Decellularized Biomaterial and Method for Formation

Abstract

Methods for developing a decellularized tissue and biomaterials for use as biomimetic grafts or in vitro cellular scaffolds formed with the decellularized tissue are described. The biomaterials are particularly well suited for use as an intervertebral disc graft. The decellularized tissue is formed from an intervertebral disc source tissue and can be substantially decellularized and substantially free of potential immunogenic material (e.g., DNA and RNA), while maintaining ECM materials including both glycosaminoglycan and collagen.

Description

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent application Ser. No. 15/758,511, which is the United States national stage entry of International Patent Application No. PCT/US2016/050689, having a filing date of Sep. 8, 2016, which claims filing benefit of U.S. Provisional Patent Application Ser. No. 62/215,475, having a filing date of Sep. 8, 2015, both of which are incorporated herein by reference.

BACKGROUND

Low back pain poses a significant socioeconomic burden with a lifetime prevalence of 84% and estimated U.S. expenditures of $85.9 billion per year. Although the cause of low back pain is difficult to pinpoint, it often originates from degenerating or herniated intervertebral discs. As shown in FIG. 1, an intervertebral disc 12 includes the nucleus pulposus 10 surrounded by the annulus fibrosus 16. A disc 12 forms a cushion between adjacent vertebrae 14 that allows for flexibility and motion and supports compressive loads during activities of daily living. The nucleus pulposus is a highly hydrated tissue composed primarily of type II collagen and the proteoglycan aggrecan. Intervertebral disc degeneration is a multifactorial process that manifests initially as biochemical degradation of the nucleus pulposus 10. Intervertebral disc herniation is commonly the result of an increase in intradiscal pressure due to abrupt loading that exceeds the strength of the restraining annulus fibrosus 16 of the disc 12 leading to extrusion of nucleus pulposus tissue 10 from the disc 12. Both pathologies can result in decreased disc height and irritation of adjacent nerve roots leading to the generation of back and leg pain, as well as limb weakness.

Current therapies for both intervertebral disc degeneration and herniation are palliative and merely delay invasive surgical management in the form of discectomy, spinal fusion, and total disc replacement. While these procedures may temporarily relieve pain, they do not attempt to replace, restore, or regenerate healthy nucleus pulposus tissue. Additionally, there are concerns with the use of surgical methodologies that may promote re-herniation, altered spinal biomechanics, and accelerated degeneration in adjacent discs.

Regenerative medicine approaches to intervertebral disc degeneration repair have been investigated both in vitro and in vivo. For instance, injection of growth factors and stem cells into the nucleus pulposus has been examined, but has demonstrated limited success due short half-life, leakage, and inability to maintain an appropriate phenotype due to the altered tissue extracellular matrix (ECM) microarchitecture.

Tissue engineering strategies combining a supporting scaffold and an alternative healthy cell source (i.e., stem cells) together provide a promising avenue for developing viable nucleus pulposus tissue constructs. However, success of such approaches relies largely upon the development of a biomaterial scaffold that mimics the biochemistry and mechanical properties of the subject and that can function to deliver, protect, and provide instructive cues to stem cells such that they attain the appropriate phenotype and produce nucleus pulposus-specific ECM. Examples of biomaterial scaffolds investigated include pre-formed and injectable hydrogels composed of type II collagen-hyaluronic acid and Crosslinked alginate.

An alternative scaffold formation method includes the decellularization of a source tissue. Decellularization attempts to remove host cells from the source tissue while maintaining intact, tissue-specific ECM. One primary advantage of this approach is that the remaining ECM provides cues that can advantageously affect migration, proliferation, differentiation, and subsequent tissue-specific ECM production by seeded cells. Successful decellularization has proven difficult however, as it requires complete removal of potential immunogenic materials while maintaining suitable levels of desirable ECM components such that function of the scaffold can be maintained. Attempts have been made to form suitable implantable graft materials from xenograft or allograft sources. While some approaches have demonstrated the ability to retain key ECM components in physiologically relevant quantities and ratios, these methods unfortunately left the source cells devitalized but sequestered within the tissue. Others have demonstrated successful removal of cell DNA via disruption of the native ECM, but the resulting scaffold material also showed significant reduction in GAG, which would significantly affect function of the resulting graft material.

What is needed in the art is a biomaterial scaffold that can recapitulate native nucleus pulposus microarchitecture, biochemistry, and mechanical properties, and that can support cells seeded thereon as a route to aiding patients with intervertebral disc degeneration and intervertebral disc herniation.

SUMMARY

According to one embodiment, disclosed is a biomaterial that includes a decellularized intervertebral disc tissue. The decellularized intervertebral disc tissue can be an allograft or xenograft intervertebral disc tissue that has been treated so as to be substantially free of cell nuclei. In addition, the decellularized intervertebral disc tissue can include nucleus pulposus tissue that has a glycosaminoglycan content of about 200 micrograms (μg) or greater per milligram (mg) dry weight of the decellularized nucleus pulposus tissue and can include nucleic acids in an amount of about 50 nanograms (ng) or less per mg dry weight of the decellularized nucleus pulposus tissue. In one embodiment, the decellularized intervertebral disc tissue can include both nucleus pulposus tissue and annulus fibrosus tissue. In another embodiment, the decellularized intervertebral disc tissue can include nucleus pulposus tissue and can be substantially free of annulus fibrosus tissue. In another embodiment, the decellularized intervertebral tissue can include annulus fibrosus tissue and can be substantially free of nucleus pulposus tissue.

Also disclosed is a method for forming a biomaterial. The method can include subjecting a source intervertebral disc tissue, e.g., an allograft or xenograft nucleus pulposus tissue, annulus fibrosus tissue, or an entire intervertebral disc to both chemical and mechanical treatments. For instance, a chemical treatment can include contacting the intervertebral disc tissue with a decellularization solution that includes one or more non-ionic surfactants and a protease inhibitor, optionally in conjunction with additional materials (e.g., antimicrobials, ionic surfactants, etc.). The decellularization solution can be agitated while the intervertebral disc tissue is held in contact with the solution. In addition, the decellularization solution containing the intervertebral disc tissue can be subjected to ultrasonication. Following, the intervertebral disc tissue can be contacted with an enzyme solution, for instance to remove remaining DNA and RNA.

BRIEF DESCRIPTION OF THE FIGURES

A full and enabling disclosure of the present subject matter, including the best mode thereof to one of ordinary skill in the art, is set forth more particularly in the remainder of the specification, including reference to the accompanying figures in which:

FIG. 1 illustrates a typical intervertebral disc and associated vertebrae.

FIG. 2 is a table (Table 1) showing details of 13 different decellularization solutions and processes.

FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D compare the DNA content of fresh bovine nucleus pulposus to tissue decellularized as disclosed herein both quantitatively (FIG. 3A) and qualitatively (FIG. 3B). Glycosaminoglycan (GAG) content of decellularized and fresh tissues are compared at FIG. 3C. Hydroxyproline (HYP) content of decellularized and fresh tissues are shown at FIG. 3D. Horizontal bars connecting groups indicate significant differences (p<0.05).

