DESCRIPTION FIELD

[0001] The disclosure relates to tissue engineering, and more particularly to the treatment of non-human cartilage, and/or fibrocartilage, to provide a biomaterial for use as a xenograft replacement for, and/or repair of, defective or damaged human cartilage.

BACKGROUND

[0002] Techniques are known in the art, for processing soft tissue from certain non-human species, to such make tissue substantially non-immunogenic with respect to humans. As such, the tissue is suitable for implantation into humans as a xenograft, for replacement and/or repair of original defective or damaged human tissue. The following patents, individually in some cases, and collectively in others, disclose processing for such non-human tissue, for example, with a- galactosidase to remove much, although not all, of the principal immunogenic component, namely a-gal epitopes, from cell membranes in the tissue:

US Patent Nos:

5865849,

5984858,

6040379,

6093204,

5902338,

6110206,

6210440,

6102783,

6063120,

5782915,

5944755,

6049025,

5922027,

5913900,

6758865,

6972041,

6267786,

6455309,

6231608,

6383782,

7594934,

7064287, and

7595377. Those patents are incorporated by reference herein.

[0003] As noted in those patents, a-galactosidase-treated porcine tissue is known to have highly reduced immunogenicity compared to untreated porcine tissue. However, in the art, such glycosidase treatments have been, and are, applied to generally intact tissue, tissue segments or other forms of bulk tissue, leading to immunogenicity which, although considerably reduced, is not sufficiently reduced for optimal use of the treated tissue as a xenograft for humans. That limitation in reduction of immunogenicity in bulk soft tissue, is primarily due to the inability to effectively treat much of the interior regions of the bulk tissue because of difficulties in applying the a-galactosidase in a desired uniform and controlled manner throughout the 3-dimensional bulk tissue. The below described techniques overcome this problem.

SUMMARY

[0004] One or more implementations of the subject disclosure are illustrated by and/or described in connection with one or more of the below figures, and are set forth in the claims, of the disclosure.

[0005] The description in this summary section may provide some illustrative examples of the disclosure. This section is not intended to be a broad overview or to identify essential elements of Hie disclosure.

[0006] Full thickness defects of human soft tissue, such as articular cartilage in the knee, and other joints, have poor capacity for repair. Often, such defects progress to osteoarthritis and require arthroplasty.

[0007] Allograft cartilage tissue, in plug form, and in micronized and lyophilized form, have shown utility to act as a scaffold for cartilage cells in attempts to regenerate hyaline cartilage in cartilage defects. This disclosure takes recognition that xenograft tissue is more plentiful in supply than allograft tissue, and has similar extracellular matrix composition, and uses a treated, substantially immunocompatible matrix from a non-human animal as a xenograft for use in treating human tissue defects and/or damaged tissue, including, but not limited to, cartilage defects. DESCRIPTION

[0008] Some forms of embodiments disclosed herein, are new and improved biomaterials, suitable for use as xenografts in humans. In a form, xenograft candidate non-human soft tissue, for example intact porcine tissue or tissue segments, or other bulk soft tissue, is treated with a- galactosidase, and other processes, in a conventional manner, and then is subjected to further processes to transform the a-galactosidase-treated tissue to a form with improved physical structure, as well as immunogenicity, so that it is substantially more suitable as a xenograft in humans. In particular, a-galactosidase-treated tissues such as those from the prior art, are further processed to be lyophilized and micronized, forming a non-immunogenic particulate-based "paste"-like structure which may be applied to tissue defects in a human. For example, a- galactosidase treated hyaline cartilage (preferably, but not necessarily, including native extracellular matrix complements and proteoglycans) may be lyophilized and then micronized, or micronized and then lyophilized, to form a non-immunogenic particulate-based "paste' ike structure for repair of a cartilage tear, or other defect, in a human. In various forms, the order of the a-galactosidase, lyophilizing and micronizing treatments may be any permutation.

[0009] Preferably, the α-galactosidase-treated bulk soft tissue forming the basis for the subject xenograft material, is first lyophilized using conventional techniques (for example, using negative pressure applied to chilled base material so that water in the material sublimates out). The lyophilization is effectively a freeze-drying process wherein the material undergoing the process loses water and it changes its physical conformation so that it has an increased surface-to-volume ratio. The resultant lyophilized tissue then is preferably micronized to particle sizes in the range 200-1000μ, and preferably in the range 300-500μ. Because the micronized particles are obtained from lyophilized material, the individual particles are themselves characterized by a relatively large surface-to-volume ratio, for example, providing a bulk density on the order of 0.2 g/cc.

