based on the results, that 1 x 30 min 1xPBS incubation followed by 1 x 24 hour concentrated DNase / RNase treatment (10 U / ml DNase) followed by 3 x 60 min 1xPBS rinses is the optimal processing method of removing DNA and was therefore incorporated into the protocol for subsequent production lots. Also, a 16-24-hour 1xPBS hold can be introduced as needed but does not lead to further removal of DNA. Supporting the quantitative data from the PicoGreen assay are histological images showing complete removal of cell nuclei in DAPI or H&E-stained tissue sections (FIG. 12).

[0092] The final selected processing method was repeated as part of Example 6 to ensure that the data is reproducible. It was determined that Example 4 group 3 (selected protocol) achieved -9.62 ng DNA per mg dry tissue, while Example 6 showed 8 11 ng DNA per mg dry tissue, using the selected protocol. The conclusion was made that the process is reproducible and that the processed tissues meet the specification of <50 ng DNA per mg dry tissue weight and, >95% DNA reduction rate (reduction rate shown is >98%).EXAMPLE 5

[0093] The following example demonstrates the peracetic acid (PAA) treatment step used to sterilize the tissue. It was determined that PAA treatment prior to further processing is not desirable for sterilization validation and that a terminal sterilization step with the device placed in its final product packaging will be most appropriate Based on known literature, PAA treatment may have potential deleterious effects on mechanical integrity of the tissue and therefore this experiment was necessary to determine if it is essential As described above, the PBS rinses are crucial for removal of residual endonucleases and DNA fragments from the tissue.

[0094] It was unclear if PAA was a necessary step for this protocol as it is known to alter tissue physical properties and it was hypothesized to potentially affect subsequent binding and effectiveness of the PGG crosslinker which stabilizes the tissue. Therefore, an experiment was designed to compare tissue properties and residual PGG content in final processed tissue using protocols with and without PAA treatment step. PBS rinses were optimized as part of Example 4 and remained unchanged. As will be evident from the results in Example 6, PAA is not a necessary protocol step.EXAMPLE 6

[0095] The following example illustrates the Penta-Galloyl Glucose (PGG) treatment PBS rinses are necessary for PGG and IPA removal. pH, residual PGG (methanol extraction of bound PGG from lyophilized tissue, followed by PGG Assay on the extract solution), resistance to degradation (elastase and collagenase challenge), mechanical testing (biaxial tensile testing in circumferential or longitudinal axes, suture pullout) were all used to measure success FIG 13 details the testing conditions for each group.

[0096] No obvious impact of rinsing methodology on pH was noticed. All groups showed nearly neutral pH very close to that of 1x PBS (range 7.19-7.32; FIG. 14). Groups in which the veins were not incubated in 0.1 % PAA showed lower residual PGG levels bound to the Groups in which veins were (FIG. 15). However, Groups 2 & 3 following the prior art rinse method showed lowest values of residual PGG bound to the tissue (-12-14 ug PGG per mg tissue) whereas Groups 4, 5, and 6 showed comparatively higher residual PGG values (18-24 ug PGG per mg tissue; FIG. 14). Based on comparison to tissue that underwent the original protocol (Group 1 ), shortening the overall vein processing protocol seemed to slightly increase the amount of residual PGG bound to tissues. It should be noted, however, that the levels of tissue-bound PGG measured were likely affected by the efficiency of the methanol extraction process and based on the levels of PGG measured in the rinse solution, it is likely that those values are underreporting the actual amount of residual PGG bound to the tissue. Because the decreasing quantity of PGG measured in sequential PBS rinse solutions (1 , 3, and 6; FIG 17), the 6 x 30-minute PBS rinses were deemed to be necessary and the Group 5 protocol was thereforeselected as the optimal protocol as it most closely mimics the original rinsing process but is more appropriate for manufacturing.

[0097] No apparent difference in resistance to elastase degradation was observed among experimental groups (FIG. 18). There were some subtle differences in collagenase degradation, with a slight increase in degradation by collagenase observed in Group 1 (PGG + PAA) and Group 2 (new PGG process) compared to the Control Process (original protocol w / PAA; FIG. 19). There seemed to be a substantial difference in susceptibility to collagenase degradation between PAA treated tissue vs tissue that was not treated with PAA, though Group 2 samples not exposed to PAA underwent slightly less degradation than Group 1 samples.