FIG. 4A through FIG. 4J illustrate several different decellularized tissues (FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4F, FIG. 4G, FIG. 4H, FIG. 4I) and fresh, untreated tissue (FIG. 4E and FIG. 4J) following Alcian blue counterstained with nuclear fast depicting GAG and cell nuclei (FIG. 4A through FIG. 4E are at 200× magnification; FIG. 4F through FIG. 4J are at 400× magnification).

FIG. 5A through FIG. 5H presents histological analysis of treated tissues (FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D) compared to fresh untreated tissue (FIG. 5E, FIG. 5F, FIG. 5G, FIG. 5H).

FIG. 6A presents a macroscopic image of fresh bovine nucleus pulposus (left) and treated tissue (right).

FIG. 6B presents representative unconfined compression stress relaxation curves for fresh bovine tissue at panel B showing relaxation profiles following the sequential application of 8, 12, and 16% strain, respectively.

FIG. 6C presents representative unconfined compression stress relaxation curves for treated bovine tissue at panel B showing relaxation profiles following the sequential application of 8, 12, and 16% strain, respectively.

FIG. 7A presents Young's modulus determined following stress relaxation testing to defined applied strain end-points for treated and fresh tissue.

FIG. 7B presents aggregate modulus determined following stress relaxation testing to defined applied strain end-points for treated and fresh tissue.

FIG. 7C presents shear modulus determined following stress relaxation testing to defined applied strain end-points for treated and fresh tissue.

FIG. 8A through FIG. 8D provide graphical representation of dynamic mechanical analysis of treated tissue (FIG. 8A and FIG. 8B) compared to fresh tissue (FIG. 8C and FIG. 8D) tested to 8% compressive strain.

FIG. 9 presents live/dead staining on scaffolds seeded on the surface with hAMSCs for 3, 7, and 14 days (insets A, B, C, respectively). The graph provides the percent cell viability. Also shown are representative live dead images of hAMSC seeded scaffolds following 3 (A), 7 (B), and 14 (C) days of culture. Representative Alcian blue stained scaffold seeded with hAMSCs (arrow heads) is shown at inset D following 14 days of culture. All images were obtained at 200× total magnification.

FIG. 10A through FIG. 10D present representative live/dead images of non-crosslinked FIG. 10A and FIG. 10C) and EDC/NHS treated (FIG. 10B and FIG. 10D) treated tissue after 3 days (FIG. 10A and FIG. 10B; 200X total magnification) and 14 days (FIG. 10C and FIG. 10D; 100X total magnification), respectively.

FIG. 10E presents a graphical representation of hAMSC viability within non-crosslinked (“ABNP”) and EDC/NHS treated (“crosslinked”) tissue after 3 and 14 days in culture. Horizontal bars connecting groups indicate statistical differences (p<0.05).

FIG. 11 provides representative images of a fresh intact intervertebral disc (left) and a decellularized intact intervertebral disc (right).

FIG. 12A through FIG. 12D illustrate the results of ethidium staining for residual DNA for fresh nucleus pulposus (FIG. 12A) and fresh annulus fibrosus (FIG. 12C) compared to decellularized nucleus pulposus (FIG. 12B) and decellularized annulus fibrosus (FIG. 12D) of intervertebral discs demonstrating the absence of residual DNA following decellularization.

Repeat use of reference characters in the present specification and drawings is intended to represent the same or analogous features or elements of the present invention.

DETAILED DESCRIPTION

Reference will now be made in detail to various embodiments of the disclosed subject matter, one or more examples of which are set forth below. Each embodiment is provided by way of explanation of the subject matter, not limitation thereof. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made in the present disclosure without departing from the scope or spirit of the subject matter. For instance, features illustrated or described as part of one embodiment, may be used in another embodiment to yield a still further embodiment.

In general, the present disclosure is directed to methods for developing a decellularized biomaterial that can be utilized in one embodiment as an implantable graft material and can be particularly suited for use as an intervertebral graft material. In particular, the biomaterial can be substantially decellularized and can be substantially free of potential immunogenic material (e.g., DNA and RNA), while maintaining ECM materials including both glycosaminoglycan and collagen in an amount so as to provide excellent cell supporting and mechanical characteristics as an intervertebral graft.

As used herein, the term “substantially free” means that the presence of a particular component is either not detected using known assays, or if it is detected, it is only present in an amount that is in accordance with the tissue regulations of the U.S. Food and Drug Administration (FDA) as set forth in Title 21 Code of Federal Regulations (CFR), Parts 1270 and 1271, herein incorporated by reference. In various aspects, “substantially free” can include a tissue that has been reduced in the amount of the component by about 90% or more, about 95% or more, about 98% or more, or about 99% or more as compared to the amount of the component by dry weight in natural, untreated tissue. In certain aspects, “substantially free” means that the tissue is completely free of the component.

As used herein “substantially decellularized” means that the tissue has been reduced in the amount of cells by about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 98% or more, or about 99% or more as compared to the amount of cells by dry weight in natural, untreated tissue. In certain aspects, “substantially decellularized” means that the tissue is completely free of cells.

Disclosed methods can produce biomimetic biomaterials through decellularization of source xenogeneic or allogeneic intervertebral disc tissue according to a process that can remove host DNA as well as other potentially immunogenic materials while maintaining native ECM components. In particular, disclosed methods can retain physiologically relevant amounts of glycosaminoglycan (GAG) and collagens, e.g., type II collagen. As such, the mechanical characteristics of the biomaterials, e.g., unconfined static and dynamic compressive mechanical properties, can approach values reported for human intervertebral disc components (e.g., nucleus pulposus). Moreover, cellular scaffolds formed of the biomaterials can maintain viability of cells, e.g., stem cells that can be planted on the biomaterials. Accordingly, the biomaterials can be particularly well suited for use as regenerative intervertebral disc graft materials. However, it should be understood that the biomaterials are not limited to utilization as intervertebral disc graft materials and can be utilized in other applications including as other graft materials (e.g., articular cartilage graft materials, osteochondral graft materials, etc.), as well as for use in in vitro or ex vivo applications. For instance, the biomaterials can be particularly well suited for use as scaffolding materials for supporting growth and development of extrinsic cells, i.e., cells loaded onto the biomaterials following formation such as stem cells, ex vivo differentiated cells, etc., that can include intervertebral disc-derived cells or any other type of cell(s) for study purposes, as well as for implantation/grafting purposes.

According to one embodiment, disclosed treatment methods can remove about 90% or more, e.g., about 93% or more, of the DNA of the source tissue, with no detectable residual base-pairs present. For instance, treated nucleus pulposus tissue can exhibit a total DNA content of about 50 ng or less DNA/mg dry tissue weight, or even lower in some embodiments, such as about 20 ng or less DNA/mg dry tissue weight, or about 10 ng or less DNA/mg dry tissue weight. Treated annulus fibrosus tissue can exhibit a total DNA content of about 70 ng or less DNA/mg dry tissue weight, or even lower in some embodiments, such as about 65 ng or less DNA/mg dry tissue weight or about 50 ng or less DNA/mg dry tissue weight. Moreover, treated tissues can have little or no DNA over about 200 base-pair fragment length, with a lack of visible nuclear material via DAPI or H&E staining and an absence of intact residual cell nuclei and of high and low molecular weight host DNA fragments.

