Nutritional joint health supplement compositions and methods for making and using same

Abstract

The present disclosure relates generally to joint health supplement compositions derived from a xenogeneic cartilaginous tissue source, methods of making such compositions, as well as methods of using such compositions, for example, to treat joint pain or other joint ailments due to arthritis in human and animal populations both at risk and actively presenting with such joint pain or other joint ailments. One aspect of the present disclosure can include a nutritional joint health supplement composition including a cartilaginous tissue material harvested from a xenogeneic tissue source and at least a trace amount of a detergent, wherein the nutritional joint health supplement composition has a beneficial component profile and reduced cellular and DNA content.

Description

PATENT APPLICATION NUTRITIONAL JOINT HEALTH SUPPLEMENT COMPOSITIONS AND METHODS FOR MAKING AND USING SAME RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No.63 / 663,783, filed June 25, 2024, the entirety of which is hereby incorporated by reference for all purposes. TECHNICAL FIELD

[0002] The present disclosure relates generally to joint health supplement compositions derived from a xenogeneic cartilaginous tissue source, methods of making such compositions, as well as methods of using such compositions, for example, to treat joint pain or other joint ailments due to arthritis in human and animal populations both at risk and actively presenting with such joint pain or other joint ailments. BACKGROUND

[0003] Joint cartilage degradation poses a major impediment for arthritic patients, active athletes, and the elder population, all of which suffer continuous pain, joint stiffness and an overall decrease in quality of life. While enormous efforts are being made to develop treatments that impede or delay cartilage degeneration, invasive procedures are far from optimal and create collateral undesired risks. Nutritional supplements are commonly used to support joint structure, function, and comfort. Among these, collagen-based supplements (e.g., containing type II collagen) have ^received significant attention due to their role in maintaining the integrity of cartilage and connective tissues.

[0004] Existing joint health supplements containing type II collagen, however, are often derived from non-porcine sources and may suffer from inconsistent bioavailability, insufficient immune-modulating activity, or degradation during processing. Furthermore, many formulations fail to adequately preserve the native structure of type II collagen, which is critical for interaction with mucosal immune structures, such as Peyer’s patches in the small intestine. These lymphoid tissues are involved in the oral tolerance pathway and are believed to mediate systemic immune responses that may reduce joint inflammation and support cartilage preservation. SUMMARY

[0005] Collagen supplements have recently arisen as an alternative to prevent joint degradation. Such commercially available products (e.g., UC-II, Bioiberica, Gelita) are based in chicken sternum cartilage or hydrolyzed collagen, which undesirably contain low type II collagen content as well as low sulfated glycosaminoglycan (sGAG) content. The nutritional joint health supplement compositions of the present disclosure, however, contain not only high type II collagen and sGAG content, but also low detergent content. Consequently, the nutritional joint health supplement compositions advantageously induce a strong immune response and promote oral tolerance (e.g., after ingestion and digestion thereof) as the type II collagen interacts with immune cells present in the intestinal microenvironment (e.g., Peyer’s patches) and thereby starts a cascade immune response that prevents or mitigates joint degradation and associated joint pain. ^

[0006] As such, one aspect of the present disclosure can include a nutritional joint health supplement composition comprising a cartilaginous tissue material harvested from a xenogeneic tissue source and at least a trace amount of a detergent, wherein the nutritional joint health supplement composition has a beneficial component profile and reduced cellular and DNA content.

[0007] Another aspect of the present disclosure can include a method for preparing a nutritional joint health supplement composition. The method can include treating a cartilaginous tissue material harvested from a xenogeneic tissue source with an acidic solution to form an acid-treated cartilaginous tissue material containing at least a trace amount of a detergent, wherein the cartilaginous tissue material has a beneficial component profile and reduced cellular and DNA content.

[0008] Another aspect of the present disclosure can include a method of treating joint pain in a subject. The method can comprise administering a nutritional joint health supplement composition to the subject, the nutritional joint health supplement composition comprising a cartilaginous tissue material harvested from a xenogeneic tissue source and at least a trace amount of a detergent, wherein the nutritional joint health supplement composition has a beneficial component profile and reduced cellular and DNA content, the nutritional joint health supplement composition being administered to the subject in a therapeutically effective amount to reduce joint pain in both a healthy subject and a subject with at least one arthritic joint.

[0009] Another aspect of the present disclosure can include a nutritional joint health supplement composition comprising a therapeutically effective amount of soluble form cartilaginous tissue material comprising type II collagen, the type II collagen being solubilized in a physiologically acceptable aqueous medium, wherein the type II collagen is harvested from a xenogeneic tissue source, is free of any ^detergent(s), and has a beneficial component profile and reduced cellular and DNA content.

[0010] Another aspect of the present disclosure can include a method for forming a nutritional joint health supplement composition comprising a therapeutically effective amount of soluble form cartilaginous tissue material comprising type II collagen, the type II collagen being solubilized in a physiologically acceptable aqueous medium, wherein the type II collagen is harvested from a xenogeneic tissue source, is free of any detergent(s), and has a beneficial component profile and reduced cellular and DNA content. The method can comprise treating a cartilaginous tissue material harvested from a xenogeneic tissue source with an acidic solution to form an acid-treated cartilaginous tissue material, wherein the cartilaginous tissue material is free of any detergent(s) and has a beneficial component profile and reduced cellular and DNA content.

[0011] Another aspect of the present disclosure can include a method of treating joint pain in a subject. The method can comprise administering, to a subject, a nutritional joint health supplement composition comprising a therapeutically effective amount of soluble form cartilaginous tissue material comprising type II collagen, the type II collagen being solubilized in a physiologically acceptable aqueous medium, wherein the type II collagen is harvested from a xenogeneic tissue source, is free of any detergent(s), and has a beneficial component profile and reduced cellular and DNA content. The nutritional joint health supplement composition can be administered to the subject in a therapeutically effective amount to reduce joint pain in both a healthy subject and a subject with at least one arthritic joint. BRIEF DESCRIPTION OF THE DRAWINGS ^

[0012] The foregoing and other features of the present disclosure will become apparent to those skilled in the art to which the present disclosure relates upon reading the following description with reference to the accompanying drawings, in which:

[0013] Fig.1 is a process flow diagram illustrating a method for preparing a cartilage-derived tissue composition in accordance with one aspect of the present disclosure;

[0014] Figs.2A-C show biochemical characterization of processed porcine septal cartilage with histology (Fig.2A) showing preservation of native structure and content of collagen type II (Fig.2B) as shown in SDS-PAGE. Biochemical characterization (Fig.2C) showed very low content of DNA and residual detergent, while showing 50-70% collagen. ELISA analysis also showed presence of native collagen type II in all the lots combined with excipients (15% of cartilage in final formulation), averaging 5 ng / mg dry powder;

[0015] Figs.3A-C show preclinical monoiodoacetate (MIA)-induced arthritis model testing in which a formulation of porcine collagen type II with excipients (cellulose, magnesium stearate) (Fig.3A) was administered daily to rats at 3 mg / kg. Porcine collagen type II dosing prevented stress-associated weight loss in rats (Fig. 3B), indicating potential pain relief. The sustained increase in the joint width (Fig. 3C) for 14 days in MIA injected knee (R) as compared to control (L) demonstrated the suitability of the model. Modest improvements in functional movement measured by Gait analysis, specifically at day 3, indicated a beneficial action on joint inflammation elicited by collagen type II;

[0016] Figs.4A-B show porcine collagen type II dosing restored basal levels of crosslinked collagen type II in serum (CTXII) in MIA rats. Similarly, the main ^cytokines involved in arthritis progression were modulated to basal levels by collagen type II dosing (Fig.4A). Porcine collagen type II dosing also decreased histological inflammatory score (Fig.4B), indicating a modulation of the MIA-induced inflammation; and

[0017] Fig.5 is a process flow diagram illustrating different collagen type II supplementation formats according to another aspect of the present disclosure. DETAILED DESCRIPTION

[0018] Definitions

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the present disclosure pertains.

[0020] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as “consisting of”), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.

[0021] In the context of the present disclosure, the term “about”, when expressed as from “about” one particular value and / or “about” another particular value, also specifically contemplated and disclosed is the range from the one particular value and / or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, ^specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub- ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these aspects are explicitly disclosed.

[0022] Optionally, in some aspects, when values or characteristics are approximated by use of the antecedents “about,” “substantially,” or “generally,” it is contemplated that values within up to 15%, up to 10%, up to 5%, or up to 1% (above or below) of the particularly stated value or characteristic can be included within the scope of those aspects.

[0023] As used herein, phrases such as “between X and Y” and “between about X and Y” can be interpreted to include X and Y.

[0024] As used herein, phrases such as “between about X and Y” can mean “between about X and about Y”.

[0025] As used herein, phrases such as “from about X to Y” can mean “from about X to about Y”.

[0026] As used herein, the term “attached” can refer to being fastened, fixed, joined, connected, bound, adhered to, or assembled with.

[0027] It will be understood that when an element is referred to as being “on”, “attached” to, “connected” to, “coupled” with, “contacting”, etc., another element, it can be directly on, attached to, connected to, coupled with or contacting the other ^element or intervening elements may also be present. In contrast, when an element is referred to as being, for example, “directly on”, “directly attached” to, “directly connected” to, “directly coupled” with or “directly contacting” another element, there are no intervening elements present. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature.