[0098] One of the goals of decellularization is to preserve the mechanical properties of the original tissue while removing all native cells Biaxial tensile testing in the circumferential or longitudinal axes and suture pull-out were performed to assess the mechanical properties of the veins as a result of the new PGG processing protocol, with or without PAA. During tensile testing of veins, stress remains low with increasing strain until reaching the elastic phase, at which point collagen fibers are stretching and the stress increases with strain. Ideally, the stress-strain curves produced during tensile testing of processed tissue should resemble those of the native tissue (FIG. 20). The greater area below the extension / relaxation stress-strain curve, the stiffer the tissue is, and less likely to return to its original state PGG treatment according to both the original protocol and the optimized protocol increased the maximum tensile stress observed at 150% extension of the tissue compared to native bovine jugular veins and stiffened the tissue in the longitudinal direction. PAA treatment before PGG treatment also caused the tissue to further stiffen and veins from Group 2 showed the highest maximum tensile stress (Group 2; FIG. 20). Similarly, the new processing protocol without PAA (Group 2) was less stiff with lower maximum tensile stress at 150% extension. Similar trends among groups were observed during the suture pull-out assay (FIG. 21).

[0099] Based on these collective results, it was decided that PAA treatment is not an essential step in the bovine jugular vein processing protocol, and the step was eliminated from the protocol. However, it should be noted that while PAA treatment made an impact, its effects were not observed to be overly deleterious to the overall material properties and the including step may be considered if warranted in the future (e.g., to reduce viral load).EXAMPLE 7

[0100] The following example illustrates processes for terminal sterilization and packaging Terminal sterilization was added to enable production of a sterilized product in its final packaging that is free of contamination from living microorganisms, including bacteria, yeasts, and viruses while maintaining biomechanical and structural properties of the scaffold. The aim of terminal sterilization is to achieve a SAL of 10-6 per the appropriate standard contained in ISO section: 11.080.01.

[0101] Two sterilization methods were proposed: E-beam sterilization and Super Critical Carbon Dioxide (SCCO2) sterilization. Two different radiation doses were compared for samples sterilized via E-beam, and two different sterilant PAA concentrations were compared for samples sterilized via SCCO2. Note that SCCO2 sterilant contains PAA. The concentration of PAA in the sterilization solution is ~10X less than the PAA concentration (1000 ppm or 0.1%) of the PAA treatment step that wasremoved from the process. Residual PGG (methanol extraction of bound PGG from lyophilized tissue, followed by PGG Assay on the extract solution), resistance to degradation (elastase and collagenase challenge), mechanical testing (biaxial tensile testing in circumferential or longitudinal axes, suture pullout) were tested. Table 1 contains the different sterilization parameters for 7 tested groups.Table 1 : Different sterilization parameters All tissue was processed using the optimized protocol

[0102] As part of this experiment, residual PGG bound to tissue and PGG concentrations in solutions were assessed during various steps. When residual PGG bound to tissue was tested, methanol extraction was used to extract first PGG from the processed tissue, following by quantification of PGG in the extract solution. When solutions were directly tested for PGG levels, results were normalized to total tissue dry weight for consistency. See the Table 2 below for obtained PGG values at various points in the process.Table 2: PGG values throughout experiments

[0103] Effect of Sterilization on Retention of Tissue-bound PGG: PGG concentrations were directly measured in storage solutions (1x PBS pre-sterilization, 1x PBS post E-beam sterilization, and sterilization solution post SCCO2 sterilization; Figure 22). SCCO2 sterilization was the only method in which the initial storage solution was changed during sterilization; therefore, the incubation period of the veins in the solution was shorter for the SCCO2 groups compared to the others. PGG concentrationsin storage solutions after E-beam sterilization were higher compared to the initial PGG concentrations in veins that were not sterilized. This suggests that E-beam sterilization increases PGG loss from tissue into the storage solution Levels of PGG in sterilization solution post SCCO2 sterilization were lower than the levels of PGG in PBS in veins not subjected to sterilization. The lower values in SCCO2 storage solution would suggest improved PGG retention, or that the solution exchange during sterilization washes out remaining unbound PGG in the precursor PBS rinse solution, causing the levels measured to be lower in the sterilization solution. Regardless, SCCO2 sterilization did not appear to not affect PGG retention in tissue as dramatically as E-beam sterilization.