Treatments can also retain glycosaminoglycan (GAG) of the source tissue. For instance, treated nucleus pulposus tissue can retain GAG in an amount of about 200 μg or more of per milligram of nucleus pulposus tissue dry weight or higher in other embodiments, such as about 210 μg or more per mg dry weight, about 220 μg or more per mg dry weight, or about 250 μg per mg dry weight in some embodiments. When considering treatment of annulus fibrosus tissue, the tissue can retain GAG in an amount of about 100 μg or more per mg dry weight, about 115 μg or more per mg dry weight, or about 130 μg per mg dry weight in some embodiments. The retention of this key ECM component, along with collagens, and in particular collagen type II, is critical for the mechanical function of the intervertebral disc tissue, as it creates the osmotic properties and swelling pressures required to support compressive loads and prevent nerve and blood vessel in-growth into the intervertebral disc.

The biomaterials can retain significant amounts of GAG as well as collagen type II from the source tissue. For instance, treated tissue can retain a hydroxyproline (HYP) content (as measure of type II collagen content) such that the treated tissue has a GAG:HYP ratio of about 15:1 or higher; for instance, from about 10:1 to about 25:1 in some embodiments. The biomaterials can retain significant levels of other collagens (e.g., collagen type IX, collagen type XI, etc.) that can improve the function of the biomaterials.

Without wishing to be bound to any particular theory, it is believed that the relatively high quantities of ECM materials retained in the biomaterials may support earlier tissue regeneration in grafting applications as less ECM would need to be produced by seeded cells in order to achieve proper mechanical function of the tissue. As such, the biomaterials can be ideally suited for use as intervertebral graft materials in lumbar as well as cervical applications; for instance, as a surrogate to regenerate nucleus pulposus tissue and/or annulus fibrosus tissue that has succumbed to degeneration or extrusion in patients with intervertebral disc degeneration or intervertebral disc herniation.

The biomaterials can also exhibit viscoelastic properties that can mimic or re-establish the native properties of the host materials, e.g., the human nucleus pulposus and/or annulus fibrosus that it can augment or replace. For instance, the biomaterials can exhibit a storage modulus of about 6 kilopascals (kPa) or greater; for instance, from about 6 kPa to about 15 kPa in some embodiments. A biomaterial can exhibit a loss modulus of about 1 kPa or greater; for instance, from about 1.5 kPa to about 5 kPa in some embodiments. A biomaterial can exhibit a complex modulus of about 6 kPa or greater; for instance, from about 6 kPa to about 15 kPa or greater, and can exhibit a phase angle of about 15° or greater; for instance, from about 15° to about 27° in some embodiments.

The biomaterials can exhibit mechanical properties that approach healthy human intervertebral disc tissue values, even in the absence of cell seeding, suggesting that the biomaterials may can function mechanically immediately upon implantation. Moreover, the viscoelastic properties can improve upon cell seeding and development of additional ECM by the transplanted cells.

In general, the source tissue material for development of the decellularized biomaterial can be obtained from any xenogeneic or allogeneic source including, without limitation, porcine, bovine, human cadaver, etc. In one embodiment, the source tissue can be bovine intervertebral disc tissue, and in one particular embodiment, caudal (tail) intervertebral disc tissue. Bovine caudal source tissue may be beneficial in some embodiments due to the similarity in size (e.g., height-to-diameter ratio) to human lumbar intervertebral disc tissue, as well as due to similarities in resting stress and biochemistry to native human intervertebral disc tissue.

The source tissue can be any portion or all of an intervertebral disc. For instance, in one embodiment, the source tissue can be essentially limited to nucleus pulposus tissue and absent of annulus fibrosus or other disc tissue. However, the disclosure is not limited to only nucleus pulposus source tissue. In other embodiments, a source tissue can include all or part of a nucleus pulposus in conjunction with other tissue of the source intervertebral disc, such as all or part of the adjoining annulus fibrosus. For instance, in one embodiment, an entire intervertebral disc, including both the annulus fibrosus and the nucleus pulposus, can be treated. Alternatively, the source tissue can be limited to annulus fibrosus tissue and all or a part of the annulus fibrosus can be decellularized, essentially absent of any nucleus pulposus or other disc tissue.

To form the biomaterial, the source tissue material (e.g., bovine caudal nucleus pulposus) can be treated according to a combination of chemical and mechanical treatments. The chemical treatment can include contacting the source tissue with a decellularization solution that includes at least one non-ionic surfactant, optionally in conjunction with one or more additional surfactants.

Optionally, prior to contact between the source tissue and the decellularization solution, the process can include an ethanol pre-wash, which can dehydrate the source tissue and encourage uptake of the components (and, in particular, the surfactant(s)) of the decellularization solution into the interstice of the tissue. For instance, the source tissue can be immersed in an ethanol solution (e.g., a 70% ethanol solution) for a period of time of about 15 minutes or more; for instance, from about 30 minutes to about 60 minutes in some embodiments, optionally in conjunction with agitation of the mixture. Inclusion of an ethanol pre-wash may be particularly beneficial in those embodiments in which the decellularization solution includes fewer and/or lower concentrations of surfactants.

The decellularization solution can include at least one non-ionic surfactant. Examples of non-ionic surfactants can include, without limitation, a polyethylene oxide-based surfactant or a polysorbate-based surfactant such as Triton™ X-100 (t-octylphenoxypolyethoxyethanol), Tween® 20 (polysorbate 20), Tween® 80 (polysorbate 80), Igepal® CA630 (ethoxylated nonylphenol), etc. In general, the decellularization solution can include a non-ionic surfactant in an amount of about 0.5 v/v %; for instance, from about 0.5 v/v % to about 2 v/v % in some embodiments. In one embodiment, the decellularization solution can include the non-ionic surfactant in a higher concentration than has been utilized in the past, particularly in those embodiments in which an ethanol pre-wash is not utilized, as discussed above. For instance, in some embodiments, the decellularization solution can include one or more non-ionic surfactants in an amount of about 1 v/v % or greater.

The decellularization solution can include additional surfactants in conjunction with one or more non-ionic surfactants. For example, a decellularization solution can include one or more anionic, zwitterionic, and/or cationic surfactants in addition to the non-ionic surfactant. Examples of anionic, zwitterionic, and cationic surfactants can include, without limitation, Triton™ X-200, sodium deoxycholate, CHAPS, sodium dodecyl sulfate (SDS), N-lauroyl-sarcosinate, Sulfobetain-10 and Sulfobetain-16. When included, an ionic surfactant can generally be present in the decellularization solution in an amount of from about 0.5 w/v % to about 2 w/v %. It should be understood, however, that the presence of ionic surfactants are not required in the decellularization solution. In fact, it has been surprisingly found that certain ionic surfactants, such as SDS, that have been considered to be necessary in the past for successful decellularization are not necessary in the disclosed decellularization solutions and can be omitted.