[0028] Spatially relative terms, such as “under”, “below”, “lower”, “over”, “upper” and the like, may be used herein for ease of description to describe one element or feature’s relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms can encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is inverted, elements described as “under” or “beneath” other elements or features would then be oriented “over” the other elements or features.

[0029] As used herein, the terms “first,” “second,” etc. should not limit the elements being described by these terms. These terms are only used to distinguish one element from another. Thus, a “first” element discussed below could also be termed a “second” element without departing from the teachings of the present disclosure. The sequence of operations (or acts / steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise.

[0030] As used herein, the terms “optionally” and “optional” can mean that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present. ^

[0031] As used herein, the term “administer” can mean to give or to apply (e.g., to a subject), either by the subject himself / herself or by another person (e.g., a medical professional).

[0032] As used herein, the term “biocompatible” can refer to causing no clinically relevant tissue irritation, injury, toxic reaction, or immunological reaction to living tissue.

[0033] As used herein, the terms “acellular” and “decellularized” can be used interchangeably and refer to a biological tissue (e.g., nasal septum cartilage) that has been treated by a process in which cells and cellular components (including DNA) are removed from the biological tissue, thereby leaving the extracellular matrix (ECM) thereof free, or essentially free, of such cells and cellular components.

[0034] As used herein, the term “improve” (or “improving”) can refer to bringing into a more desirable or excellent condition.

[0035] As used herein, the terms “reduced” or “to reduce” can refer to a diminishing, a decrease in, an attenuation or abatement of the degree, intensity, extent, size, amount, density, or number.

[0036] As used herein, the term “substantially similar” can mean that a first value, aspect, trait, feature, number, or amount is of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of a second value, aspect, trait, feature, number, or amount.

[0037] As used herein, the terms “subject” and “patient” can be used interchangeably and refer to a vertebrate, such as a mammal (e.g., a human). Mammals can include, but are not limited to, humans, dogs, cats, horses, cows, and pigs. ^

[0038] As used herein, the phrase “subject in need thereof” can refer to a patient that: (i) will be administered a joint health supplement composition of the present disclosure; (ii) is receiving a joint health supplement composition of the present disclosure; or (iii) has received a joint health supplement composition of the present disclosure; unless the context and usage of the phrase indicates otherwise.

[0039] As used herein, the term “lacking substantially all viable cells” can mean that the concentration of viable cells in a joint health supplement composition of the present disclosure is less than about 1% (e.g., 1%; 0.9%; 0.8%; 0.7%; 0.6%; 0.5%; 0.4%; 0.3%; 0.2%; 0.1%; 0.01%; 0.001%; 0.0001%; 0.00001%; 0.000001%, or any value between) of that in the porcine cartilage source from which the joint health supplement composition was made.

[0040] As used herein, a joint health supplement composition lacking “substantially all DNA” can refer to a joint health supplement composition in which the concentration of DNA is less than about 1% (e.g., less than: 1%; 0.9%; 0.8%; 0.7%; 0.6%; 0.5%; 0.4%; 0.3%; 0.2%; 0.1%; 0.01%; 0.001%; 0.0001%; 0.00001%; or 0.000001%) of that in the porcine cartilage source from which the joint health supplement composition was made. This includes all DNA (e.g., DNA within intact cells (alive or dead cells)) or residual DNA material left from the decellularization process.

[0041] As used herein, the term “therapeutically effective amount”, when referring to a joint health supplement composition of the present disclosure, can refer to an amount effective thereof, when administered to a patient, to provide a therapeutic effect, such as an amelioration of symptoms, reduced disease progression, and / or causing or increasing disease regression. A quantity of a specified joint health supplement composition is sufficient to achieve a desired effect in a subject being ^treated, such as to maintain joint cartilage, minimize cartilage degradation, promote healthy joints by protecting cartilage integrity, diminish the action of enzymes that affect joint health, improve joint movement and / or function, support joint function, alleviate joint pain, alleviate joint discomfort, alleviate joint stiffness, improve joint range of motion, improve joint flexibility, improve joint range of motion and flexibility, promote mobility, or the like. A therapeutically effective amount can be administered orally, for example, and can be administered in a single application, or in several applications over time. One skilled in the art will appreciate that the effective amount can be dependent on the preparation applied, the subject being treated, the severity and type of the affliction, and the manner of administration.

[0042] As used herein, the term “therapeutic effect” can refer to a consequence of treatment with a joint health supplement composition of the present application, the results of which are judged to be desirable and beneficial. A therapeutic effect may include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation. A therapeutic effect also may include, directly or indirectly, the arrest reduction or elimination of the progression of a disease manifestation.

[0043] As used herein the terms “nasal septal tissue” and “nasal septum tissue” can refer to the tissue portion which separates the nasal cavity into the two nostrils, particularly the cartilaginous portion, including the quadrangular cartilage, vomeronasal cartilage, lateral nasal cartilage, greater alar cartilage, and / or accessory nasal cartilage. In some instances, the joint health supplement compositions of the present disclosure can be derived from hyaline cartilage. The joint health supplement compositions of the present disclosure can comprise collagen, preferably type II collagen. In some forms, the joint health supplement ^compositions of the present disclosure can comprise predominantly (e.g., greater than 50%) type II collagen.

[0044] As used herein, the terms “sulfated glycosaminoglycan” or “sGAG” can refer to one or more of the following: heparin, heparin sulfate, chondroitin sulfate, dermatan sulfate, keratin sulfate, and / or hyaluronic acid. sGAGs help guide cellular differentiation, as their strong negative charge binds positively charged growth factors. sGAGs are highly polar and attract water, thus increasing the lubricity and shock absorbance of the joint health supplement compositions as disclosed herein. As discussed herein, a Dimethylmethylene Blue (DMMB) Assay may be utilized to quantify the sGAG content of the joint health supplement compositions of the present disclosure.

[0045] As used herein, the term “nutritional supplement” refers to a food product formulated as a dietary or nutritional supplement to be used as part of a diet.

[0046] As used herein, the terms “nutraceutical” or “nutraceutical composition” can be used interchangeably to refer to, without limitation, food compositions, food additives, food compositions in bulk, food additives in bulk, dietary supplements, medical foods, and foods for special dietary use, of the present disclosure. In some forms, a nutraceutical composition of the present disclosure can comprise one or more consumable vehicles, carriers, excipients, or fillers in addition to a joint health supplement composition.

[0047] As used herein, the terms “dietary supplement” or “nutritional supplement” can refer to a product (other than tobacco) intended to supplement the diet that bears or contains one or more of the following dietary ingredients (in addition to a joint health supplement composition of the present disclosure): a vitamin; a mineral; an herb or other botanical; an amino acid; a dietary supplement used by man to ^supplement the diet by increasing the total dietary intake; or a concentrate, metabolite, constituent, extract, or a combination of any of the ingredients. Typically, a dietary supplement is a product that is labeled as a dietary supplement and is not represented for use as a conventional food or as a sole item of a meal or the diet. A dietary supplement can be consumed by a subject independent of any food, unlike a food additive which is incorporated into a food composition during the processing, manufacture, preparation, or delivery of the food composition, or just before its consumption.

[0048] The terms “supplement” or “supplement”, as used herein, can refer to a product that improves, promotes, supports, increases, regulates, manages, controls, maintains, optimizes, modifies, reduces, inhibits, or prevents a particular condition, structure or function associated with a natural state or biological process. In certain aspects, a supplement is a dietary supplement. For example, with regard to joint health-related conditions, dietary supplements may be used to maintain joint cartilage, minimize cartilage degradation, promote healthy joints by protecting cartilage integrity, diminish the action of enzymes that affect joint health, improve joint movement and / or function, support joint function, alleviate joint pain, alleviate joint discomfort, alleviate joint stiffness, improve joint range of motion, improve joint flexibility, improve joint range of motion and flexibility, promote mobility, or the like.

[0049] As used herein the term “treating” or “treatment” of joint deterioration or a joint-health related condition includes: (1) preventing joint deterioration; (2) reducing the rate and / or the amount of joint deterioration; (3) relieving the joint deterioration, i.e., causing regression of joint deterioration. By “preventing”, it will be appreciated that compositions of the present disclosure can be used prophylactically, e.g., in ^apparently healthy subjects; that is, subjects with no apparent or reported symptoms or diagnosis joint disease, arthritis, etc.

[0050] As used herein, the terms “supporting” or “support”, when referring to joint health, can include: (1) beneficial effects on bone formation and quality; (2) improving mineralization process in bone; (3) contributing to healthy bone cortex and bone matrix; (4) aiding in formation and / or preservation of collagen found in ligaments, tendons, cartilage and bone; and (5) decreasing incidence of bone-related injuries.

[0051] As used herein, the term “arthritic joint” can refer to a joint affected by arthritis, a condition characterized by inflammation and pain in one or more joints. This inflammation can lead to joint pain, stiffness and swelling, and can be caused by various diseases or conditions. Examples of joints that can be affected by arthritis include hands (e.g., finger joints, wrists and thumbs), hips, knees, feet, shoulders, ankles, elbows and spine.

[0052] As used herein, the term “xenogeneic” can refer to cells or tissues derived from individuals of different species, including, but not limited to, porcine, bovine, ovine, caprine, avian, equine, canine, lapine, feline, and / or other non-human mammals.