[0104] Tissue assessment: To observe the impact of sterilization on tissue-bound PGG, veins were lyophilized, tissue was digested enzymatically using papain, and methanol was used to extract PGG from tissue to enable calculation of bound PGG per dry tissue weight (FIG 23). It is worth noting that methanol extraction may have interfered with the assay results and further validation of the test method is required. Collected data was used to observe trends that will inform further work.

[0105] Compared with the non-sterilized tissue (Group 3: 29.2 pg PGG per mg dry tissue weight), lower PGG amounts were measured in post-sterilized tissue (ranging between 17.71 -22.74 pg PGG per mg dry tissue weight across Groups 4-7). Although, statistical analysis hasn’t been performed, it is unlikely that there is any statistically significant difference between the four sterilization groups based on comparative means and relatively high standard deviations observed (Groups 4-7) The amount of PGG extracted from tissue in each group was different than the estimated amount of PGG initially bound to tissue. Therefore, results of PGG in this assay may be impacted by how efficiently the methanol was able to extract PGG from the digested tissue.

[0106] As mentioned above, no specification currently exists for acceptable PGG levels bound to tissue for this product. Data collected was used for exploratory purposes to assess general trends of the impact of sterilization method and processing conditions on PGG binding. Further PGG quantification using a more robust method needs to be developed for accurate and precise conclusions to be made (for example, full tissue digestion followed by liquid chromatography or mass spectrometry assessment).

[0107] Resistance to Enzymatic Degradation: Native tissue degraded at a higher rate compared to the processed samples, indicating that treatment with PGG improved tissue resistance to degradation by either elastase (40% mass loss in controls vs -18-22% mass loss in processed tissue groups; FIG. 24) or collagenase (83% mass loss vs -7-11% mass loss in processed tissues; FIG. 25). There was no apparent impact of sterilization method or sterilization processing conditions on degradation by elastase, and sterilizing the tissue did not affect susceptibility to elastase degradation (see Group 3 vs Groups 4-7; FIG. 25). Sterilization did appear to slightly increase % mass loss after collagenase degradation (see Group 3 vs. Groups 4-7; FIG. 25). There was a higher % higher mass loss in SCCO2 sterilized samples compared to E-beam sterilized samples. While statistical analysis (ANOVA and post- hoc testing) is required to compare individual groups, the relatively low sample number (n=6) and relatively high standard deviations suggests there would not likely be any statistically significant differences observed.

[0108] Mechanical testing (biaxial tensile testing in circumferential or longitudinal axes and suture pullout) was performed on sterilized veins and compared to both unprocessed bovine jugular veins and processed veins that did not undergo sterilization Ideally, the mechanical properties of the final product should resemble those of native pulmonary arteries, and literature searching and / or mechanical testing may be warranted to better define these metrics.

[0109] Compared to native tissue, maximum stress observed at 150% extension and stiffness were higher in processed but not sterilized tissue (Group 3) in the longitudinal direction only (FIG. 26). E- beam and SCCO2 sterilization appeared to increase both maximum stress observed at 150% extension and stiffness (Young’s modulus) compared to non-sterilized tissue. The highest level of stress and stiffness were observed in 27 kGy E-beam treated veins which was higher than all other groups. Based on the increased stiffness and higher area under the extension / relaxation stress- strain curve in veins exposed to 27 kGy E-beam, 16.5 kGy E-beam sterilization or SCCO2 sterilization with either 75 or 150 ppm PAA were determined to be preferable. However, a radiation dosage of only 16.5 kGy is less likely to efficiently and reproducibly sterilize the devices, and further work is required to validate that the sterilization process can ultimately achieve a SAL of 10-6 for this device.