In addition to the surfactant(s), the decellularization solution can include one or more protease inhibitors. Protease inhibitors can be included in the solution to prevent degradation of the extracellular matrix. Collagen-based connective tissues such as nucleus pulposus contain proteases and collagenases as endogenous enzymes in the extracellular protein matrix. Additionally, certain cell types that may be present in the source tissue, including smooth muscle cells, fibroblasts, and endothelial cells, contain a number of these enzymes inside the lysosomes. When these cells are damaged during the decellularization process, the lysosomes are ruptured and their contents released. As a result, the extracellular matrix can undergo severe damage from protein, proteoglycan, and collagen breakdown. As such, the decellularization solution can include one or more protease inhibitors. Suitable protease inhibitors can include, without limitation, N-ethylmaleimide (NEM), phenylmethylsulfonylfluoride (PMSF), ethylenediamine tetraacetic acid (EDTA), ethylene glycol-bis-(2-aminoethyl(ether)NNN′N′-tetraacetic acid, ammonium chloride, elevated pH, aprotinin, and leupeptin. In general, the decellularization solution can include a protease inhibitor in an amount of from about 0.1 w/v % to about 1 w/v %; for instance, from about 0.2 w/v % to about 0.6 w/v %.

The decellularization solution can include additional components as are generally known in the art including, without limitation, one or more salts (e.g., KCl, NaCl), one or more organic or inorganic buffers, one or more antibiotics/antimycotics (e.g., penicillin, vancomycin, streptomycin, gentamycin, kanamycin, neomycin, sodium azide (NaN₃)) with or without anti-fungal agents (e.g., Amphotericin B, Nystatin)), etc.

The tissue can be held in the decellularization solution with agitation so as to encourage contact between the tissue and the solution components. Periodically, e.g., every 12-24 hours, the solution can be changed out for fresh solution.

In conjunction with contact (e.g., immersion) of the tissue with the decellularization solution, the solution can be periodically subjected to ultrasonication for a period of time, generally from about 5 minutes to about 60 minutes; for instance, from about 10 minutes to about 30 minutes in some embodiments. The ultrasonication can generally be at a power level of about 10 W or greater; for instance, about 50 W or greater, or about 100 W or greater; for instance, from about 10 W to about 1000 W in some embodiments.

The frequency of the ultrasonication can generally be about 20 kHz or greater, for instance about 50 kHz or greater, or about 100 kHz or greater in some embodiments, such as from about 20 kHz to about 200 kHz. By way of example, a suitable ultrasonication condition may be from about 50 W to about 100 W at about 20 kHz to about 50 kHz for about 10 minutes.

The ultrasonication can be carried out periodically over the course of a treatment regimen. For instance, ultrasonication can be carried out once every 12-24 hours. In one embodiment, the ultrasonication can be carried out after contacting the tissue with fresh decellularization solution. For instance, following any pretreatment, a nucleus pulposus source tissue can be immersed in a decellularization solution and subjected to a sonication treatment. Following a period of ultrasonication, the tissue can be agitated in the decellularization solution for a period of time (e.g., 12-24 hours). Following a contact period under agitation, the tissue can be placed in a fresh decellularization solution and the process can be repeated (ultrasonication followed by contact under agitation). The total treatment time can generally be from about 1 day to about 7 days in order to obtain essentially complete decellularization; for instance, from about 2 days to about 5 days in some embodiments.

Following this portion of the decellularization treatment, the tissue can be contacted with an enzyme solution to produce an enzyme-treated biomaterial. A nuclease enzyme can generally be used to breakdown any remaining nucleic and ribonucleic acids. Enzymes can include nucleases such as endonucleases (e.g., DNAse, RNAse, Benzonase®). An enzyme treatment can generally be carried out according to standard procedures as are known in the art.

Additional treatments can be optionally carried out to modify the physical characteristics of the biomaterials. For example, in one embodiment, biomaterial can be crosslinked with collagen and/or elastin crosslinking agents. Crosslinking can be utilized to affect multiple characteristics of a biomaterial. For example, the level of crosslinking can influence the porosity and various strength characteristics of the biomaterial. Crosslinking of the biomaterial can also be utilized to control the degradation characteristics of the material following implantation. Degradation of collagen is a naturally occurring phenomenon prevalent in intervertebral disc pathology. Collagen is rapidly broken down by collagenases, e.g., matrix metalloproteinases (MMPs), produced in the area. Accordingly, crosslinking of a biomaterial can be of benefit to slow the natural degradation processes of the material, particularly when considering the biomaterials for use as a nucleus pulposus graft material.

Any suitable crosslinking agent can be utilized. For example, collagen fixatives such a glutaraldehyde, carbodiimide, polyepoxides, etc. and/or elastin fixatives including polyphenolic compounds (tannic acid, pentagalloyl glucose, etc.) and the like can be utilized to crosslink the structural proteins of the multi-layer construct.

The decellularization procedure can effectively and efficiently be applied to intervertebral disc source tissue to provide a mechanically competent biomimetic biomaterial capable of supporting stem cell viability. Beneficially, the biomaterials may be used in one embodiment in conjunction with stem cells for regenerative medicine approaches in treatment of intervertebral disc degeneration and intervertebral disc herniation in order to mitigate progressive dysfunction of the intervertebral disc.

The present disclosure may be better understood with reference to the Examples set forth below.

Example 1

Tissue Harvest

Bovine tails were harvested from 2-3 years old cows at a local abattoir and were transported on wet ice to the lab. Betadine® was applied to the exterior of the tail to remove bacteria. Excess fascia and muscle were removed from the tails with a scalpel; the location of intervertebral discs was visually confirmed prior to excision immediately adjacent to the endplates via bone cutters. An 8 mm biopsy punch was used to remove nucleus pulposus tissue from the center of the intervertebral disc. Fresh nucleus pulposus samples were stored at −20° C. and served as controls for experiments. Samples for decellularization were immediately placed either in ethanol or various decellularization solutions.

Decellularization Methods

Thirteen different decellularization treatment methods were examined as summarized in the table presented in FIG. 2. The methods differed by chemical decellularization solution, number of ultrasonication (40 kHz) treatments, total treatment duration, and inclusion of ethanol pre-treatment. All decellularization solutions (except sample no. 12) were made in 50 mM Tris buffer (pH 7.5) containing 2% antibiotic/antimycotic. Ten tissue samples (10 samples/100 mL of each solution) were placed in their respective solutions and ultrasonicated for 10 minutes prior to undergoing constant agitation (150 RPM) on an orbital shaker at room temperature. Every 24 hours thereafter decellularization solutions were changed for fresh solution and samples were ultrasonicated for an additional 10 minutes. This process was repeated for a total of either 3 or 6 days. At the completion of the process, all samples were rinsed in sequential changes of distilled water, 70% ethanol, and distilled water again, each for 30 minutes under orbital agitation (150 RPM) at room temperature prior to undergoing nuclease treatment at 37° C. for 48 hours. All nuclease solutions were made in 1×PBS (pH 7.5) containing 5 mM magnesium chloride. Following nuclease treatment, all samples were thoroughly rinsed in distilled water for one hour under agitation.

Decellularization Efficacy Screening

The efficacy of each treatment method was initially screened via quantitative biochemical assays used to measure residual bovine DNA and glycosaminoglycan (GAG) content via PicoGreen® (Thermo Fisher™ Scientific) and DMMB assays, respectively. Additionally, histological analyses were performed on OCT-embedded cryosections to detect the presence of residual bovine cell nuclei via Alcian blue counterstained with nuclear fast red. Acceptance criteria were established a priori to indicate successful decellularization and included: 1) an average residual DNA content of less than or equal to 50 ng DNA/mg dry tissue; 2) an average residual GAG content of greater than or equal to 200 μg GAG/mg dry tissue; and 3) no histological evidence of intact cell nuclei within the de-celled NP tissue. All samples that met the acceptance criteria were further evaluated for residual DNA via agarose gel electrophoresis.