[0053] As used herein, the term “undenatured collagen” can refer to collagen that retains its original molecular weight and amino acid sequence and optionally its original three-dimensional (tertiary) structure. Undenatured collagen has not been hydrolyzed into its component amino acids and may or may not be associated with proteoglycans or carbohydrate polymers such as, for example, chondroitin sulfate or hyaluronic acid, that are also typically found in cartilage. “Undenatured collagen” ^may also be referred to as “native” collagen, referring to the fact that undenatured proteins retain their native folds.

[0054] Overview

[0055] As discussed in more detail below, one aspect of the present disclosure includes a process for obtaining high purity xenogeneic cartilage powder, which content of collagen type II and certain other cartilage components, such as sGAGs advantageously induce a strong immune response and therefore a better oral tolerance effect, resulting in greater prevention of joint degradation as compared to conventional cartilage-based products (e.g., those produced with chicken sternum cartilage or hydrolyzed collagen). As exemplified in the Examples below, the xenogeneic (porcine) cartilage powder of the present disclosure contains higher total collagen content and more collagen type II. When compared to chicken sternum cartilage, the xenogeneic (porcine) cartilage powder of the present disclosure unexpectedly showed: higher total collagen content; higher native collagen type II content; prevention of weight loss in arthritis model; reduction in inflammatory cytokines; and a higher response at restoring histological structure / regeneration of native cartilage.

[0056] Based at least in part of this discovery, the present disclosure provides joint health supplement compositions derived from a xenogeneic (e.g., porcine) cartilaginous tissue source, methods of making such compositions, as well as methods of using such compositions, for example, to treat joint pain or other joint ailments due to arthritis in human and animal populations both at risk and actively presenting with such joint pain or other joint ailments.

[0057] Without wishing to be bound by theory, it is believed that the advantageous and unexpected features of the present disclosure are realized via the ^interaction between undenatured type II collagen and Peyer's patches, which leads to the induction of oral tolerance through the production of type II collagen-specific Treg cells. This process, described in more detail below, helps to regulate the immune system’s response towards its own type II collagen, ultimately supporting joint health by reducing collagen degradation and inflammation.

[0058] When undenatured type II collagen is administered orally, it interacts with Peyer’s patches in the gut, which are specialized lymphoid tissues in the small intestine responsible for immune surveillance. This interaction triggers a process called oral tolerance, where the immune system learns to recognize and tolerate type II collagen as a harmless substance. Undenatured type II collagen in its native, undenatured form interacts with Peyer’s patches. This form retains its specific 3D structure which is necessary for the immune interaction. Peyer’s patches act as an immune gateway in the gut, sampling the contents of the digestive system and deciding whether to mount an immune response. The undenatured type II collagen interacts with immune cells within the Peyer’s patches, prompting the activation and transformation of naive T-cells into T-regulatory (Treg) cells. These Treg cells are specifically programmed to recognize type II collagen. By inducing Treg cells, the body develops oral tolerance towards type II collagen. This means the immune system's response to type II collagen is diminished. The Treg cells circulate through the body and, when they encounter type II collagen in the joints, they help to suppress the immune response that might otherwise cause inflammation and degrade the cartilage. This anti-inflammatory action is mediated by the secretion of cytokines like IL-10 by the Treg cells. In summary, the interaction between undenatured type II collagen and Peyer’s patches leads to the induction of oral tolerance through the production of type II collagen-specific Treg cells. This process ^helps to regulate the immune system’s response towards its own type II collagen, ultimately supporting joint health by reducing collagen degradation and inflammation.

[0059] Advantageously, joint health supplement compositions of the present disclosure provide an improved joint health supplement that delivers bioactive type II collagen in a form that can engage the gut-associated lymphoid tissue and promote beneficial immunomodulatory effects. The joint health supplement compositions address the shortcomings of conventional, cartilage-based supplements by providing a nutritional supplement comprising type II collagen, derived from a xenogeneic (e.g., porcine) cartilage source, that is processed to retain its native triple-helical structure and biological activity. By targeting Peyer’s patches through the gut- immune axis, the joint health supplement compositions of the present disclosure are uniquely prepared and formulated to support joint comfort and function through both nutritional and immunological mechanisms, offering a more effective alternative to existing joint health products.

[0060] One aspect of the present disclosure includes a processing method to produce a xenogeneic (e.g., porcine) cartilage powder, which final use is intended as nutritional supplement to improve joint health. Cartilage can be sourced from septum, auricular, or costal tissues, for example. In each case, the sourced cartilage is isolated, cleaned, and sized for processing. Advantageously, processing of the sourced cartilage has been significantly improved to maximize collagen type II retention and diminish content of detergent after extensive rinses.

[0061] The method of processing xenogeneic cartilage is shown in Fig.1 and briefly described below. Processing to decellularize harvested cartilage can include an initial treatment with peracetic acid, a first detergent, PBS and HEPES, a second detergent, and IPA with purified water washes included. After decellularizing the ^harvested cartilage, a 24-72 hour freeze drying cycle can be performed to remove water content, followed by cryomilling. The result is a fine white powder that yields approximately 10g of final dry powder per 100g of wet tissue.

[0062] Figs.2A-C show the results and characteristics of several lots produced according to the present disclosure. Overall, testing after decellularization demonstrated preservation of native type II collagen structure and low DNA content. The level of detergent (Triton-X) remained low, and the content of type II collagen is elevated. Moreover, the powder obtained by the method of the present disclosure exhibits significant content of native type II collagen.

[0063] Referring to Figs.3A-C, porcine cartilage powder was combined with excipients and administered orally to rats suffering monoiodoacetate (MIA)-induced arthritis (Fig.3A). The MIA model utilized was sufficient in creating a local inflammatory response that caused significant swelling in all groups in the MIA- injected right knee joint when compared to their respective contralateral control joints (Fig.3C). Compared to MIA groups, administration of porcine cartilage prepared according to the present disclosure resulted in a significant increase in body weight (Fig.3B).

[0064] Overall, an increase in CTX-II serum concentrations in animals treated with the porcine cartilage was observed (Fig.4A). Porcine cartilage showed a tendency to restore basal levels of IL-17A, IL-12, eotaxin, MCP3, and RANTES compared to no treatment MIA group. Similarly, histopathological assessment of articular cartilage showed an overall trend of decreased cartilage damage when animals were administered the porcine cartilage (Fig.4B). Unexpectedly, results showed an overall trend of increased body weight, higher CTX-II serum levels, and ^differential expression of inflammatory mediators after treatment with the porcine cartilage.

[0065] As shown in Fig.5 and discussed in more detail below, the present disclosure can include various formulations comprising a nutritional joint health supplement composition. Such formulations can include freeze dried powder for tablets, capsules, or incorporation to other insoluble supplements. Also contemplated is a nutritional joint health supplement composition in solubilized form after mild acid treatment to maintain native type II collagen structure. After solubilization, the composition can be used in liquid form and incorporated into pre- cast gels and / or freeze dried for use as a soluble powder for further solubilization in liquids.

[0066] Nutritional Joint Health Supplement Compositions

[0067] One aspect of the present disclosure can include a nutritional joint health supplement composition. The nutritional joint health supplement composition can comprise a cartilaginous tissue material harvested from a xenogeneic tissue source and at least a trace amount of a detergent. The nutritional joint health supplement composition can have a beneficial component profile and reduced cellular and DNA content.

[0068] In one aspect, the xenogeneic tissue source can include a non-human mammal. In some instances, the non-human mammal can be a genetically-modified organism or a native (non-genetically-modified) organism. In one example, the xenogeneic tissue source can be a pig.

[0069] In another aspect, the xenogeneic tissue source can include a nasal septum, auricular cartilage, costal cartilage, or articular cartilage. In one example, the xenogeneic tissue source can comprise a porcine nasal septum. ^

[0070] In another aspect, the cartilaginous tissue material can comprise predominantly type II collagen. For example, the cartilaginous tissue material can comprise greater than about 50% by weight (wt. %) type II collagen, greater than about 60% by weight (wt. %) type II collagen, greater than about 70% by weight (wt. %) type II collagen, greater than about 80% by weight (wt. %) type II collagen, or greater than about 90% by weight (wt. %) type II collagen.

[0071] In another aspect, the type II collagen comprising the cartilaginous tissue material can be substantially undenatured. For example, the cartilaginous tissue material can comprise at least about 50% by weight (wt. %) undenatured type II collagen, about 55% by weight (wt. %) undenatured type II collagen, about 60% by weight (wt. %) undenatured type II collagen, about 65% by weight (wt. %) undenatured type II collagen, about 70% by weight (wt. %) undenatured type II collagen, about 75% by weight (wt. %) undenatured type II collagen, about 80% by weight (wt. %) undenatured type II collagen, about 85% by weight (wt. %) undenatured type II collagen, about 90% by weight (wt. %) undenatured type II collagen, about 95% by weight (wt. %) undenatured type II collagen, or about 99% by weight (wt. %) undenatured type II collagen. In some instances, the type II collagen comprising the cartilaginous tissue material can be entirely (100%) undenatured.