[0110] An increase in maximum stress at suture pullout was observed in processed but not sterilized tissue (Group 3) compared to native tissue (FIG. 27), likely due to PGG treatment. Sterilization overall appeared to slightly decrease the maximum stress observed at pull-out and lowered the Young’s modulus / stiffness (in contrast to its impact on biaxial mechanical testing; FIG. 28). SCCO2-treated tissue appeared more elastic and less stiff than E-beam sterilized tissue. Relatively similar mean stresses and high standard deviations prevent any substantial conclusions from being drawn among sterilization treatments.

[0111] Although E-beam treatment has commonly been used for terminal sterilization of other medical devices and therefore is better established and more familiar to the FDA, there is currently no data showing its long-term impact on the pulmonary valved graft performance. Subject matter experts have advocated against using E-beam processing for biological tissues due to its deleterious effects on durability. Based on the data generated in Example 7, a lower E-beam radiation dose (16.5 kGy) would likely be preferred over the higher dose (27 kGy) because it had a less significant impact on mechanical properties of the final device. Unfortunately, there is no data to support that the lower dose achieves the desired sterility and it was suggested that the range of E-beam dosage would have to be expanded to -15-25 kGy for transfer to production.

[0112] Compared to E-beam processing, SCCO2 testing at the two levels of sterilant (75 or 150 ppm PAA) had a less adverse impact on tissue mechanical properties and the treatment method itself was tentatively chosen as more favorable for this product To accurately choose an appropriate sterilant level during SCCO2 processing, microbial analysis / bioburden testing will have to be conducted in addition to evaluating the mechanical and physical properties of the processed device. Although SCCO2 sterilization has been approved by the FDA for instruments and tissue banking, SCCO2 is relatively new in medical device sterilization, and more work is likely required to validate the process for terminal sterilization.

[0113] The purpose of this series of experiments was to determine the processing protocol for a pulmonary valved graft. Based on the improvements implemented from the lessons learned through this series of experiments, the total process has been reduced from 19 days to 8 days of active labor, excluding hold steps (overnights and weekends) and terminal sterilization.

[0114] Major improvements performed include:- Optimization of various in-process rinses for removal of residuals from tissue- Reduction in Decellularization Solution treatment from 144 hours to 24-48 hours- Reduction of 70% EtOH incubation (for removal of residual SDS) from 8 to 4 hours- Reduction in DNase / RNase step processing time from 96±8 hours to 24±2 hours (by increasing working DNase concentration from 1.6 U / mL to 10 U / mL)- Removal of 0 1% PAA treatment step- Introduction of terminal sterilization

[0115] The final Optimized Process Flow chart for Examples 1-7 is outlined in FIG. 37A-D. For the Examples above, the referenced testing steps are detailed in Table 3.Table 3: Test Methods PerformedEXAMPLE 8

[0116] The following example details improved processes for manufacturing decellularized biological scaffolds with physical stabilization for "in situ" tissue engineering applications. The current processing aims to achieve terminal sterilization utilizing Supercritical carbon-dioxide (SCCO2) for PGG stabilized decellularized tissues with low concentrations of Peracetic acid (PAA) (75-100 ppm) as the additive for 60-120 minutes. A 95% bioburden reduction (4-5 logs) was observed in previous studies with these conditions, and the aim of the optimization was to achieve 6 logs of reduction (Sterility Assurance Levels of 10-6; SAL6) of bioburden as currently required by the FDA The optimization work in this example aims at understanding and optimizing the parameters involved in effective sterilization with supercritical carbon-dioxide with additives like hydrogen peroxide (H2O2) and peracetic acid (PAA) to achieve acceptable Sterility Assurance Levels.

[0117] H2O2 and PAA were compared as additives to SCCO2 to achieve the require reduction in bioburden, without altering the mechanical and chemical properties of the PGG treated scaffolds. Initial studies were performed in a small scale, with tissue sections

[0118] Three separate modes of contact of the SCCO2 and additives and the tissues were tested, including:- Saturating supercritical CO2 in additives before contact with wet tissue at constant pressure and constant flow rate of 002 separately.- Adding additive in the chamber at a constant volume ratio during SCCO2 contact with wet tissue at constant pressure of C02.- Adding additive and tissues in a liquid media during the CO2 contact at constant pressure.T able 4 summarizes the methods and additives tested for sterilizing tissues with SCC02.

