Compositions and methods for intraarticular treatments

Abstract

Methods and compositions for treating or the preventative treatment of intraarticular injury in a subject. The compositions can include a carrier composition and a gas entrapped within the carrier composition. The gas can include carbon monoxide.

Description

[0001] COMPOSITIONS AND METHODS FOR INTRAARTICULAR TREATMENTS

[0002] CROSS REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 653,089, filed May 29, 2024, the entire content of which is incorporated by reference in its entirety .

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under CA086862 and CA276908 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0006] BACKGROUND

[0007] Early articular cartilage damage contributes to post-traumatic osteoarthritis (PTOA) via oxidation and mitochondrial dysfunction. There are also mitochondrial abnormalities in chondrocytes later in disease progression. PTOA is a debilitating joint disease that can develop after a joint injury. Injuries to articular cartilage are known to initiate oxidative damage to resident articular chondrocyte mitochondria over the first few hours after injury. Similar damage to chondrocytes can result from intra-articular surgical exposures. Treatments for articular cartilage damage or injury to mitigate PTOA are investigated.

[0008] SUMMARY

[0009] In one aspect, a method of treatment or preventative treatment of intraarticular injury or damage in a subject is provided. The method can include applying a gas-entrapped composition to cartilage and / or a joint in a subject.

[0010] In another aspect, formulations and compositions that comprise a carrier composition and a gas for the treatment or preventative treatment of intraarticular injury or damage are provided.

[0011] BRIEF DESCRIPTION OF THE FIGURES

[0012] FIGS. 1A-1F. CO Rapidly Induces Chondrocyte HO-1. mitofusin-1, and Increases Mitochondrial Function. Western blots of tissue homogenates from explants at various timepoints after CO exposure show increasing presence of HOI within 1 h after CO compared to control specimens exposed to normal 5% O2 gassed solutions (FIG. 1A). This cohered with increased presence of mitofusin-1. Mitochondrial stress tests of chondrocytes extracted from similarly treated cartilage showed increased per-cell basal respiration (FIG. IB), ATP-linked respiration (FIG. 1C), and no indications of damage to the mitochondrial electron transport chain as indicated by a lack of change in proton leakage (FIG. ID). Densitometric analyses after CO exposure (FIG. IE). Mean OCR from mitochondrial stress tests (FIG. IF), n = 1 1, * p < 0.05 b, ** p < 0.01 by t-test.

[0013] FIGS. 2A-2C. HO-1 overexpression in articular chondrocytes increases mitochondrial metabolism and decreases pro-inflammatory signaling. Immunohistochemical staining of HOI in articular cartilage shows increased staining in articular chondrocytes after 2 days of doxycycline feeding. These increases are not observed elsewhere in the joint. Western blotting of protein homogenates from the joints of these animals show induction of electron transport chain complexes II, III, and IV, suggesting increased respiratory capacity (FIG. 2B) and decreased total p65 (FIG. 2B). Lastly, these samples also showed increased sirtuin 1 (SIRT1), supporting the hypothesis that mitochondrial biogenesis was stimulated.

[0014] FIGS. 3A and 3B. CO augments articular chondrocyte GSH metabolism to prevent damage. Osteochondral tissue was impacted with 2.0 J / cm2impacts immediately after CO or control foam treatments. Control foams incurred losses in MCB that suggest redox injury (representative images shown in FIG. 3A, quantified in FIG. 3B). With impact, control foams did not prevent loss of MCB staining, while CO prevented oxidation of GSH indicated by this stain, n = 5, p < 0.05 by two way ANOVA.

[0015] FIGS. 4A-4F. Intraarticular CO is non-toxic to chondrocytes and improves cartilage health in situ. Fluoroscopy of iodinated COF shows how intraarticular injection can fill the stifle joints of rabbits (FIG. 4A) and mice (FIG. 4B). Rabbit joints exposed to CO in this manner show increased MCB staining compared to contralateral joints exposed to RAF (shown in FIG. 4C, quantified in FIG. 4D alongside biochemical analyses of GSH oxidation to glutathione disulfide (GSSG))( FIG. 4E). n = 5, p = 0.0007 by pairwise t-test for MCB analyses. The viability of hock (ankle) injections during these experiments (FIG. 4F) was also confirmed.