[0072] In another example, the cartilaginous tissue material can have an undenatured type II collagen content of about 10 ng / mg to about 40 ng / mg on a dry weight basis, about 15 ng / mg to about 35 ng / mg on a dry weight basis, or about 20 ng / mg to about 30 ng / mg on a dry weight basis. In yet another example, the cartilaginous tissue material can have an undenatured type II collagen content of about 10 ng / mg on a dry weight basis, about 15 ng / mg on a dry weight basis, about ^20 ng / mg on a dry weight basis, about 25 ng / mg on a dry weight basis, about 30 ng / mg on a dry weight basis, about 35 ng / mg on a dry weight basis, or about 40 ng / mg on a dry weight basis.

[0073] In another example, the cartilaginous tissue material can have an undenatured type II collagen content of about 1 ng / mg to about 10 ng / mg on a dry weight basis, about 2 ng / mg to about 9 ng / mg on a dry weight basis, about 3 ng / mg to about 8 ng / mg on a dry weight basis, about 4 ng / mg to about 7 ng / mg on a dry weight basis, or about 5 ng / mg to about 6 ng / mg on a dry weight basis. In yet another example, the cartilaginous tissue material can have a type II collagen content of about 1 ng / mg on a dry weight basis, about 2 ng / mg on a dry weight basis, about 3 ng / mg on a dry weight basis, about 4 ng / mg on a dry weight basis, about 5 ng / mg on a dry weight basis, about 6 ng / mg on a dry weight basis, about 7 ng / mg on a dry weight basis, about 8 ng / mg on a dry weight basis, about 9 ng / mg on a dry weight basis, or about 10 ng / mg on a dry weight basis. In a further example, the cartilaginous tissue material can have a type II collagen content of 5 ng / mg on a dry weight basis.

[0074] In another aspect, the cartilaginous tissue material can retain at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the type II collagen and have a water content of less than about 10%, less than about 8%, less than about 6%, less than about 4%, less than about 2% by weight of the total composition, or be void of any water. In one example, the cartilaginous tissue material can retain at least 80% of the type II collagen and have a water content of less than 10% by weight (wt. %) of the total composition.

[0075] In another aspect, the cartilaginous tissue material can lack substantially all DNA; that is, have a low or depleted DNA content relative to that of the ^xenogeneic tissue source. For example, the cartilaginous tissue material can have a DNA content, on a dry weight basis, of less than about 20 ng / mg, e.g., about 0 ng / mg to about 20 ng / mg, about 5 ng / mg to about 15 ng / mg, or about 10 ng / mg to about 15 ng / mg. In another example, the cartilaginous tissue material can have a DNA content, on a dry weight basis, of less than about 18 ng / mg, less than about 16 ng / mg, less than about 14 ng / mg, less than about 12 ng / mg, less than about 10 ng / mg, less than about 8 ng / mg, less than about 6 ng / mg, less than about 4 ng / mg, or less than about 2 ng / mg. In yet another example, the cartilaginous tissue material can have a DNA content, on a dry weight basis, of about 9 ng / mg or about 10 ng / mg.

[0076] In some instances, the cartilaginous tissue material can lack substantially all viable cells and / or cellular remnants; that is, is void (0%) or essentially void of living cells (e.g., of the donor or source organism) and / or cellular remnants. As used herein, “essentially void” and similar terms can refer to undetectable amounts (by one or more, or all, current detection methods). In addition, “essentially void” and similar terms can refer to less than 0.01 %, less than 0.1%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, or less than 35% (wt. %) compared to an original amount or control amount of the component present in the xenogeneic tissue source.

[0077] In some embodiments, the cartilage-derived implant comprises no living cells and / or cellular remnants or a reduced amount of living cells and / or cellular remnants (e.g., compared to the xenogeneic tissue source). As used herein, “reduced amount” and similar terms can refer to any statistically significant decrease in an amount compared to a standard (e.g., the xenogeneic tissue source from which the cartilaginous tissue material was derived). For instance, the cartilaginous tissue ^material can comprise or retain less than 0.0001%, less than 0.001 %, less than 0.01 %, less than 0.1%, less than 1 %, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, or less than 35% (wt. %) of living cells and / or living cells of the donor or source organism. As used herein, “cellular remnants” and similar terms can refer to DNA, RNA, soluble proteins, immunogenic proteins, immunogenic effectors, major histocompatibility complex (MHC) proteins, galactose-alpha-l,3-galactose, blood, and / or cellular vestiges or debris of the donor or source organism (e.g., not including structural components of cartilage and / or the extra-cellular matrix (ECM) of the donor or source organism).

[0078] In another aspect, the cartilaginous tissue material can have a sulfated glycosaminoglycan (sGAG) content, on a dry weight basis, of at least about 30 µg / mg, at least about 40 µg / mg, at least about 50 µg / mg, at least about 60 µg / mg, at least about 70 µg / mg, or at least about 80 µg / mg. For example, the cartilaginous tissue material can have a sGAG content, on a dry weight basis, of about 30 µg / mg to about 80 µg / mg, about 35 µg / mg to about 75 µg / mg, about 40 µg / mg to about 70 µg / mg, about 45 µg / mg to about 65 µg / mg, about 50 µg / mg to about 60 µg / mg, or about 55 µg / mg to about 60 µg / mg.

[0079] In another aspect, the beneficial component profile can comprise: the cartilaginous tissue material having a less than 20 ng / mg DNA content on a dry weight basis; the cartilaginous tissue material having a 30 ng / mg undenatured collagen type II content on a dry weight basis; and / or the cartilaginous tissue material having a sGAG content of about 30 µg / mg to about 80 µg / mg on a dry weight basis. ^

[0080] In another aspect, the detergent comprising the trace amount of detergent present in the nutritional joint health supplement composition can comprise a non- ionic detergent, such as Triton X (e.g., Triton X-100). The detergent is neither an anionic detergent nor a cationic detergent. The trace amount of detergent can be, on a dry weight basis, less than about 20 ng / mg but greater than about 0.5 ng / mg. For example, the trace amount of detergent can be, on a dry weight basis, less than about 18 ng / mg but greater than about 1.5 ng / mg, less than about 16 ng / mg but greater than about 3 ng / mg, less than about 14 ng / mg but greater than about 4.5 ng / mg, less than about 12 ng / mg but greater than about 6 ng / mg, or less than about 10 ng / mg but greater than about 7.5 ng / mg. In another example, the trace amount of detergent can be, on a dry weight basis, about 10 ng / mg to about 20 ng / mg, or about 12 ng / mg to about 16 ng / mg.

[0081] In another aspect, the nutritional joint health supplement composition can be in the form of a particulate or a powder. In some instances, the particulate or the powder can be formulated into an individual dosage vessel selected from the group consisting of a tablet, a capsule, a pill, a suppository, a liquid, a drink, an intravenous or percutaneous injection, a topical cream, a gel, an ointment, an emulsion, a paste, and combinations thereof.

[0082] In another aspect, the nutritional joint health supplement composition can be formulated as a nutraceutical composition, a processed food product, or a pharmaceutical composition.

[0083] In some instances, a particulate or powder nutritional joint health supplement composition can be provided in a mixture with one or more additional components as discussed herein. In some forms, the particulate or powder can ^have an average particle cross section in the range of about 20 to about 350 microns.

[0084] In some instances, nutritional joint health supplement compositions can also include other nutritional supplements, such as vitamins (e.g., vitamin C, vitamin D, Vitamin B's, etc.), as well as minerals, etc. Exemplary minerals that can be present in compositions of the invention include, but are not limited to, calcium, zinc, iron, copper and magnesium, and their pharmaceutically accepted salts. Other desirable additives can additionally or alternatively be included, such as, but not limited to, starches, sugars, fats, antioxidants, amino acids, proteins, derivatives thereof or combinations thereof. Inclusion of additives that assist in formulating the final composition may also be desirable.

[0085] In other aspects, nutritional joint health supplement compositions, together with one or more conventional adjuvants, carriers or diluents, can be placed into the form of nutraceutical / pharmaceutical compositions and unit dosages. The nutraceutical / pharmaceutical compositions and unit dosage forms can be comprised of conventional ingredients in conventional proportions, with or without additional active compounds or principles, and the unit dosage forms can contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed. Nutritional joint health supplement compositions can be employed as solids, such as tablets or filled capsules, semisolids, powders, sustained release formulations, or liquids such as solutions, suspensions, emulsions, elixirs, or filled capsules for oral use; or in the form of sterile injectable solutions for parenteral use.

[0086] Where a nutritional joint health supplement composition is formulated as a solid or semi-solid, an associated carrier can be one or more substances which can ^also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. In powders, the carrier generally is a finely divided solid which is a mixture with the finely divided active component. In tablets, the active component generally is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. Suitable carriers for formulating a preparation of a nutritional joint health supplement composition can include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatine, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term “preparation” is intended to include the formulation of the active composition (i.e., a nutritional joint health supplement composition) with encapsulating material as carrier, providing a capsule in which the active composition, with or without carriers, is surrounded by a carrier, which is in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be as solid forms suitable for oral administration.

[0087] Nutritional joint health supplement compositions can also be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and can be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. Such compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Examples of oily or nonaqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic ^esters (e.g., ethyl oleate), and can contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilizing and / or dispersing agents.

[0088] When desired, formulations can be prepared with enteric coatings adapted for sustained or controlled release administration of the active ingredients (i.e., nutritional joint health supplement composition of the present disclosure).