DMMB Analysis for GAG

Fresh (n=6) and treated tissue samples (n=6/treatment) were frozen for 24 hours at −80° C. prior to lyophilization. Sample dry weights were recorded for data normalization prior to complete digestion in PBE buffer containing papain (5 mM L-cysteine, 100 mM dibasic phosphate buffer, 5 mM EDTA, 12.50 μg/mL papain, pH 7.5) for 24 hours at 65° C. Fresh and treated samples were diluted by 1000× and 100×, respectively, in PBE buffer prior to combining 50 μL of each sample with 200 μL of DMMB reagent (40 mM NaCl, 40 mM Glycine, 46 μM DMMB, pH 3.0) in a 96-well plate. Absorbance was read at 525 nm and GAG content was determined from a standard curve developed from known concentrations of chondroitin-6-sulfate.

PicoGreen® Analysis for dsDNA

Total double stranded DNA content of fresh (n=6) and treated tissue samples (n=6/decell treatment) were quantified using a PicoGreen® assay according to manufacturer's instructions. Briefly, papain-digested samples were thawed and diluted 2× in a 1× TE buffer prior to combining 100 μL of sample with 100 μL of PicoGreen® reagent in a black-walled 96-well plate. Fluorescence was detected using excitation and emission wavelengths of 480 nm and 520 nm, respectively. DNA content was determined from a standard curve developed from known concentrations of A DNA supplied by the assay manufacturer.

Agarose Gel Electrophoresis

DNA was purified from aliquots of papain digested samples of both fresh (n=3) and treated tissue samples (n=3/treatment) using a Qiagen DNeasy® Blood & Tissue extraction kit according to manufacturer's instructions. A total of 30 μl of purified DNA sample was loaded into each well of an agarose gel containing ethidium bromide. Two concentrations of agarose gels were used in order to detect high and low molecular weight ranges of residual DNA. A wide range DNA ladder (300-24,000 bp, exACTGene® 24 kb) was ran on a 1% agarose gel at 100V for 60 minutes. A low range DNA ladder (10-300 bp, O'GeneRuler™ Low Range DNA) was run on a 5% agarose gel at 75V for 60 minutes. Gels were imaged in a bio-imager (Bio-Rad) using an ethidium bromide filter to detect the presence of DNA bands. All samples were run against a DNA standard. Decellularization treatment no. 13 was selected as an example treatment protocol that provided a treated tissue devoid of intact cell nuclei and residual DNA while maintaining an average GAG content of 200 μg/mg dry sample. This treated biomaterial scaffold material is hereafter referred to as the acellular bovine nucleus pulposus (ABNP) scaffold, and was further characterized as follows:

Water Content Analysis of ABNP Scaffold

Percent water content was determined by the following equation: [(W_wet−W_dry)/W_wet]×100, where W_wet was sample wet weights and W_dry was the sample dry weight determined following lyophilization.

Hydroxyproline Analysis of ABNP Scaffold

Collagen content of fresh bovine NP (n=3) and ABNP scaffolds (n=6) were quantified using a hydroxyproline (HYP) assay according to the manufacturer's instructions (MAK008, Sigma-Aldrich). Hydrolyzed samples from both fresh and ABNP scaffolds were diluted 100× in 12M HCl and 10 μL of each sample were analyzed at 550 nm. HYP content was calculated using a standard curve developed from serial dilutions of a known concentration of HYP. HYP Values were normalized to sample dry weight.

Histology and Immunohistochemistry (IHC) of ABNP Scaffold

Samples of fresh bovine NP and ABNP scaffolds were fixed in 10% neutral buffered formalin for 24 hours. Fixed samples were then washed in 1×PBS with 0.01% sodium azide for 30 minutes and then soaked in a 15% sucrose solution at 4° C. overnight. Subsequently, samples were placed in a 30% sucrose solution for 2 hours at 4° C., then placed in a 50:50 30% sucrose and OCT solution for 2 hours at room temperature. Finally, samples were placed in OCT for 2 hours, then transferred to OCT molds for cryosectioning at −20° C. until solid. Cryosections of 8 μm thickness were collected on positively charged slides and stored at −20° C. until staining. All imaging was performed using a ZEISS AxioVert.A1 inverted microscope with AxioVision software.

Immunohistochemistry for Type II Collagen

Frozen sections of fresh bovine NP (n=3) and ABNP scaffolds (n=3) were fixed for 20 minutes in cold acetone. Slides were rinsed twice in TBS for 5 minutes, permeabilized in 0.025% Triton X-100, non-specific binding and endogenous peroxidases were blocked with normal serum and a solution of 0.3% hydrogen peroxide in 0.3% normal serum, respectively. A rabbit polyclonal antibody towards bovine type II collagen (ab78482, Abcam; 1:50 dilution) was incubated overnight at 4° C. prior to thorough rinsing and incubation at room temperature for 30 minutes with a secondary biotinylated antibody and avidin biotin complex according to manufacturer's instructions (VECTASTAIN® ABC Elite® Kit Rabbit IgG, Vector Labs). A DAB substrate kit (SK4100, Vector Labs) was used to visualize positive staining according to manufacturer's instructions prior to counterstaining with a dilute hematoxylin solution for 30 seconds. To account for non-specific binding of the secondary antibody, select samples did not receive primary antibody. Additional positive and negative controls for type II collagen staining included chondrogenically-induced hAMSC cell pellets and decellularized porcine pericardium, respectively (data not shown).

Alcian Blue and Nuclear Fast Red Histology Staining

Frozen sections of fresh bovine NP (n=6) and ABNP scaffolds (n=6) were rinsed in tap water prior to being placed in 3% acetic acid for 1 minute followed by a 1% Alcian blue in 3% acetic acid (pH 2.5) solution for 3 minutes. Slides were rinsed in tap water for 1 minute and counterstained in 0.1% nuclear fast red for 2 minutes.

DAPI Nuclear Staining

Frozen slides of fresh bovine NP (n=3) and ABNP scaffolds (n=3) were rinsed in 1×PBS for 1 minute and placed in a 300 mM DAPI solution for 5 minutes in the dark. Slides were washed in PBS and then immediately transported for imaging under a fluorescent microscope fitted with a 460 nm filter.

Ethidium Bromide DNA Staining

Samples of fresh bovine NP (n=3) and ABNP scaffolds (n=3) were placed in a solution of 2 mM ethidium bromide in PBS for 30 minutes. Tissues were then thinly sectioned using a scalpel blade and imaged under a fluorescent microscope.

Sample Preparation and Testing Conditions

Fresh and ABNP samples were frozen at −20° C., embedded in OCT and sectioned to a thickness of approximately 6 mm for all mechanical testing. Prior to removing samples from OCT, a 6 mm diameter biopsy punch was used to ensure consistent sample diameters, which resulted in a 1:1 diameter to height ratio. Subsequently, all samples (both fresh and ABNP) were allowed to thaw and free swell in the testing tank (which contained a solution of 1×PBS and 1% protease inhibitor) for at least one hour at room temperature (25° C.). All samples were tested while submerged in a solution of 1×PBS and protease inhibitor using a Bose® ElectroForce® 3200 Series mechanical test frame equipped with a 1000 g load cell fitted with a testing tank according to standard practice.