[0016] FIGS. 5A-5E. Intraarticular COF injection is Safe for Synovia. Representative images of synovia captured from rabbits either 1 week or 4 weeks after COF or RAF injection demonstrate no indications of thickening or increased infiltration by immune cells (FIG. 5A, quantified in FIG. 5B and FIG. 5C). No significant differences were observed, n = 10, analyzed via two way ANOVA. Analyses of live cell densities from rabbits 1 week or 4 weeks after COF or RAF injection demonstrated no indications of any changes in live cell density at either time point or using volumetric analyses (FIG. 5D) or viable cell counts per field (FIG. 5E). FIGS. 6A-6C. Activation of HOI in Articular Chondrocytes Prevents PTOA. Mice given destabilization of the medial meniscus (DMM) reliably develop PTOA by 8-10 weeks. TgHOl mice given doxycycline containing chow (625 mg / kg) for 1 week during the fourth week after injury show decreased average (FIG. 6B) and maximum (FIG. 6C) OARSI scores, suggesting HOI prevents PTOA. N = 4, p = 0.0857 by Kruskal Wallis.

[0017] FIG. 7. A schematic diagram depicting various targets for a joint damage reversal.

[0018] FIGS. 8A and 8B. Schematic representations of constructs used in the method of cartilagespecific HOI overexpression described in the Examples.

[0019] FIGS. 9A-B. Representative images of bovine articular cartilage 24 h after RAF or COF treatment stained with live cell dyes for ATP (red) and mitotracker (blue) colocalization (magenta) demonstrated that COF treatment has increased ATP-positive staining, magnification 20 x. (FIG. 9A) Quantitation of ATP staining demonstrated COF staining was significantly increased compared to paired RAF controls (FIG. 9B); paired -test * p < 0.05.

[0020] FIGS. 10A-D. CO pretreatment is mitochondrial protective and decreases oxidative damage after injury'. Representative images of mitochondrial marker TOMM20 (purple, yellow arrows highlight intracellular staining) and merger with Saf-O, 20x (FIG. 10A). Quantitation of superficial zone demonstrates that RAF + impact significantly decreased TOMM20. The COF + impact had significant more TOMM20 positivity compared to RAF + impact; n = 4, nonparametric Mann-Whitney test (FIG. 10B). Representative images of 3NT staining (purple, yellow arrows) and counterstained of articular cartilage with Saf-0 (red), 20x (FIG. 10C). Quantitation of 3NT staining demonstrated trend towards decreased 3NT positivity 24h after COF + impact; n = 4, non-parametric Mann- Whitney test (FIG. I0D).

[0021] FIGS. 11A-11B. Molecular iron released as a direct result of biochemical activity of intraarticular COF injection. Adding DFO (deferoxamine) abrogates mitochondrial protection, as viewed by staining mitochondria of the articular surface in FIG. 1 1A imaging via confocal microscope, and quantified in FIG. 1 IB. n=4; p=0.04.

[0022] FIG. 12. CO concentrations retained in stifle joints after COF injection. Intact mouse stifle joints were analyzed for CO content 24 and 72 h after intra-articular COF injection, n = 6. DETAILED DESCRIPTION

[0023] Disclosed are compositions, formulations, and methods for treatment or preventative treatment of intraarticular injury or damage in a subject. Compositions and formulation may include gas entrapped in a carrier composition, for example a foam or thickening agent. The compositions may comprise a carrier composition and a gas, and in aspects the gas may be entrapped in the carrier composition The method can include applying the disclosed compositions or formulation, such as a gas-entrapped composition, to cartilage and / or ajoint in a subject.

[0024] Definitions

[0025] The disclosed subject matter may be further described using definitions and terminology' as follows. The definitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0026] As used in this specification and the claims, the singular forms “a,” "an." and ‘‘the” include plural forms unless the context clearly dictates otherwise. For example, the term “a substituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise.

[0027] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary' skill in the art and will vary' to some extent on the context in which they' are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0028] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0029] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to beter illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0030] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc / ’ is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0031] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0032] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this later context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0033] Compositions

[0034] In aspects disclosed, gas-entrapped compositions and / or therapeutic compositions are disclosed herein. The compositions can be used for treatment and / or the preventative treatment of an intraarticular injury or damage, in certain aspects. In various aspects, the compositions disclosed herein can include a carrier composition and a gas entrapped in the carrier composition. As used throughout this disclosure, “intraarticular’" refers to being situated within, occurring within, or administered by entry into a joint. Further, “intraarticular injury” or intraarticular damage” refers to any injury, damage, or fracture that occurs to or in a joint or cartilage in a subject, including to orin surfaces of bone of ajoint or cartilage. Intraarticular injury or damage can include, but is not limited to, traumatic injury7of a joint or associated cartilage, chronic joint diseases, degenerative joint diseases, one or more arthritic diseases such as rheumatoid arthritis and / or osteoarthritis, degenerative disc(s). meniscus tear or other damage, surgical intervention to a joint, surgical joint replacement, and the like. The intraarticular injury can include injury' to one or more of articular cartilage, hyaline cartilage, or fibrocartilage, or injury due to osteophytes.