[0089] Nutritional joint health supplement compositions can be prepared in unit dosage forms, often in a single unit dosage form. As used herein, the term “single unit dosage form” can refer to a unit dosage form that is designed to be administered once a day. In unit dosage forms, the preparation is often subdivided into unit doses containing appropriate quantities of the composition of the invention. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

[0090] Other suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton, Pa.

[0091] In another aspect, a nutritional joint health supplement composition can comprise a therapeutically effective amount of soluble form collagenous tissue material comprising type II collagen. The type II collagen can be solubilized in a physiologically acceptable aqueous medium (e.g., sterile saline or water). As described above, the type II collagen can be harvested from a xenogeneic tissue source and have a beneficial component profile and reduced cellular and DNA content. In such instances, the nutritional joint health supplement composition is free of any detergent(s). ^

[0092] In certain aspects, a solubilized nutritional joint health supplement composition can be suitable for oral administration and include, for example, liquid form preparations including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, semi-solid forms, or solid form preparations, which are intended to be converted shortly before use to liquid form preparations. Emulsions can be prepared in solutions, for example, in aqueous propylene glycol solutions or may contain emulsifying agents, for example, such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the nutritional joint health supplement composition in water and adding suitable colorants, flavors, stabilizers, and thickening agents. Aqueous suspensions can be prepared by dispersing the nutritional joint health supplement composition in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well known suspending agents.

[0093] Therapeutic Methods

[0094] Another aspect of the present disclosure can include a method of treating, reducing, or preventing joint pain in a subject. The method can comprise administering a nutritional joint health supplement composition to the subject. The nutritional joint health supplement composition can comprise a cartilaginous tissue material harvested from a xenogeneic tissue source and at least a trace amount of a detergent. The nutritional joint health supplement composition can have a beneficial component profile and reduced cellular and DNA content. The nutritional joint health supplement composition can be administered to the subject in a therapeutically effective amount, thereby reducing or eliminating joint pain in both a healthy subject and a subject with at least one arthritic joint. ^

[0095] In one example, the nutritional joint health supplement composition can be formulated in a single unit dosage form and be consumed orally by a subject in need thereof.

[0096] In another example, the nutritional joint health supplement composition can be solubilized (as described above) and orally administered to or by a subject in need thereof, e.g., as a liquid solution or emulsion.

[0097] In other instances, the therapeutically effective amount of the nutritional joint health supplement composition can be effective to improve or maintain joint health or reduce, alleviate, and / or slow the progression of at least one symptom in the subject as shown by improvement in the subject’s joint health, as measured by: changes in baseline versus end point scores in a 30 second chair stand test (30SCST); a Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score (WOMAC A Pain), (WOMAC B Stiffness), and / or (WOMAC C Physical function);^range of motion of a body part (e.g., an increased range of motion in knee flexion); radiographic minimum joint space width; and / or serum calcium level.

[0098] Moreover, nutritional joint health supplement compositions of the present disclosure are useful in supporting joint health and treating joint deterioration. As used herein the term “treating” or “treatment” of joint deterioration can include: (1) preventing joint deterioration; (2) reducing the rate and / or the amount of joint deterioration; (3) relieving the joint deterioration, i.e., causing regression of joint deterioration. As used herein the term “supporting” or “support” of joint health can include aiding in formation of collagen found in ligaments, tendons, cartilage, and bone.

[0099] Methods of Production ^

[0100] One aspect of the present disclosure can include a method for preparing a nutritional joint health supplement composition. Broadly, the method can comprise treating a cartilaginous tissue material harvested from a xenogeneic tissue source with an acidic solution to form an acid-treated cartilaginous tissue material containing at least a trace amount of a detergent, wherein the cartilaginous tissue material has a beneficial component profile and reduced cellular and DNA content.

[0101] One example of a method for preparing a nutritional joint health supplement composition is illustrated in Fig.1. The method 10 is illustrated as a processes flow diagram with flowchart illustrations that can be implemented by a combination of steps. For purposes of simplicity, the method 10 is shown and described as being executed serially; however, it is to be understood and appreciated that the present disclosure is not limited by the illustrated order as some steps could occur in different orders and / or concurrently with other steps shown and described herein. Moreover, not all illustrated aspects may be required to implement method 10.

[0102] As shown in Fig.1, the method 10 can include the following Steps: (1) cartilage harvesting, cleaning and slicing (Step 12); (2) peracetic acid decontamination (Step 14); (3) detergent and osmotic treatment (Step 16); (4) salt balance treatment (Step 18); (5) detergent cellular removal (Step 20); (6) water and alcohol rinses for detergent removal (Step 22); (7) freezing and lyophilization (Step 24); and (8) cryomilling (Step 26).

[0103] Although not shown in Fig.1, it will be appreciated that multiple washes (e.g., at least 2, at least 3, at least 4, or at least 5 washes using pH2O at room temperature for about 30-60 minutes) can be applied between any one or combination of the Steps comprising the method 10. ^

[0104] At Step 12, one or more samples of a cartilaginous tissue material from the same or different xenogeneic tissue source(s) can be obtained by harvesting from a non-mammalian source (e.g., a pig). The harvested cartilage tissue material can be cleaned and sliced. In one example, porcine nasal septal cartilage can be harvested, the perichondrium removed during cleaning, and sections thereof sliced at a desired thickness (e.g., 2 mm thickness). The prepared cartilaginous tissue material can then be further processed according to method 10; or, alternatively, frozen in high purity water and stored for subsequent processing.

[0105] At Step 14, the harvested and prepared cartilaginous tissue material can be contacted with an amount peracetic acid at a desired concentration and for a desired period of time sufficient to decontaminate the cartilaginous tissue material, i.e., to remove any microorganisms, such as viruses and fungal spores as well as partially solubilize cells comprising the cartilaginous tissue material. In one example, a harvested and prepared cartilaginous tissue material (e.g., porcine nasal septum) can be treated with about 2% w / w to about 5% w / w peracetic acid (e.g., 3.2% w / w) for about 30-90 minutes (e.g., about 45-75 minutes) at room temperature. Step 14 does not include the use of other (strong) acids (i.e., an acid that completely ionizes or dissociates into ions when dissolved in a solvent, such as water) at high concentrations, such as HCl, nor highly concentrated weak acids, such as acetic acid, citric acid, etc.) because application of such acids would negatively affect collagen type II structure, i.e., result in undesirable denaturation.

[0106] At Step 16, the acid-treated cartilaginous tissue material of Step 14 is subjected to detergent and osmotic treatment using a solution. Advantageously, the solution is formulated to balance the need to maximize ECM native structure with at least partial decellularization of the cartilaginous tissue material. As such, the ^solution can comprise a non-ionic detergent, a basic buffer, an acidic buffer, a chelating agent, and a mineral salt. In one example, the solution can comprise KCl (e.g., about 100-120 g / L, for example, 111.41 g / L), EDTA (e.g., about 1-3 g / L, for example, 1.456 g / L), TrisHCl (e.g., about 2-4 g / L, for example, 3.2 g / L), Tris (e.g., about 0.1-1 g / L, for example, 0.5 g / L), and Triton X-100 (e.g., about 5-15 mL / L, for example, 10 mL / L) and be applied for about 10-20 hours (e.g., 16 hours) at room temperature.

[0107] At Step 18, the cartilaginous tissue material from Step 16 can be treated with a balanced salt solution. In one example, the balanced salt solution can comprise HEPES (e.g., about 1-3 g / L, for example, 2.37 g / L) in 1X PBS for about 15- 90 minutes (e.g., 30-60 minutes) at room temperature.

[0108] At Step 20, the cartilaginous tissue material from Step 18 is treated with a further detergent to decellularize the material. In one example, the further detergent can comprise a non-ionic detergent, such as Triton X-100 (e.g., about 20-40 ml / L, for example, 30 ml / L), which can be applied for about 10-20 hours (e.g., 16 hours) at room temperature.

[0109] At Step 22, the decellularized cartilaginous tissue material from Step 20 is contacted with an alcohol solution and, optionally, one or more rinses (e.g., using pH2O). In one example, the alcohol solution can comprise isopropyl alcohol (e.g., about 50-80%, for example, 70%), which is applied for about 15-90 minutes (e.g., 30-60 minutes) at room temperature.

[0110] At Step 24, the cartilaginous tissue material from Step 22 can be frozen and lyophilized. For example, the cartilaginous tissue material can be frozen at - 80°C. for at least 4 hours. Thereafter, the cartilaginous tissue material can be dried. Drying can be conducted sufficiently to stabilize the processed cartilaginous tissue ^material. Drying of the processed cartilaginous tissue material can involve lyophilization (or freeze drying) or vacuum drying at ambient or elevated temperatures.

[0111] At Step 26, the frozen and lyophilized cartilaginous tissue material of Step 24 can be formed into a powder using one or a combination of techniques, such as grinding, a hammer mill, or cryomilling (e.g., milled with liquid nitrogen). In one example, the frozen and lyophilized cartilaginous tissue material of Step 24 can be formed into a powder by liquid nitrogen milling at -300°F. for about 5-30 minutes (e.g., 15 minutes).

[0112] In some instances, the powder cartilaginous tissue material formed by the method 10 can be refrozen and / or sterilized (e.g., by irradiation, ethylene oxide gas, or any other suitable sterilization technique).