[0119] Pressure, additive concentrations, and time of SCCO2 contact were varied to obtain the optimum conditions enabling bioburden reduction without changes to the tissue, and excess residual additive Based on the results, PAA added in a liquid media at a constant pressure was selected as an optimum additive with SCC02 to achieve required sterilization. The optimum temperature, pressure, and time of treatments were found to be 35-37»C, 1400-1500 psi, and 120-180 minutes respectively. The PAA concentration used (100-200 PPM) was not found to alter the properties off the tissue. The primary packaging devices for the TxGuard PVG conduit - vented filter caps and Pyrex bottles were tested to optimize the liquid levels and filter type to allow SCCO2 permeability into the containers without overflowing of storage liquid. PGG treated tissue conduits were sterilized at the determined range of PAA concentration (150-200 PPM), pressure, temperature, and contact time in a Nova2200™ unit at Novasterilis, NY. The processed samples were found to be unaltered in their physical and chemical properties, while achieving required reduction in bioburden levels. Table 4 outlines the results of the methods tested, and Table 5 lists the spore counts from the varying PAA concentrations.Table 4: Summary of methods and additives utilized for sterilizing tissues with SCCO2Table 5: Spores recovered from Control and Processed samples in Nova2200 RunEXAMPLE 9

[0120] The following example details tested decellularized PGG treated bovine jugular vein valved conduits (BJV) implantation in sheep. Three sheep were implanted with PGG-treated BJV tricuspid conduits for one month and two sheep for three months. These implants were placed in sheep using a cardiopulmonary bypass to resect the native pulmonary valve leaflets and main pulmonary artery and then to insert the decellularized PGG treated bovine jugular vein valved conduit to replace the resectednative tissues The valves were functional, and no tissue overgrowth or scarring was seen on the leaflets. Von Kossa staining showed no calcification.

[0121] Significant cellularization of the wall of the conduit was found, and no thrombus or calcification development at three months. To identify if the cells are remodeling the conduit matrix, Heat shock protein 47 (HSP-47) IHC was performed HSP-47 is a collagen-specific molecular chaperone that is required for molecular maturation of several types of collagens. It was observed that most of the infiltrated cells were positive for HSP-47, and this suggests they are actively producing collagen (FIG. 33). Alpha-smooth muscle actin (a-SMA) and vimentin staining were also performed. More than 90% of cells were positive for both a-SMA and vimentin, clearly suggesting these were myofibroblasts and / or smooth muscle cells and not inflammatory cells (FIG 33) Von Willebrand factor (VWF) FITS staining showed uniform endothelial covering the luminal side of the conduit (FIG. 33). Most importantly, conduits grew by 10% in three months when outer diameter and length were measured at implant and at explant

[0122] The present disclosure further optimized the decellularization and stabilization method, reducing the production process from 19 to 8 active production days (excluding sterilization) (FIG. 34) with a contract company capable of GMP manufacturing. The process has been optimized with bovine jugular veins to maximize removal of native cellular and nuclear material and minimize residual components from the decellularization process that might have deleterious effects on the mechanical properties of the tissues, or potentially be cytotoxic after implantation. The optimized process has been shown to effectively remove cells from the native tissues, with a final DNA concentration of <50 ng / mg dry weight of decellularized tissues. Bovine Jugular vein conduits developed using the optimized process have shown comparable chemical and mechanical characteristics to those developed previously with the extended treatment. Preclinical studies are in progress for these scaffolds prepared with the optimized process to confirm their functionality These studies include extended mechanical testing (hydrodynamic testing and Accelerated Wear Testing (AWT)) 150-day subcutaneous implantation in rats, and 150-day extended implantation of conduits in sheep as valved pulmonary conduit replacement units.