[0035] The terms “damage” and “injury” may be used interchangeably throughout this disclosure and refer to an alteration in cellular or molecular integrity, activity, level, robustness, state, or other alteration, which may be traceable to an event. For example, an injury includes a physical, mechanical, chemical, biological, functional, infectious, or other modulator of cellular or molecular characteristics.

[0036] A gas entrapped in a carrier composition or entrapped in a composition refers to a gas that is present in pockets or as bubbles within the carrier composition. The pockets or bubbles can be any size and the size can be modulated based on the pressure used to generate the bubbles. In one aspect, the pockets or bubbles can include varying relative sizes. Without being bound by any7particular theory, the size of the bubbles may at least partly affect the rate of entrapped gas release, whereby larger pockets or bubbles may release gas at a faster rate than smaller pockets or bubbles. In one or more aspects, the pockets or bubbles can have a maximum dimension of about 1 pm to about 700 pm, or about 5 pm to about 500pm.

[0037] The gas can be present in the carrier composition in any amount that is suitable for use in the methods disclosed herein. In various aspects, the gas can be present in an amount of about 0.001 mg / g of carrier composition or more, about 0.01 mg / g or more, about 0.1 mg / g or more, about 0.001 mg / g to about 5 mg / g, about 0.01 mg / g to about 5 mg / g, or about 0. 1 mg / g to about 3 mg / g.

[0038] In various aspects, the compositions disclosed herein are formulated such that the gas releases from the earner composition over a period of about 15 minutes or more, about 30 minutes or more, about 1 hour or more, about 6 hours or more, about 12 hours or more, about 24 hours or more, about 2 days or more, about 3 days or more, about 4 days or more, about 5 days or more, about 6 days or more, about 7 days or more, about 15 minutes to about 7 days, about 15 minutes to about 5 days, about 15 minutes to about 3 days, about 15 minutes to about 2 days, about 15 minutes to about 36 hours, about 15 minutes to about 24 hours, or about 15 minutes to about 16 hours.

[0039] In disclosed aspects, gas can include carbon monoxide, nitric oxide, oxygen, nitrogen, hydrogen sulfide, one or more noble gases, or any combinations thereof. In various aspects, the gas is carbon monoxide. In the same or alternative aspects, the gas is selected from carbon monoxide, nitric oxide, oxygen, nitrogen, hydrogen sulfide, one or more noble gases, and any combinations thereof. In various aspects, the gas, for example carbon monoxide, is not conjugated to a heavy metal and / or the gas-entrapped composition does not include a heavy metal. In such aspects, the heavy metal not included within the composition or conjugated to the gas (e.g., carbon monoxide) may be nickel, iron, molybdenum, cobalt, and / or manganese.

[0040] In certain aspects, the gas can comprise, consist essentially of, or consist of carbon monoxide. In various aspects, the gas entrapped in the carrier composition can comprise about 25% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 99% or more, or 100% carbon monoxide.

[0041] In various aspects, the carrier composition can include one or more thickeners and / or one or more foaming agents. In certain aspects, the one or more thickeners and / or the one or more foaming agents can include any compounds that are Generally Recognized As Safe (GRAS), e.g., by the United States Food and Drug Administration. In one or more aspects, the thickeners and / or foaming agents can include one or more of hyaluronic acid, sodium alginate, methylcellulose, xanthan gum, guar gum, gellan gum, carrageenin gum, polyethylene glycol, decyl glucoside, coco glucoside, hydroxypropyl methylcellulose, hydroxylpropyl cellulose, ethyl hydroxyethyl cellulose, cellulose, ethylcellulose, gelatin, lecithin, or locust bean. In certain aspects, the one or more thickeners and / or one or more foaming agents can include one or more of methyl cellulose, xanthan gum, or hyaluronic acid. In one aspect, the one or more thickeners or foaming agents, and / or the compositions, do not include maltodextrin.

[0042] In various aspects, it may be desirable to formulate the composition to be isotonic, e.g., with respect to tissue being treated within the subject. In certain aspects, any suitable salt and / or buffering system can be used in the compositions disclosed herein to form an isotonic solution. In one example aspect, the compositions disclosed herein can include Phosphate Buffer Saline (PBS) or other saline solution. In various aspects, the compositions disclosed herein can be formulated and / or prepared as foams or hydrogels. In various aspects, a foam can be a composition that includes gas entrapped or present as bubbles within a liquid or semi-solid material, such as a carrier composition. In one or more aspects, a foam may generally include an increased volume of gas as compared to the carrier composition. In various aspects, the hydrogels can be a cross-linked hydrophilic polymer that can maintain a defined structure. In one aspect, the composition can be a combination of a foam and hydrogel.