Unconfined Compression

Unconfined compressive stress relaxation experiments were performed on fresh (n=5) and ABNP scaffolds (n=5) to determine the compressive properties of the specimens once equilibrium relaxation was attained. Briefly, a compression platen was brought in contact with a stationary testing platform and the displacement measurement was zeroed prior to pre-loading. Pre-loading consisted of compressing the specimens in displacement control until a 0.05N compressive load was achieved (which resulted in approximately 12% total strain). Platen displacement was held constant for 20 minutes allowing for the sample to equilibrate. Sample heights were determined at the end of pre-loading based on the initial zeroed displacement by the test software. Subsequently, stress relaxation testing was conducted which consisted of subjecting the preconditioned samples to a final strain of 16% (based on sample heights determined after preconditioning) using 4% strain increments (starting at 8%) held for 20 minutes each until the change in load was less than 0.025N/minute. Data was collected in WinTest 7 using level crossing with each change of 0.025N being recorded. NIH's ImageJ software was used to measure and confirm sample height and diameter at the applied peak strain and at equilibrium. Intrinsic equilibrium Young's modulus of the solid matrix was determined through the equilibrium response during stress relaxation at 8%, 12%, and 16% applied strains by dividing the equilibrium stress by the respective applied strain. Percent relaxation was determined at each applied strain using the following equation: [(σ_p−σ_e)/σ_p]×100, where σ_p=peak stress and σ_e=equilibrium stress. Poisson's ratio (u), shear modulus (μ), and aggregate modulus (HA) were determined assuming isotropic, homogeneous biphasic theory for cartilaginous materials as previously reported. Briefly, Poisson's ratio was measured directly from unconfined compression testing using an optical method. Briefly, direct lateral images of specimens were obtained at equilibrium following compression to 8 and 12% strain, respectively. Images of each specimen were obtained using a digital camera and a stationary metric calibrator located inside the testing tank. ImageJ software was used in conjunction with the metric calibrator in order to determine equilibrium lateral expansion of the specimen immediately adjacent to the lower testing platform. Shear and aggregate moduli were determined through their relationships with both intrinsic equilibrium Young's modulus and Poisson's ratio via the following equations, respectively:

$\mu =\frac{E}{2\left(1+v\right)}$ $H_A=\frac{E\left(1-v\right)}{\left(1+v\right)\left(1-2v\right)}$

Unconfined Dynamic Mechanical Analysis (DMA)

DMA testing was used to determine the complex, storage, and loss modulus, as well as phase angle of a second set of fresh bovine NP (n=6) and ABNP scaffolds (n=6). Similar to unconfined compression testing, samples were pre-loaded to 0.05N under displacement control and were then allowed to equilibrate for 20 minutes with the platen displacement being held constant. The height and width of each sample was confirmed from a direct lateral digital image taken at the end of the preconditioning period. Height data was input into the DMA testing software within WinTest 7. Samples were then preconditioned to 5% strain for 10 cycles prior to initiation of the test program, which was set to apply a cyclic strain of 8% (based on initial sample height) at a frequency of 0.01, 0.1, 1, and 10 Hz with a 20-minute relaxation between each test frequency.

ABNP Scaffold Cytotoxicity

Scaffolds were sterilized in 0.01% peracetic acid followed by washing three times for 15 minutes each in 1× sterile PBS. Scaffolds were then placed in a solution containing 50% FBS, 48% DMEM, and 2% AB/AM for 24 hours. Human amniotic mesenchymal stem cells (hAMSCs; passage 2) were obtained from consenting patients immediately following the delivery via elective cesarean section of full-term babies. Stem cells were isolated within 4 hours of delivery. Stem cells were seeded via drop-wise addition to the surface of the scaffolds at a density of 2,000 hAMSCs/mm². Seeded scaffolds were incubated at 37° C. for 3 hours to allow time for cells to attached prior to the addition of media (DMEM containing 10% FBS and 1% Ab/Am). Media was refreshed every 3 days. On days 3, 7, and 14, a live/dead assay was performed (n=3 per time point) was performed according to manufacturer's instructions (Biotium). ABNP scaffolds were sectioned using a scalpel blade and imaged under a fluorescent microscope. Three representative images of both live and dead staining were taken for each scaffold. Quantification of cells was performed by manually counting live and dead cells in each image. Percent viability was determined by dividing the number of live cells by the total number of cells.

ABNP Scaffold Crosslinking for Stem Cell Injection Studies

ABNP scaffolds were crosslinked in a solution of 30 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCL (EDC)/6 mM n-hydroxysuccinimide (NHS) in 50 mM beta-morpholinoethansulfonurea hydrate (MES) buffer (pH 5.5) for 30 minutes. Scaffolds were subsequently washed three times for 15 minutes each, sterilized in 0.1% peracetic acid, and washed again three times at 15 minutes each in 1× sterile PBS; they were then placed in a solution of 50% FBS, 48% DMEM, and 2% AB/AM for 24 hours prior to cell seeding/injection.

ABNP Scaffold Cell Injection

Injection of hAMSCs into ABNP scaffolds was investigated due to the observation that hAMSCs were not able to infiltrate the scaffold after 14 days of culture following seeding on the scaffold surface. Thus, a pilot study was initiated to determine if hAMSCs could be injected into the ABNP scaffolds. Preliminary studies were performed to confirm appropriate needle bore size for injection (i.e., what was the smallest bore needle diameter that could be used to inject stem cells without killing them due to shearing). A 28G needle was selected based on the results of the preliminary hAMSC viability studies after injection through various syringe needle diameters (data not shown). Scaffolds were injected with 150 μL of hAMSC suspension (4×10⁶ cells/mL). Cell-injected scaffolds were cultured for 3 and 14 days, and cell viability was assessed using live/dead staining and fluorescent microscopy.

Statistical Analysis

Results are represented as a mean±standard error of the mean (SEM). For quantitative biochemical data obtained during decellularization screening studies, viability comparisons and mechanical data comparing across strain levels within each study group (ABNP vs Fresh) were obtained by utilizing a one-way analysis of variance (ANOVA) using a Tukey's honest significant difference post hoc analysis for all pairwise comparisons. Mechanical data was also analyzed via a student's two-tailed t-test, assuming equal variance in order to determine if differences between study groups (i.e., ABNP vs Fresh) existed within each applied strain (for stress relaxation testing) or test frequency (for DMA testing), respectively. Significance was defined in all cases as p<0.05.

Results

The effectiveness of decellularization procedures was initially screened by evaluating residual bovine DNA concomitant with the retention of GAG. Optimal decellularization procedures were defined as those that resulted in a tissue that contained less than 50 ng/mg DNA and maintained at least 200 μg/mg of GAG. Three decellularization procedures were identified based on these criteria: treatment nos. 7, 10, and 13, respectively (FIG. 2).

Evaluation of residual double-stranded bovine DNA demonstrated significant reductions (p<0.01) in treatment nos. 7, 10, & 13 as compared to fresh tissue. These treatment groups contained 33.61±9.49 ng, 33.08±11.57 ng, and 9.07±1.95 ng DNA per mg sample dry weight, respectively (FIG. 3 at A). Interestingly, samples treated with SDS, a method commonly employed for tissue decellularization, did not show statistical reductions in DNA content in treated tissue (71.31±30.59 ng DNA per mg dry weight). Compared to fresh bovine NP tissue (125.45±11.81 ng DNA per mg sample dry weight), there was a 73.2±7.56%, 73.63±9.22%, & 92.77±1.54% reduction in DNA content for nos. 7, 10, and 13, respectively.