[0123] The optimized process utilizes a terminal sterilization step with Supercritical CO2 (SCCO2) with low concentrations of peracetic acid (PAA) (75-150 ppm) as the sterilant for 60-120 minutes. This sterilization method has not been used previously for stabilized tissue products and is the first known application for the sterilization of PGG stabilized decellularized tissues. Our studies with the SCCO2 processing have shown no significant alterations in the mechanical or chemical properties of the scaffolds induced by the treatment (FIG. 35). Additionally, a 95% reduction in the bioburden levels tested with controlled contamination has been achieved with the current procedure Optimization is in progress to achieve 6 logs reduction of bioburden as currently required by the FDA.

[0124] Phase II research efforts have also been dedicated towards optimizing final packaging for PGG treated decellularized scaffolds. A terminal sterile package has been designed, utilizing a glass bottle with semi-porous PTFE filter fitted cap (See FIG. 37). The semi-porosity of the cap allows gas during SCCO2 sterilization to diffuse into the bottle with the tissues stored in saline buffer, while maintaining liquid inside the bottle An additional Tyvek header pouch has been investigated as a secondarypackaging to maintain sterility. This packaging has been confirmed to maintain sterility and be capable of aseptic handling in a clinical set-up Eliminating the secondary pouch and incorporating an additional lid with the semi-porous cap may improve the ease of aseptically handling the tissue-product.EXAMPLE 10

[0125] The following example details the manufacture and use of decellularized xenograft valved conduits for pediatric patients. Congenital Heart Disease (CHD) affects approximately 40,000 newborns each year in the US. Out of the new patients found to have CHD each year, an estimated 2500 patients have a defect that requires the use of a substitute, non-native conduit artery to replace structures that are congenitally absent or hypoplastic. Materials in current use for conduit replacement involve glutaraldehyde crosslinked tissues with varying degrees of stiffness and flexibility, durability, calcification, thrombosis, susceptibility to infection, and lack of growth potential. To overcome valve calcification, stenosis, and thrombosis, and to allow conduits to grow with patients, a superior valved conduit can be produced with acellular Bovine valved Jugular Veins (BJV) that are physically crosslinked with Penta Galloyl Glucose (PGG). Such a replacement graft would allow cellular ingrowth of host cells and potentially enable regenerative growth and remodeling of the graft, an essential feature for pediatric patients, thereby reducing the need for reoperations required with current devices

[0126] BJV conduits were decellularized using 0.25% Sodium Dodecyl Sulfate (SDS), 0 5% Sodium Deoxycholate (DOC), 0 5% Triton X100, and 0 2% Ethylene Diamine Tetra-Acetic acid (EDTA), followed by treatment with DNase and RNase solutions. They were treated with PGG to physically stabilize elastin and collagen within the tissue extracellular matrix. DNA analysis was performed using pico-green reagent to quantify the reduction in DNA content of the tissues after the decellularization process. The stability of the PGG crosslinked tissues were analyzed by treatment with elastase and collagenase enzymes. Elastase challenge was also performed on PGG treated tissues stored for up to 24 months to determine the long-term storage potential of these conduits. Conduit wall and leaflet tissues were implanted subdermally in rats to study biocompatibility and calcification PGG treated tricuspid conduits were implanted in sheep, using a cardiopulmonary bypass procedure that replaced the native pulmonary valve leaflets and pulmonary artery, for up to three months to analyze the functionality of the conduits and assess the cellular infiltration as well as growth potential of the grafts.

[0127] Histology and DNA analysis showed that the decellularization protocol removed most of the cells from the scaffolds, with >97% reduction in the DNA content. The PGG treated scaffolds retained the ability to resist degradation without any PGG loss over the period of 24 months. Sheep implant studies showed significant cellularization of the conduit wall and leaflets, with no thrombus or calcification development. Most of the infiltrated cells were positive for HSP-47, suggesting they are actively producing collagen. A 10% increase in the outer diameter and length of the conduits was also observed on explanting after three months.