[0010] More particularly, in a form, an improved article of manufacture is provided as a substantially non-immunogenic xenograft tissue, for example, an articular cartilage xenograft, for implantation into humans. An exemplary method for preparing such an articular cartilage xenorograft uses the steps of removing at least a portion of an articular cartilage from a non- human animal to provide a heterograft; washing the heterograft in hypotonic solutions, detergents, hydrogen peroxide, and alcohol; subjecting the heterograft to at treatment including, but not limited to exposure to ionizing radiation, immersion in alcohol, freeze/thaw cycling, and optionally to chemical cross-linking. In addition to or in lieu of some of the above treatments, the method includes a cellular disruption treatment and glycosidase digestion of carbohydrate moieties of the heterograft. The graft is then processed to form, as needed, sheets or other custom sizes and shapes for corresponding defects or plugs, as in the prior art. Alternatively, the processed tissue is subjected to micronization and lyophilization, in any order, to form a novel and improved xenograft biomaterial.

[0011] In a particular form, as an example, the invention provides an articular cartilage xenograft for implantation into a human including a portion of an articular or fibrocartilage cartilage, or cartilage particles, from a non-human animal, wherein the portion includes extracellular matrix and substantially only dead cells. The matrix and dead cells have substantially no surface a- galactosyl epitopes, and have been lyophilized and/or micronized, and presented as an immunocompatible and sterile implant.

EXAMPLES

Example 1: Assessment Of Primate Response to Implanted Porcine Cartilage Treated with a-galactosidase

[0012] In this example, porcine cartilage implants are treated with a-galactosidase to eliminate a-galactosyl epitopes, and then the implants are transplanted into cynomolgus monkeys, and the primate responoo to those cartilage implants is assessed.

[0013] Porcine stifle joints are sterilely prepared and the articular cartilage and surrounding attached soft tissues surgically removed from the monkeys. The cartilage specimens are washed with an alcohol, such as ethanol or isopropanol, to remove synovial fluid and lipid soluble contaminants.

[0014] The cartilage specimens are then frozen at a temperature ranging from about -35°C to about -90°C, and preferably at a temperature up to about -70°C, to disrupt, that, is to kill, the specimens' native cells.

[0015] The cartilage is then cut into two portions. A first cartilage portion is immersed in a buffer solution containing α-galactosidase at a predetermined concentration. The specimens are allowed to incubate in the buffer solution for a predetermined time period at a predetermined temperature. A second cartilage portion is incubated under similar conditions as the first cartilage portion, in a buffer solution in the absence of α-galactosidase and serves as a control. [0016] At the end of the incubation, the cartilage is washed under conditions which allow the enzyme to diffuse out. Assays are performed to confirm the complete removal of the a-gal epitopes.

[0017] Each cartilage sample is implanted in the supra patellar pouch of six cynomolgus monkeys. With the animals under general inhalation anesthesia, an incision of about 1 cm is made directly into the supra patellar pouch at the superior medial border of the patella extending proximally. A piece of the porcine cartilage of about 0.5 cm to about 1 cm in length is placed into the pouch with a single 3-0 nylon stitch as a marking tag. The procedure is performed under sterile surgical technique, and the wounds are closed with 3-0 vicryl or a suitable equivalent. The animals are permitted unrestricted cage activity and monitored for any sign of discomfort, swelling, infection, or rejection. Blood samples (e.g., 2 ml) are drawn periodically (e.g., every two weeks) for monitoring of antibodies.

[0018] The occurrence of an immune response against the xenograft is assessed by determining anti-Gal and non-anti-Gal anti-cartilage antibodies (i.e., antibodies binding to cartilage antigens other than the a-gal epitopes) in serum samples from the transplanted monkeys. At least two ml blood samples are drawn from the transplanted monkeys on the day of implant surgery and at periodic (e.g., two week) intervals post-transplantation. The blood samples are centrifuged and the serum samples are frozen and evaluated for the anti-Gal and other non-anti-Gal anti-cartilage antibody activity.

[0019] Anti-Gal activity is determined in the serum samples in ELISA with a-gal-BSA as solid phase antigen, according to methods known in the art, such as, for example, the methods described in Galili et al., Porcine and Bovine Cartilage Transplants in Cynomolgus Monkey: II. Changes in anti-Gal response during chronic rejection, 63 Transplantation 645-651 (1997).