[0113] It will be appreciated that additional washing steps can be included as part of the method 10 where the nutritional joint health supplement composition is to be solubilized to ensure no trace amount(s) of detergent(s) are present in the composition. In one example, an amount of powdered nutritional joint health supplement composition (e.g., lyophilized) that is free of any detergent(s) can be dissolved in a mild acid (e.g., 0.01-0.05 N HCl or 1.0-0.25 M acetic acid) at a range of about 5 mg to about 50 mg / ml (e.g., 10 mg / ml to 30 mg / ml) at room temperature or colder, with agitation, for about 24-72 hours (e.g., 48 hours). Optionally, dialysis can be performed using a 14 kDa cut-off cellulose dialysis membrane overnight, at 4°C., with 0.1 mN HCl or 0.1 mM acetic acid.

[0114] It will also be appreciated that the method 10 does not include any heating steps (e.g., performed above 37°C.) or steps that include application or use of a basic solution (e.g., a pH greater than 8.5). ^

[0115] Exemplary Aspects

[0116] In view of the described compositions, devices, and methods and variations thereof, herein below are certain more particularly described aspects of the present disclosure. These particularly recited aspects should not, however, be interpreted to have any limiting effect on any different claims containing different or more general teachings described herein, or that the “particular” aspects are somehow limited in some way other than the inherent meanings of the language literally used therein.

[0117] Aspect 1: A nutritional joint health supplement composition comprising a cartilaginous tissue material harvested from a xenogeneic tissue source and at least a trace amount of a detergent, wherein the nutritional joint health supplement composition has a beneficial component profile and reduced cellular and DNA content.

[0118] Aspect 2: The nutritional joint health supplement composition of Aspect 1, wherein the xenogeneic tissue source is a non-human mammal.

[0119] Aspect 3: The nutritional joint health supplement composition of any one of Aspects 1-2, wherein the xenogeneic tissue source is one of a nasal septum, auricular cartilage, costal cartilage, or articular cartilage.

[0120] Aspect 4: The nutritional joint health supplement composition of any one of Aspects 1-3, wherein the xenogeneic tissue source is a porcine nasal septum.

[0121] Aspect 5: The nutritional joint health supplement composition of any one of Aspects 1-4, wherein the cartilaginous tissue material comprises predominantly type II collagen.

[0122] Aspect 6: The nutritional joint health supplement composition of any one of Aspects 1-5, wherein the type II collagen is substantially undenatured. ^

[0123] Aspect 7: The nutritional joint health supplement composition of any one of Aspects 1-6, wherein a type II collagen content of the cartilaginous tissue material is 25 wt. % or greater, 50 wt. % or greater, or 70 wt. % or greater.

[0124] Aspect 8: The nutritional joint health supplement composition of any one of Aspects 1-7, retaining at least 80% of the type II collagen and having a water content of less than 10% by weight of the total composition.

[0125] Aspect 9: The nutritional joint health supplement composition of any one of Aspects 1-8, wherein the beneficial component profile comprises: the cartilaginous tissue material having a less than 20 ng / mg DNA content on a dry weight basis; and wherein the cartilaginous tissue material having a 30 ng / mg undenatured collagen type II content on a dry weight basis.

[0126] Aspect 10: The nutritional joint health supplement composition of any one of Aspects 1-9, wherein the beneficial component profile further comprises the cartilaginous tissue material having a sulfated glycosaminoglycan (sGAG) content of about 30 µg / mg to about 80 µg / mg on a dry weight basis.

[0127] Aspect 11: The nutritional joint health supplement composition of any one of Aspects 1-10, wherein the detergent is a non-ionic detergent.

[0128] Aspect 12: The nutritional joint health supplement composition of any one of Aspects 1-11, wherein the trace amount of the detergent is less than about 20 ng / mg on a dry weight basis but greater than about 0.5 ng / mg on a dry weight basis.

[0129] Aspect 13: The nutritional joint health supplement composition of any one of Aspects 1-12, wherein the detergent is neither an anionic detergent nor a cationic detergent.

[0130] Aspect 14: The nutritional joint health supplement composition of any one of Aspects 1-13, being in the form of a particulate or a powder. ^

[0131] Aspect 15: The nutritional joint health supplement composition of any one of Aspects 1-14, wherein the particulate or the powder is formulated into an individual dosage vessel selected from the group consisting of a tablet, a capsule, a pill, a suppository, a liquid, a drink, an intravenous or percutaneous injection, a topical cream, a gel, an ointment, an emulsion, a paste, and combinations thereof.

[0132] Aspect 16: The nutritional joint health supplement composition of any one of Aspects 1-15, being formulated as a nutraceutical composition, a processed food product, or a pharmaceutical composition.

[0133] Aspect 17: A method for preparing a nutritional joint health supplement composition, the method comprising: treating a cartilaginous tissue material harvested from a xenogeneic tissue source with an acidic solution to form an acid- treated cartilaginous tissue material containing at least a trace amount of a detergent, wherein the cartilaginous tissue material has a beneficial component profile and reduced cellular and DNA content.

[0134] Aspect 18: The method of Aspect 17, wherein the acidic solution comprises about 1.5 % w / w to about 3.5% w / w peracetic acid.

[0135] Aspect 19: The method of any one of Aspects 17-18, wherein the acidic solution does not comprise hydrochloric acid, acetic acid and / or citric acid.

[0136] Aspect 20: The method of any one of Aspects 17-19, further comprising removing perichondrium from the cartilaginous tissue material harvested from porcine nasal septal tissue.

[0137] Aspect 21: The method of any one of Aspects 17-20, further comprising, after contacting the cartilaginous tissue material with the acidic solution, subjecting the acid-treated cartilaginous tissue material to a detergent and osmotic treatment. ^

[0138] Aspect 22: The method of any one of Aspects 17-21 wherein subjecting the acid-treated cartilaginous tissue material to a detergent and osmotic treatment includes applying a solution comprising a non-ionic detergent, a basic buffer, an acidic buffer, a chelating agent, and a mineral salt.

[0139] Aspect 23: The method of any one of Aspects 17-22, further comprising, after applying the solution, the steps of: freezing and lyophilizing the cartilaginous tissue material; and cryomilling the cartilaginous tissue material into a powder.

[0140] Aspect 24: The method of any one of Aspects 17-23, wherein neither heat nor a basic solution is applied to any step thereof.

[0141] Aspect 25: A nutritional joint health supplement composition formed by the method of any one of Aspects 17-24.

[0142] Aspect 26: A method of treating joint pain in a subject comprising administering a nutritional joint health supplement composition to the subject, the nutritional joint health supplement composition comprising a cartilaginous tissue material harvested from a xenogeneic tissue source and at least a trace amount of a detergent, wherein the nutritional joint health supplement composition has a beneficial component profile and reduced cellular and DNA content, the nutritional joint health supplement composition being administered to the subject in a therapeutically effective amount to reduce joint pain in both a healthy subject and a subject with at least one arthritic joint.

[0143] Aspect 27: The method of Aspect 26,^wherein the nutritional joint health supplement composition is consumed orally.

[0144] Aspect 28: The method of any one of Aspects 26-27, wherein the therapeutically effective amount of the nutritional joint health supplement composition is effective to improve or maintain joint health or reduce, alleviate, ^and / or slow the progression of at least one symptom in the subject as shown by improvement in the subject’s joint health, as measured by: changes in baseline versus end point scores in a 30 second chair stand test (30SCST); a Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score (WOMAC A Pain), (WOMAC B Stiffness), and / or (WOMAC C Physical function);^range of motion of a body part; radiographic minimum joint space width; and / or serum calcium level.

[0145] Aspect 29: The method of any one of Aspects 26-28, wherein the improvement in the subject’s joint health is an increased range of motion in knee flexion.

[0146] Aspect 30: A nutritional joint health supplement composition comprising a therapeutically effective amount of soluble form collagenous tissue material comprising type II collagen, the type II collagen being solubilized in a physiologically acceptable aqueous medium, wherein the type II collagen is harvested from a xenogeneic tissue source, is free of any detergent(s), and has a beneficial component profile and reduced cellular and DNA content.

[0147] Aspect 31: A method for forming the nutritional joint health supplement composition of Aspect 30, comprising treating a cartilaginous tissue material harvested from a xenogeneic tissue source with an acidic solution to form an acid- treated cartilaginous tissue material, wherein the cartilaginous tissue material is free of any detergent(s) and has a beneficial component profile and reduced cellular and DNA content.

[0148] Aspect 32: A method of treating joint pain in a subject comprising administering the nutritional joint health supplement composition of Aspect 30 to the subject, the nutritional joint health supplement composition being administered to the ^subject in a therapeutically effective amount to reduce joint pain in both a healthy subject and a subject with at least one arthritic joint.

[0149] The following Examples are for the purpose of illustration only and are not intended to limit the scope of the claims, which are appended hereto. Example 1

[0150] Experiments were performed to biochemically characterize a nutritional joint health supplement composition formulated according to one aspect of the present disclosure.

[0151] Methods

[0152] Histology and SDS-PAGE

[0153] Several pieces of the porcine nasal septum cartilage, having been processed according to the present disclosure, were collected and analyzed though histology. Briefly, porcine cartilage was fixed in 4% formalin in PBS for at minimum 48 hours. Then, it was paraffinized, sectioned on glass slides, and stained for Hematoxylin and Eosin. Pictures were taken with a slide scanner.