No residual DNA fragments between 10-24,000 base pairs were detected in sample nos. 10 and 13, while residual fragments (indicated by light bands) appeared in the range of 300 bp for samples in treatment group 7 (FIG. 3 at B).

GAG quantification of decellularized tissue demonstrated the retention of 216.98±31.06 μg, 208.93±24.96 μg, and 205.5±16.00 μg GAG per mg sample dry weight using methods 7, 10, and 13 respectively (FIG. 3 at C). Expectedly, these values were statistically lower than values obtained for fresh tissue (677.66±93.22 μg GAG per mg sample dry weight) (FIG. 3 at C). Decellularization resulted in a reduction in average GAG content of 67.98±4.58%, 69.17±3.68%, & 69.68±2.36% in treatment nos. 7, 10, and 13 respectively. These findings were corroborated by Alcian blue staining (FIG. 4) which illustrated that the ECM in all decellularization samples was stained less intensely blue, thus indicating a reduction in GAG and a disrupted microarchitecture compared to fresh tissue sample. In FIG. 4, panels A and F illustrate sample no. 7, panels B and G illustrate sample no. 10, panels C and H illustrate sample no. 12, panels D and I illustrate sample no. 13, and panels E and J illustrate fresh, untreated tissue. The top panels in FIG. 4 are at 200× magnification and the lower panels are at 400× magnification. Further biochemical, histological and mechanical analysis was performed on samples subjected to decellularization method no. 13, and hereafter, these samples were termed the “acellular bovine nucleus pulposus” (ABNP) scaffold.

ABNP scaffolds had significantly higher HYP content per dry mass when compared to fresh tissue (p<0.05) (FIG. 3 at D). More specifically, ABNP scaffolds contained nearly double the average amount of HYP as compared to fresh tissue (13.87±1.14 μg HYP/mg sample dry weight and 8.63±0.24 μg HYP/mg sample dry weight, respectively). Additionally, the GAG-to-HYP ratio was calculated to be approximately 15:1 for ABNP sample, whereas fresh bovine NP tissue had an apparent ratio of 79:1.

IHC for collagen type II confirmed the presence of this ECM protein within both ABNP scaffolds and fresh tissue. Histological images (FIG. 5) illustrated intense brown (positive) staining in both the ABNP tissues (top panels A-D) and the fresh untreated tissue (panels E-H) throughout the ECM and pericellular regions as compared to negative controls. Immunohistochemistry for collagen type II is shown at panels A and E. Alcian blue counterstained with nuclear fast red depicting GAG and cell nuclei is shown at panels B and F. Black circles in panels B and F indicate empty lacunae; black arrowheads in fresh tissue images indicate cell nuclei. Ethidium bromide staining is shown at C and G indicating a lack of DNA fragments in the treated as compared to fresh tissue. DAPI staining is shown at D and H indicating a lack of nuclei in treated as compared to fresh tissue (arrow heads point to intact cell nuclei).

As can be seen in FIG. 6 (top panel) macroscopically, both fresh tissue (left) and the ABNP scaffolds (right) maintained their firm shape. Fresh tissue and ABNP exhibited an average water content of 76.58±1.47% and 94.56±0.37%, respectively. Qualitative histology of the fresh tissue depicted the presence of cells in clusters/islands. The fresh tissue also stained more intensely with Alcian blue (indicating the presence of GAG within the ECM) as illustrated by a deeper blue staining as compared to ABNP scaffolds (panels B and F, FIG. 5), which corroborated DMMB results. Additionally, empty lacunae were visible throughout the ECM of the ABNP scaffolds and ethidium bromide staining confirmed the absence of nucleic acids in ABNP samples (FIG. 5, panel D). However, nuclear material was present in the fresh tissue, as indicated by positive DAPI staining (FIG. 5 panel H).

All mechanical testing results, with the exception of Poisson's ratio, were determined with respect to 8%, 12%, and 16% applied strain. Mean unconfined intrinsic equilibrium Young's modulus of ABNP scaffolds (10.13±1.58 kPa, 7.59±1.00 kPa and 5.85±0.90 kPa) were significantly different at only the 8% strain increment (p<0.05) compared to fresh tissue scaffolds (42.18±13.36 kPa, 29.87±9.79 kPa and 24.76±8.65 kPa) (FIG. 7, panel A). Percent relaxation of ABNP scaffolds (91.86±1.49%, 83.56±2.78%, and 84.67±2.74%) was not significantly different compared to fresh tissue (86.82±3.72%, 77.20±4.02%, and 77.75±4.10%) (FIG. 6, panel B and C, respectively). Apparently, Poisson's ratio from 8-12% strain of ABNP scaffolds (0.142±0.026) was not significantly different compared to values obtained for fresh bovine NP (0.126±0.059). Shear modulus (FIG. 7, panel C) (4.44±0.69 kPa, 3.33±0.44 kPa, and 2.56±0.40 kPa) and aggregate modulus (FIG. 7, panel B) (10.62±1.66 kPa, 7.97±1.05 kPa, and 6.14±0.95 kPa) values for ABNP scaffolds were significantly different at only the 8% strain increment (p<0.05) compared to fresh tissue scaffolds (18.73±5.93 kPa, 13.26±4.35 kPa, and 11.00±3.84 kPa) and (43.77±13.86 kPa, 30.99±10.15 kPa, and 25.69±8.98 kPa), respectively.

Complex, storage, and loss moduli, as well as the phase angle, were evaluated across a range of frequencies using DMA (Table 2). No statistical differences were observed between the two groups at any of the frequencies tested. Moduli values for each study group demonstrated a test frequency dependent relationship. The range of values observed for ABNP scaffold for the complex, storage, and loss moduli (FIG. 8 panel A) and phase angle (FIG. 8 panel B) were 6.59-13.52 kPa, 6.35-12.11 kPa, 1.68-4.80 kPa, and 14.99-26.44°, respectively over the frequencies tested. Fresh tissue values were found within the ranges of 6.36-13.99 kPa, 5.99-13.13 kPa, 2.12-4.55 kPa (FIG. 8, panel C), and 15.36-20.16° (FIG. 8, panel D) respectively.