[0020] A33a s arc conducted lu determine whether a-galactosidase-treated xenografts induce the formation of anti-cartilage antibodies. For measuring anti-cartilage antibody activity, an ELISA assay is performed according to methods known in the art, such as, for example, the methods described in K.R.Stone et al., Porcine and Bovine Cartilage Transplants in Cynomolgus Monkey: I. A model for chronic xenograft rejection, 63 Transplantation 640-645 (1997).

[0021] The cartilage treatment with alpha-galactosidase, effectively removes Gal epitopes, attenuated anti-Gal antibody response to minimal levels. [0022] The cartilage is optionally explanted at one to two months post-transplantation, sectioned and stained for histological evaluation of inflammatory infiltrates. Post-transplantation changes in anti-Gal and other anti-cartilage antibody activities, are correlated with the inflammatory histologic characteristics (i.e., granulocytes or mononuclear cell infiltrates) within the explanted cartilage, one to two months post-transplantation, using methods known in the art, as, for example, the methods described in K.R.Stone et al., Porcine and Bovine Cartilage Transplants in Cynomolgus Monkey: I. A model for chronic xenograft rejection, 63 Transplantation 640-645 (1997).

Example 2: Soft Tissue Grafts

[0023] Introduction: Dermis, pericardium, vascular graft, bladder, articular cartilage, and fibrocartilage all have utility as grafts and biomaterials for human reconstruction procedures. These materials may vary in anatomical architecture, but are primarily composed of collagen with associated cells and extra-cellular matrix. The antigenic a-galactosyl epitope is associated with both cells and extra-cellular matrix of all these tissue types at native levels associated with acute rejection when implanted in humans. Reducing the the epitope level in a stepwise manner through cellular inactivation, decellularization, delipidation, enzymatic cleavage by a- galactosidase enzyme, and sterilization yields an immunocompatible graft suitable for human implantation.

[0024] Materials and Methods: Porcine dermis, pericardium, vascular graft, and bladder were harvested and surface sanitized with 80% EtOH followed by rinse in distilled water and or normal saline. Pericardium and bladder were initially processed with SDS and 1M sodium chloride to facilitate membrane harvest. Decellularization is accomplished by sonicating the grafts in a 0.3% combined weight solution of Triton X-100 and SDS. After washing in normal saline, grafts were further decellularized and delipidated by sonicatinn in 0.3% sodium deoxycholatc. This combination of steps presents one embodiment for decellularization. After rinsing the graft in phosphate buffered saline, grafts were subjected to alpha-galactosidsae enzyme at 30 U/mL for 12 hours at 24-26°C. Washout of the enzyme was accomplished by serial washes ending in phosphate buffered saline.

[0025] Both glutaraldehyde and carbodiiimide are utilized to cross-link the materials.

Glutaraldehyde is used at 0.10% and 0.01% and carbodiimide at 20 and 5mM concentration using standard cross-link methods. For both cross-link agents, unreacted function groups are quenched with glycine followed by exhaustive washout with phosphate buffered saline.

Results:

[0026] Tissue types untreated, post-decellularization, and post enzyme were homogenized in PBS and subjected to an M-86 antibody inhibition assay for determination of residual galactosyl epitope remaining. Untreated tissues were harvested to isolate the membranous or sheet like structure without futher processing. Post-decellularization tissues were sampled after all detergent treatments and washout. Post-enzyme tissues were samples after enzymatic treatment and washout. Epitope residual data is normalized to untreated as 100% epitope content with 0% referenced to human tissue homogenates, Error bars represent N=4 and percent error of standard deviation, (see Table 1 and Figure 1 below)

Table 1 : Epitope Level Post-Tissue Treatment

Tissue Type, Treatment and Residual Galactosyl Epitope

Tissue Type Untreated Post-Decell Post-Enzyme

Bladder 100 ± 13.5 % 34.7 _± 4.4 % 0.0 ± 3.9 %

Pericardium 100 ± 3.0 % 66.2 ± 12.0 % 0.0 ± 7.3 %

Vascular graft 100 ± 4.4 % 76.3 ± 1 1.0 % 0.0 ± 3.8 %

Dermis 100 ± 4.7 % 69.2 ± 8.6 % 0.0 ± 4.2 %

EMBODIMENTS

Device Preparation and Device Description:

[0027] Native xenograft tissues, including but not limited to tendon, articular cartilage, meniscus, bladddcr, pericardium, vascular graft, or dermis, are subject to, in no specific order, delipidation, decellularization, glycoside digestion of galactosyl epitopes, cross-linking, and terminal sterilization with ionizing radiation. In order to take advantage of greater suface area-to- mass ratios, treatments are performed on micronized tissues, wherein the particulate size is 100 to 1000 microns, preferably 200 to 500 microns. Micronizing is performed using a rotary sheer homogenizer, preferably under temperature control at less than 10 degrees C.