[0154] For SDS-PAGE, processed powdered cartilage was incubated in 0.5 M acetic acid and 1 mg / ml pepsin for 24 hours at 37°C. and agitation. After degradation, samples were centrifuged and supernatant collected, combined with loading buffer (4X) and NaOH for neutralization, and loaded in a 4-15% precast SDS-PAGE plate. After running the electrophoresis for 2 hours at 120 V, gel was detached from the plate and stained with Coomassie blue for band observation. A collagen type I standard was used as reference.

[0155] Biochemical analysis

[0156] Processed powdered collagen was analyzed for detergent content, DNA content, total collagen content, and native collagen type II content. ^

[0157] For DNA analysis, the powder was digested in proteinase K degradation buffer at 55°C overnight in agitation. The degraded solution was centrifuged, and DNA content quantified employing the QuantiFluor dsDNA kit (Promega, USA).

[0158] For detergent (Triton-X100) quantification, the powder was incubated in water at 37°C overnight under agitation. Solution was then centrifuged, and detergent quantified in the supernatant using a CMC-535 detergent assay (G- Biosciences, USA).

[0159] Hydroxyproline assay was used to quantify total collagen in the powder. Hydrolysis of the powder was achieved at 10 M NaOH overnight at 120°C. Hydrolysates were neutralized with 6 N HCl and used for hydroxyproline quantification using a Hydroxyproline assay kit (Cell Biolabs, Inc., USA).

[0160] For native collagen type II quantification, the powder was incubated in 0.5 M acetic acid and 1 mg / ml pepsin for 24 hours at 37°C. and agitation. After degradation, samples were centrifuged and supernatant neutralized with 1M NaOH. Then, samples were analyzed using a native type II collagen ELISA (6018, Chondrex, USA).

[0161] Results

[0162] As shown in Figs.2A-C, characterization of processed cartilage with histology showed the preservation of native ECM cartilage structure, although some nuclei were still present. The presence of collagen type II was also observed by SDS-PAGE, where presence of a2 band was lower than in collagen type I pure standard, demonstrating the predominance of collagen type II in the porcine collagen as prepared by the present disclosure

[0163] As also shown in Figs.2A-C, biochemical characterization showed a very low content of DNA and residual detergent, showing safety from an oral consumption ^standpoint. Collagen content in the dry powder varied between 50-70% of total powder dry weight. ELISA analysis also showed presence of native (undenatured) collagen type II in all the lots analyzed, including excipients, averaging 5 ng / mg dry powder. Example 2

[0164] Experiments were performed to investigate efficacy of a nutritional joint health supplement composition (Fig.3A) in a preclinical monoiodoacetate (MIA)- induced arthritis rat model.

[0165] Methods

[0166] Animals

[0167] All procedures were approved by and performed according to the guidelines of the Institutional Animal Care and Use Committee at the University of Pittsburgh. For all the experiments described below, adult female Sprague Dawley rats (Envigo, Indianapolis, Indiana) weighing approximately 200-250g at the beginning of the experimental procedure were used. The rats were group-housed in a temperature- and humidity-controlled room on a regular 12 h light / dark cycle. Standard diet and water were provided ad libitum.

[0168] Monosodium iodoacetate-induced arthritis model

[0169] The monosodium iodoacetate (MIA) rat model was conducted as previously described (see Di Cesare Mannelli et al., BMC musculoskeletal disorders. 2013;14:1-9). A total of 15 rats were randomly assigned into two groups: (i) MIA + saline control and (ii) MIA + sample B type II collagen (porcine cartilage collagen). Inventors were blinded to the identity of samples. To induce arthritis, rats were anesthetized with isoflurane, the right knee was shaved and disinfected with 70% ethanol, and 3 mg / kg of MIA (Sigma Aldrich, I2512) dissolved in 25 ^l PBS were ^injected into the articular cavity of the right knee joints using a 30-G needle. Following injection with MIA on day 0, 3 mg / kg of type II collagen supplements was administered by oral gavage to group (ii), while group (i) received saline by oral gavage daily until day 14. Rats were observed daily to assess knee joint swelling. At the 14-day timepoint the rats were sacrificed, and the knee joints and blood samples were collected for downstream analyses.

[0170] Gait analysis and assessment of joint swelling

[0171] The hind paws of the rats were brushed with dark blue ink to perform gait analysis as previously described (see Orhan C. et al., Frontiers in veterinary science. 2021;8:617789). The rats were allowed to walk on a white paper (60 cm-long, 7 cm- wide). The footprints of the rats were scanned at 300 dpi and paw area (cm2), paw width (cm), and stride length (cm) measurements were calculated with Image J software (National Institutes of Health, USA). The diameter (mm) of joints was measured in triplicate daily using a digital caliper to assess joint swelling.

[0172] Histology and immunohistochemistry

[0173] Rat knee joints were collected at euthanasia and fixed in 10% formalin, decalcified using 5% formic acid, and embedded in paraffin wax. Tissue sections were stained with Safranin O for microscopic assessment of cartilage histopathology. Macrophages and T cells in the periarticular area were assessed by immunolabeling. M1-like- and M2-like macrophages were identified by dual-positive staining with CD68 and CD86, or CD68 and CD206, respectively. The local lymphocyte response was evaluated by immunolabeling with antibodies against CD4 and CD8. Slides were imaged using a MoticEasyScan (Schertz, TX, USA) brightfield digital slide scanner or a Zeiss Axio-observer Z1 fluorescent microscope. Cell Profiler software ^was used to quantify the percentage of M1 and M2 macrophages or T cells per field of view (FOV).

[0174] Cartilage degradation scoring (Safranin-O)

[0175] Histological scoring of Safranin O-stained sections was performed to assess osteoarthritis (OA) severity. Each section was prepared from posterior sectioning in the frontal plane of every single knee, including the medial femoral condyle, medial tibial plateau, lateral femoral condyle, and lateral tibial plateau. The section extended horizontally across the entire joint surface and vertically from the articular surface to below the subchondral bone plate. Microscopic evaluation was conducted at 5x magnification, and cartilage scoring was performed according to the Osteoarthritis Research Society International (OARSI) guidelines for histopathological grading (Table 1) (see Pritzker KP et al., Osteoarthritis and cartilage.2006;14(1):13-29; and Gerwin N et al., Osteoarthritis and cartilage. 2010;18:S24-S34). Each FOV was assessed by three blinded, independent scorers, and consensus scores were tabulated. Averaged scores for each treatment group were reported and performed statistical analysis. Table 1. OA cartilage histopathology grade assessment—grading methodology^

[0176] Serum cytokine and collagen type II assessment

[0177] Blood samples were collected on day 14. Serum cytokine and chemokine concentrations were assessed using rat-specific, bead-based, multiplex ELISA (Thermo Fisher Scientific, EPX220-30122-901) and the rat crosslinked collagen type II telopeptide ELISA (Antibodies Online ABIN6963322).

[0178] Statistical Analysis

[0179] To prevent bias during experimentation and / or analysis, the inventors were blinded to the identity of control and sample throughout the study. Following study completion, sample identity was revealed. Data analysis was performed on the percentage difference between intraday and initial mechanical threshold values (daily body weight, and daily joint width; normalized values in order to control for unwanted sources of variation) or on the absolute threshold values (stride length, paw length, paw area, cytokines and chemokines concentrations and OARSI scores; crude values). The host immune response (macrophage staining / T-cell staining) will be evaluated using CellProfiler software. Total number of macrophages and T-cells will be quantified. The percentage of total M1-like and M2-like macrophages will be calculated to determine macrophage polarization, and the ratio of M2:M1 macrophages is determined. The percentage of total helper T cells (CD4) and cytotoxic T-cells (CD8) is calculated to determine ratio of CD4:CD8 T-cells. Data are presented as mean ± standard error of five biologic replicates. Significant differences were determined by one-way model ANOVA, with P<0.05 accepted as significant (GraphPad Prism 10.0.3).

[0180] Results

[0181] Animals treated with porcine cartilage did not show any adverse effects to oral supplementation through the study. Weight follow-up revealed a decrease in ^rats injected with MIA and supplemented with saline, while porcine collagen prevented weight loss, indicating a likely prevention of pain and associated distress on animals (Fig.3B). The sustained inflammation in animals MIA-treated knee as compared to the untreated knee for the 14 days of the study supported the suitability of the model. Functional movement analysis was analyzed using a Gait test. Some differences were observed amongst treatment groups for paw width and stride length (Fig.3C).

[0182] As shown in Fig.4A, analysis of C-terminal cross-linked telopeptides of type II collagen (CTX-II) was performed in serum. In the MIA group, serum CTX-II concentration showed a slight decrease while porcine collagen group exhibited an increase in serum CTX-II levels from baseline. These results suggest treatment appeared to elevate serum CTX-II concentrations and therefore an intestinal absorption was taking place. Regarding serum cytokine levels, porcine collagen showed a tendency to restore basal levels of IL-17A, IL-12, eotaxin, MCP3 and RANTES.