[0128] BJV conduits were decellularized with >97% reduction in DNA content. PGG treatment of conduits improved their ability to resist rapid degradation by Collagenase and Elastase enzymes for up to 24 months. PGG treated tissues were biocompatible and did not calcify when implanted subdermally in rats. PGG treated conduits implanted in sheep showed cellular infiltration with a potential to grow without any thrombosis or calcification

[0129] While the various systems described above are separate implementations, any of the individual components, mechanisms, or devices, and related features and functionality, within the various system embodiments described in detail above can be incorporated into any of the other system embodiments herein.

[0130] The terms “about” and “substantially,” as used herein, refers to variation that can occur (including in numerical quantity or structure), for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to, mass, volume, time, distance, wave length, frequency, voltage, current, and electromagnetic field. Further, there is certain inadvertent error and variation in the real world that is likely through differences in the manufacture, source, or precision of the components used to make the various components or carry out the methods and the like The terms “about” and “substantially” also encompass these variations The term “about” and “substantially” can include any variation of 5% or 10%, or any amount - including any integer - between 0% and 10%. Further, whether or not modified by the term “about” or “substantially,” the claims include equivalents to the quantities or amounts.

[0131] Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Throughout this disclosure, various aspects of this disclosure are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges, fractions, and individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 3, 4, 5, and 6, and decimals and fractions, for example, 1.2, 3.8, 11 , and 4% This applies regardless of the breadth of the range. Although the various embodiments have been described with reference to preferred implementations, persons skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope thereof.

[0132] Although the various embodiments have been described with reference to preferred implementations, persons skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope thereof.

Claims

ClaimsWhat is claimed is:

1. A method for sterilizing a specimen tissue comprising contacting the specimen tissue to a solution comprising supercritical carbon dioxide and peracetic acid.2 The method of claim 1 wherein the peracetic acid is added to the solution at a constant pressure.

3. The method of claim 2 wherein the constant pressure is between 1300-1600 psi.

4. The method of claim 1 wherein the solution is at a temperature of between 30-40 degrees Celsius.

5. The method of claim 1 wherein the solution and specimen tissue are in contact for a time of 100-200 minutes.

6. The method of claim 1 wherein the specimen tissue is a bovine jugular valve.

7. A method for stabilizing ex vivo or in vitro a tissue sample for use in biological scaffolds for in situ tissue engineering, comprising: a) a first step including contacting the tissue sample with a sodium hydroxide treatment for 45-90 minutes; b) a second step carried out subsequent to the first step, the second step including contacting the tissue sample with a solution comprising a decellularization solution for 18-32 hours; and c) a third step carried out subsequent to the second step, the third step including contacting the tissue sample with a solution comprising pentagalloylglucose for 16-24 hours8. The method of claim 7 wherein a fourth step carried out subsequent to the third step, the fourth step including sterilizing the tissue sample with a radiation dose.

9. The method of claim 7 wherein a fourth step carried out subsequent to the third step, the fourth step including sterilizing the tissue sample with supercritical CO2.

10. The method of claim 7 wherein the final DNA concentration in the jugular valve is less than 50ng / mg dry weight of the tissue.11 . The method of claim 7 wherein the tissue sample is a jugular vein, a pulmonary artery, an aortic valve, a pulmonary valve, pericardium, vena cava, urinary bladder tissue, peritoneum, or fascia.

12. The method from claim 7 wherein the tissue is a jugular vein.

13. The method from claim 12 wherein the jugular vein is a bovine jugular vein.

14. A kit for use in biological scaffolding, the kit comprising a glass bottle with a semi porous cap, wherein the glass bottle contains a solution comprising a sample tissue and 1X PBS.

15. The kit of claim 14 wherein the semi porous cap is a polytetrafluoroethylene polymer filter fitted cap16. The kit of claim 14 wherein the glass bottle is inside a sterile pouch.

17. The kit of claim 14 wherein the sample tissue is a jugular vein, a pulmonary artery, an aortic valve, a pulmonary valve, pericardium, vena cava, urinary bladder tissue, peritoneum, or fascia.

18. The kit of claim 14 wherein the sample tissue is a jugular vein.

19. The kit of claim 18 wherein the jugular vein is a bovine jugular vein.

20. The kit of claim 14 wherein a second cap is overlaid on the semi porous cap.

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