TABLE 2
Complex Modulus (kPa)
	Frequency (Hz)
	0.01	0.1	1	10
ABNP	6.59 ± 1.64	8.02 ± 1.96	9.96 ± 2.22	13.52 ± 2.82
Fresh	6.36 ± 1.13	8.56 ± 1.40	10.92 ± 1.59	13.99 ± 2.00
	Frequency
	0.01	0.1	1	10
Storage Modulus (kPa)
ABNP	6.35 ± 1.62	7.85 ± 1.92	10.25 ± 2.65	12.11 ± 2.85
Fresh	5.99 ± 1.11	8.23 ± 1.38	10.51 ± 156	13.13 ± 1.90
Loss Modulus (kPa)
ABNP	1.68 ± 0.29	1.83 ± 0.32	2.45 ± 0.41	4.80 ± 0.89
Fresh	2.12 ± 0.30	2.29 ± 0.39	2.86 ± 0.48	4.55 ± 0.83
Phase angle (°)
ABNP	17.3 ± 1.34	14.99 ± 1.17	16.66 ± 1.59	26.44 ± 3.53
Fresh	20.16 ± 1.61	15.93 ± 1.77	15.36 ± 1.96	18.9 ± 2.37

ABNP scaffold cytotoxicity was evaluated by seeding human amniotic mesenchymal stem cells (hAMSCs) onto the surface of ABNP scaffolds and culturing to 14 days. Live/dead fluorescence indicated cell viability to be 93.97±1.90%, 87.14±4.30%, and 95±1.46% at days 3, 7, and 14, respectively (FIG. 9, graph and panels A, B, C), indicating that the scaffold itself was not cytotoxic. There was no noticeable cell migration/penetration into the scaffolds throughout the duration of the study, as hAMSCs appeared to attach and spread on the surface of the scaffolds with increasing time in culture (FIG. 9, panel D).

Cell injection into non-crosslinked and EDC/NHS-treated ABNP scaffolds was investigated since the hAMSCs did not infiltrate the scaffolds when seeded onto the surface. The stem cells were injected via a 28G syringe in order to minimize scaffold disruption due to needle stick. Viable cells were clearly visible within and around the injection path (FIG. 10, panels A-D). Viability of hAMSCs injected within non-crosslinked ABNP scaffolds were 91±4.99% and 97±1.44%, at day 3 and 14, respectively. Comparatively, viability on EDC/NHS crosslinked samples was 73±7.92% and 65±8.12%, respectively at day 3 and 14 (FIG. 10 panel E). In general, viability was maintained in both treatment groups over time in culture (i.e., there were no statistical significant differences in viability with respect to time within each study group); however, viability was significantly decreased (p<0.05) in the crosslinked scaffolds as compared to non-crosslinked ABNP at day 14. Of note, all injected hAMSCs maintained a round morphology reminiscent of NP-like cells within the scaffolds.

Example 2

Complete bovine caudal tail intervertebral discs were chosen as the source tissue material, as they have been shown to have similar size, biochemistry, and resting stress as compared to human lumbar intervertebral discs. Intact intervertebral discs were subjected to decellularization solutions and methods as described above and subsequently evaluated at defined regions around the disc for residual cell nuclei, DNA, and glycosaminoglycan (GAG) content via histology, PicoGreen® and dimethylmethylene blue assays, respectively. Results were obtained from five intervertebral discs subjected to decellularization method no. 13 as described in Example 1. Results are expressed as an average±standard error of the mean (SEM). Significance was determined by comparing decellularized discs to fresh discs using paired t-tests with p<0.05 (*).

The macro- and microscopic architecture of the intervertebral discs remained intact following decellularization (FIG. 11), although swelling commonly occurred. Alcian blue staining confirmed the absence of residual bovine cell nuclei and DNA content in both the central nucleus pulposus and outer annulus fibrosis, which were reduced by 85% and 73.03%, respectively, as compared to fresh controls (Table 3). Qualitative fluorescence staining with ethidium (FIG. 12) demonstrated the absence of residual cell DNA in decellularized nucleus pulposus and annular fibrosus tissues within the intervertebral discs. GAG content in both the nucleus pulposus and annulus fibrosus regions of the decellularized intervertebral discs were significantly reduced when compared to fresh discs.

TABLE 3
DNA/GAG	Tissue type	Decell/Fresh	Average ± SEM
DNA	Nucleus pulposus	Decell	47.12 ± 14.66
(ng/mg dry weight)		Fresh	314.28 ± 65.4
	Annulus fibrosus	Decell	65.99 ± 4.07
		Fresh	244.67 ± 25.68
GAG	Nucleus pulposus	Decell	258.21 ± 59.3
(ng/mg dry weight)		Fresh	677.20 ± 95.04
	Annulus fibrosus	Decell	132.71 ± 11.44
		Fresh	252.70 ± 23.17

As shown, the method was successful in fully decellularizing intact intervertebral discs while maintaining their structure and extracellular matrix architecture. While there was a significant reduction in the amount of GAG in both the nucleus pulposus and annulus fibrosus regions of the decellularized discs, these quantities approached values found in the human intervertebral discs. Furthermore, it is expected that these values will increase upon seeding of human stem cells and their subsequent differentiation on the biomimetic scaffold.

While certain embodiments of the disclosed subject matter have been described using specific terms, such description is for illustrative purposes only, and it is to be understood that changes and variations may be made without departing from the spirit or scope of the subject matter.

Claims

1. A method for forming a biomaterial, the method comprising:

contacting a tissue with a decellularization solution, the decellularization solution include a non-ionic surfactant and a protease inhibitor;

subjecting the tissue while in contact with the decellularization solution to ultrasonication;

agitating the tissue while in contact with the decellularization solution; and

contacting the tissue with an enzyme solution.

2. The method of claim 1, wherein the non-ionic surfactant is a polyethylene oxide based surfactant or a polysorbate based surfactant.

3. The method of claim 1, wherein the decellularization solution includes the non-ionic surfactant in an amount of from about 0.5 v/v % to about 2 v/v %.

4. The method of claim 1, wherein the protease inhibitor comprises N-ethylmaleimide, phenylmethylsulfonylfluoride, ethylenediamine tetraacetic acid, ethylene glycol-bis-(2-aminoethyl(ether)NNN′N′-tetraacetic acid, ammonium chloride, elevated pH, aprotinin or leupeptin.

5. The method of claim 1, the decellularization solution further comprising one or more of an ionic surfactant, a salt, a buffer, an antibiotic, or an antimycotic.

6. The method of claim 1, wherein the ultrasonication is carried out for a period of from about 5 minutes to about 60 minutes.

7. The method of claim 1, wherein the ultrasonication is carried out at a power level of about 10 Watts or greater and at a frequency of about 20 kilohertz or greater.

8. The method of claim 1, further comprising repeating the ultrasonication step periodically.

9. The method of claim 8, further comprising replacing the decellularization solution with a fresh decellularization solution periodically in conjunction with each ultrasonication step.

10. The method of claim 1, wherein the enzyme solution comprises one or more nucleases.

11. The method of claim 1, further comprising crosslinking one or more components of the tissue.

12. The method of claim 1, wherein the tissue comprises intervertebral disc tissue.

13. The method of claim 12, wherein the tissue comprises nucleus pulposus tissue.

14. The method of claim 12, wherein the tissue comprises annulus fibrosus tissue.

15. The method of claim 12, wherein the tissue comprises an entire intervertebral disc.

16. The method of claim 1, the tissue comprising glycosaminoglycan and nucleic acid, wherein following them method, the tissue has a glycosaminoglycan content of about 200 micrograms or greater per milligram dry weight of the tissue and has a nucleic acid content of about 50 nanograms or less per milligram dry weight of the tissue.

17. The method of claim 1, further comprising seeding the tissue with extrinsic cells.

18. The method of claim 1, wherein the method forms an implantable graft biomaterial.

19. The method of claim 18, wherein the implantable graft biomaterial is an intervertebral disc graft biomaterial, an articular cartilage graft biomaterial, or an osteochondral graft biomaterial.

20. The method of claim 1, wherein the method forms a scaffolding biomaterial.