[0183] To visualize articular cartilage for histologic scoring, tissue sections were stained with Safranin-O (orange / red color; used to detect cartilage proteoglycans), Fast Green (blue / green; stains bone / connective tissues), and Iron Hematoxylin (black; stains nuclei), while cartilage scoring was performed according to the Osteoarthritis Research Society International (OARSI). Analysis of the stained joints showed that porcine cartilage had a lower mean pathology score (Fig.4B). Macrophage infiltration into the synovium was also quantified (data not shown). Example 3 ^

[0184] This Example illustrates an experiment in which sGAG content of porcine nasal septum cartilage (pNSC), prepared by the method 10 of the present disclosure, is determined.

[0185] The sGAG content of processed (as prepared by the method 10 of the present disclosure) and unprocessed pNSC samples is determined using the Dimethylmethylene Blue (DMMB) assay. Triplicate samples, each approximately 25 mg, are taken from both processed and unprocessed pNSC. The samples are placed in microfuge tubes and 180 ^l of PBS is added. A 180 ^l sample of HPW serves as a negative control and blank solution. 20 ^l of Proteinase K (600 units / ml HPW) is added to each sample before brief mixing with a vortex mixer. The sample tubes are then placed in a 56°C. incubator with constant orbital shaking and left to digest. After 120 minutes the samples are determined to be completely digested and cooled to room temperature. A 1:750 dilution (2 ^L of digest in 1498 ^L of HPW) of the unprocessed samples is used.

[0186] A heparin standard curve is created from a stock solution of 1200 ^g / ml heparin sodium salt (0.0024 g in 2 mL HPW) diluted with HPW to concentrations of 12, 24, and 120 ^g / ml. The DMMB solution is made by adding 95 ml of 0.1 M HCl to 500 ml HPW followed by the addition of 0.0162 g DMMB, 3.0446 g glycine, and 2.3739 g NaCl. The solution is then diluted to 1000 mL with HPW and stirred until completely dissolved. The pH is adjusted to 3.08 using 1 M NaOH and / or 1M HCl. The DMMB is prepared fresh before the assay and protected from exposure to light.

[0187] For each sample, control, and standard, 200 ^L is aliquoted into three corresponding 15 mL centrifuge tubes. These serve as sample triplicates. 2.5 mL of DMMB solution, as above, is added to each tube and briefly mixed. 200 ^L of the solution is immediately aliquoted into a 96-well microplate and the absorbance is ^read at 600 nm. The reported absorbance values are averaged for each set of triplicate aliquots. The average absorbance values are then blank corrected using the average absorbance of the HPW control. The blank corrected average absorbance values for the heparin standards are used to create a standard curve. The results of the assay, listed in Table 2, indicate retention of at least 50-60% of the original sGAG content after processing, which is unexpected as decellularized porcine septum typically contains 15-20 µg / mg tissue dry weight (see Tluczynski and Breiter, Xenotransplantation, Vol.26, No.6, 20 September 2022). Table 2: Average sGAG Content of Processed and Unprocessed pNSC Samples

[0188] From the above description of the present disclosure, those skilled in the art will perceive improvements, changes, and modifications. Such improvements, changes, and modifications are within the skill of those in the art and are intended to be covered by the appended claims. All patents, patent applications, and publications cited herein are incorporated by reference in their entirety. ^

Claims

CLAIMS The following is claimed:

1. A nutritional joint health supplement composition comprising a cartilaginous tissue material harvested from a xenogeneic tissue source and at least a trace amount of a detergent, wherein the nutritional joint health supplement composition has a beneficial component profile and reduced cellular and DNA content.

2. The nutritional joint health supplement composition of claim 1, wherein the xenogeneic tissue source is a non-human mammal.

3. The nutritional joint health supplement composition of claim 2, wherein the xenogeneic tissue source is one of a nasal septum, auricular cartilage, costal cartilage, or articular cartilage.

4. The nutritional joint health supplement composition of claim 3, wherein the xenogeneic tissue source is a porcine nasal septum.

5. The nutritional joint health supplement composition of claim 1, wherein the cartilaginous tissue material comprises predominantly type II collagen.

6. The nutritional joint health supplement composition of claim 5, wherein the type II collagen is substantially undenatured. ^7. The nutritional joint health supplement composition of claim 6, wherein a type II collagen content of the cartilaginous tissue material is 25 wt. % or greater, 50 wt. % or greater, or 70 wt. % or greater.

8. The nutritional joint health supplement composition of claim 6, retaining at least 80% of the type II collagen and having a water content of less than 10% by weight (wt. %) of the total composition.

9. The nutritional joint health supplement composition of claim 6, wherein the beneficial component profile comprises: the cartilaginous tissue material having a less than 20 ng / mg DNA content on a dry weight basis; and wherein the cartilaginous tissue material having a 30 ng / mg undenatured collagen type II content on a dry weight basis.

10. The nutritional joint health supplement composition of claim 6, wherein the beneficial component profile further comprises the cartilaginous tissue material having a sulfated glycosaminoglycan (sGAG) content of about 30 µg / mg to about 80 µg / mg on a dry weight basis.

11. The nutritional joint health supplement composition of claim 1, wherein the detergent is a non-ionic detergent. ^12. The nutritional joint health supplement composition of claim 11, wherein the trace amount of the detergent is less than about 20 ng / mg on a dry weight basis but greater than about 0.5 ng / mg on a dry weight basis.

13. The nutritional joint health supplement composition of claim 1, wherein the detergent is neither an anionic detergent nor a cationic detergent.

14. The nutritional joint health supplement composition of claim 1, being in the form of a particulate or a powder.

15. The nutritional joint health supplement composition of claim 14, wherein the particulate or the powder is formulated into an individual dosage vessel selected from the group consisting of a tablet, a capsule, a pill, a suppository, a liquid, a drink, an intravenous or percutaneous injection, a topical cream, a gel, an ointment, an emulsion, a paste, and combinations thereof.

16. The nutritional joint health supplement composition of any one of claims 1-15, being formulated as a nutraceutical composition, a processed food product, or a pharmaceutical composition.

17. A method for preparing a nutritional joint health supplement composition, the method comprising: treating a cartilaginous tissue material harvested from a xenogeneic tissue source with an acidic solution to form an acid-treated cartilaginous tissue material ^containing at least a trace amount of a detergent, wherein the cartilaginous tissue material has a beneficial component profile and reduced cellular and DNA content.

18. The method of claim 17, wherein the acidic solution comprises about 1.5 % w / w to about 3.5% w / w peracetic acid.

19. The method of claim 17, wherein the acidic solution does not comprise hydrochloric acid, acetic acid and / or citric acid.

20. The method of claim 17, further comprising removing perichondrium from the cartilaginous tissue material harvested from porcine nasal septal tissue.

21. The method of claim 17, further comprising, after contacting the cartilaginous tissue material with the acidic solution, subjecting the acid-treated cartilaginous tissue material to a detergent and osmotic treatment.

22. The method of claim 21 wherein subjecting the acid-treated cartilaginous tissue material to a detergent and osmotic treatment includes applying a solution comprising a non-ionic detergent, a basic buffer, an acidic buffer, a chelating agent, and a mineral salt.

23. The method of claim 22, further comprising, after applying the solution, the steps of: freezing and lyophilizing the cartilaginous tissue material; and cryomilling the cartilaginous tissue material into a powder. ^24. The method of claim 17, wherein neither heat nor a basic solution is applied to any step thereof.

25. A nutritional joint health supplement composition formed by the method of any one of claims 17-24.

26. A method of treating joint pain in a subject comprising administering a nutritional joint health supplement composition to the subject, the nutritional joint health supplement composition comprising a cartilaginous tissue material harvested from a xenogeneic tissue source and at least a trace amount of a detergent, wherein the nutritional joint health supplement composition has a beneficial component profile and reduced cellular and DNA content, the nutritional joint health supplement composition being administered to the subject in a therapeutically effective amount to reduce joint pain in both a healthy subject and a subject with at least one arthritic joint.

27. The method of claim 26,^wherein the nutritional joint health supplement composition is consumed orally.

28. The method of claim 26, wherein the therapeutically effective amount of the nutritional joint health supplement composition is effective to improve or maintain joint health or reduce, alleviate, and / or slow the progression of at least one symptom in the subject as shown by improvement in the subject’s joint health, as measured by: changes in baseline versus end point scores in a 30 second chair ^stand test (30SCST); a Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score (WOMAC A Pain), (WOMAC B Stiffness), and / or (WOMAC C Physical function);^range of motion of a body part; radiographic minimum joint space width; and / or serum calcium level.

29. The method of claim 28, wherein the improvement in the subject’s joint health is an increased range of motion in knee flexion.

30. A nutritional joint health supplement composition comprising a therapeutically effective amount of soluble form cartilaginous tissue material comprising type II collagen, the type II collagen being solubilized in a physiologically acceptable aqueous medium, wherein the type II collagen is harvested from a xenogeneic tissue source, is free of any detergent(s), and has a beneficial component profile and reduced cellular and DNA content.

31. A method for forming the nutritional joint health supplement composition of claim 30, comprising treating a cartilaginous tissue material harvested from a xenogeneic tissue source with an acidic solution to form an acid-treated cartilaginous tissue material, wherein the cartilaginous tissue material is free of any detergent(s) and has a beneficial component profile and reduced cellular and DNA content.

32. A method of treating joint pain in a subject comprising administering the nutritional joint health supplement composition of claim 30 to the subject, the nutritional joint health supplement composition being administered to the subject in a ^therapeutically effective amount to reduce joint pain in both a healthy subject and a subject with at least one arthritic joint. ^

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