[0028] After micronization and treatment, the particulate product is subjected to lyophillization, both primary and secondary drying. The target percent moisture is less than 10%, preferably less than 7%. as determined by Karl-Fisher titration. In an exemplary form, the bulk density of the dried final product ranges from 0.1 to 0.4 grams per cubic centimeter.

Preparation of Micronized Material

1. Native tissue is dissected out and manually minced into 10-20 mm pieces.

2. Pieces are suspend resulting in 70% IPA 0.1 % Tween 20 and/or 3% hydrogen peroxide, and stirred 4-6 hours at 4° C.

3. Solution is exchanged with water for injection for three washes.

4. Solution of suspended minced pieces is homogenized at 4 to 1° C, until the intended particle size (e.g., 200 to 500 microns) is achieved. Filter out particles under 200 micron appropriate membrane.

5. Decant supernatant and add a-galactosidase solution for 4-12 hours at 4-26° C.

6. Decant enzyme and perform three rinses with water for injection.

7. Decant water for injection and rinse three times with phosphate buffer, pH 7.0.

8. Aliquot slurry into glass vial w/ stopper.

9. Lyophilize 24-48 hours until a final moisture content of appx. 7%. 10 Rar.k fill vials with nitrogen.

1 1. Irradiate with 1.5 to 2.5 mRad under temperature control (< 0° C).

12. Store at 4° C or room temperature until use.

Growth Factor Delivery Scaffold:

[0029] Another form provides an improved bio-compatible and resorbable matrix for sequestering and delivery of growth factors. Examples of relevant factors include, but are not limited to, fibroblast growth factors, epidermal growth factors, kertinocyte growth factors, vascular endothelial growth factors, platelet derived growth factors, transforming growth factors, bone morphogenic proteins, parathyroid hormone, calcitonin, prostaglandins, ascorbic acid, or multiple combinations of the above.

[0030] Growth factors are applied aseptically in the operating room, by distributing solubalized factors on the device with a syringe or soaking in a sterile vessel. Concentrations vary according to the specific growth factor with ug/ml to mg/ml having the preferred utility. Alternately, PDGF enriched blood fractions, plasma, marrow or blood, as autologously harvested, is applied to the device. An incubation time of 5 to 60 minutes in optimally used to enhance matrix factor interactions. The factor laden device is implanted, used in reconstruction/augmentation or used in wound management using standard surgical techniques.

Cell Delivery Scaffold:

[0031] A further form functions as a immunocompatible scaffold for cellular seeding therapies. Cells can either be cultured on the grafts or matrices for a period of time pre-implantation or distributed on the grafts in-situ at the time of implantation. Cells to be co-implanted with the grafts include, but are not limited to, adult or embryonic mesenchymal stem cells, embryonic stem cells, adipocyte derived stem cells, fibroblasts, chorndrocytes, chondroblasts, pro- chondroblasts, osteocytes, osteocytes, osteoclasts, pro-osteoblasts, monocytes, pro- cardiomyocytes, pericytes, cardiomyoblasts, cardiomyocytes, myocytes or multiple combinations of the above.

[0032] Cells are either autologous or allogeneically sourced and seeded onto the device in vitro for standardized culture expansion for an extended period of time pre-implantation or applied in situ in the operating room environment. Seeding density is optimally 0.1 to 1 million cells per mL of device for culture conditions and may vary for in situ seeding. The cell laden device is implanted, used in reconstruction/augmentation or used in wound management using standard surgical techniques.

[0033] Although the invention has been described in terms of certain embodiments, other embodiments that are apparent to those of ordinary skill in the art, including embodiments which do not provide all of the benefits and features set forth herein, are also within the scope of this invention. Accordingly, the scope of the present invention is defined only by reference to the appended claims.