[0043] In various aspects, the compositions can be present in a pharmaceutical formulation. In one or more aspects, the pharmaceutical composition can be for administration to a subject for intraarticular application. In certain aspects, the pharmaceutical composition is formulated for intraarticular injection.

[0044] In various aspects, the compositions and / or pharmaceutical compositions exhibit one or more physical properties suitable for injection, e.g., intraarticular injection. For instance, in certain aspects, the compositions and / or pharmaceutical compositions exhibit a viscosity range suitable for injection.

[0045] In certain aspects, the compositions provided herein may further contain buffers and / or pharmaceutically acceptable excipients and / or pharmaceutically acceptable carriers. As is known in the art, pharmaceutically acceptable excipients and / or carriers are relatively inert substances that facilitate administration of a pharmacologically effective substance and can be supplied as liquid solutions or suspensions, as emulsions, or as solid forms suitable for dissolution or suspension in liquid prior to use. Suitable excipients include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, pH buffering substances, and buffers. Suitably the pharmaceutically acceptable carrier helps maintain the construct or viral particle integrity of the viral vector prior to administration, e.g.. provide a suitable pH balanced solution. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, any of the various TWEEN compounds, and liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Methods

[0046] In various aspects, methods are disclosed for the treatment and / or preventative treatment of an intraarticular injury or damage in a subject.

[0047] In one or more aspects, the intraarticular injury or damage can be any type of injury or damage to the cartilage and / or tissues associated with a joint. In one aspect, the intraarticular injury or damage can include, but is not limited to, traumatic injury of a joint or associated cartilage, chronic joint diseases, degenerative joint diseases, one or more arthritic diseases such as rheumatoid arthritis and / or osteoarthritis, degenerative disc(s), meniscus tear or other damage, surgical intervention to a joint, surgical joint replacement, and the like. In the same or alternative aspects, the intraarticular injury can include injury to one or more of articular cartilage, hyaline cartilage, or fibrocartilage, or injury due to osteophytes.

[0048] In various aspects, the subject can be diagnosed with or at risk of developing rheumatoid arthritis, osteoarthritis, or other arthritic condition.

[0049] In one or more aspects, the methods can include delivering or administering one or more compositions disclosed herein to the cartilage or joint of a subject. In various aspects, the compositions can be administered via injection. In one aspect, the one or more compositions disclosed herein are administered to one or more of articular cartilage, hyaline cartilage, fibrocartilage, or osteophytes. In the same or alternative aspects, one or more of the compositions disclosed herein can be applied to, e.g., injected into, at least a portion of one or more of a knee, ankle, elbow, shoulder, wrist, hip, metatarsals, metacarpals, a temporomandibular joint, or a spinal disc. In certain aspects, a therapeutically effective amount of the compositions disclosed herein is administered.

[0050] In various aspects, one or more of the compositions disclosed herein can be delivered or administered once or more than once to a subj ect. For instance, in one aspect, the compositions can be administered to the subject periodically, e.g., once a week, once a month, once every 2, 3, 4, 6, or 9 months, or yearly.

[0051] In one or more aspects, the methods and compositions disclosed herein can be particularly efficacious after a wide variety of traumatic and surgical incidents independent of the stage of disease at the time of intervention. For example, the specific activities of carbon monoxide within the cartilage, synovium, adipose, and surrounding tissues that comprise the joint are ideally suited to combating intraarticular injury, in various aspects. These effects are distinct from existing treatments because of the unique combination of antioxidant, pro-metabolic, and anti-inflammatory effects throughout the entire joint that are coordinated through the specific chemical activity of the carbon monoxide gas released from the compositions disclosed herein. These effects appear beneficial anywhere along the continuum of care for orthopedic surgery patients. In certain aspects, the compositions and methods disclosed herein can provide additional lubricating benefits.

[0052] In various aspects, the administration of one or more of the compositions disclosed herein can elicit specific activities upon some or all of the tissues of articular joints that provide specific but sometimes distinct effects in each tissue that are efficacious in reducing the burden of osteoarthritis when applied any time during disease or efficacious in preventive efforts in conjunction with any intraarticular procedure including injections, irrigations, arthroscopic procedures of all types, open procedures of all types, repair procedures, implantation procedures, debridement procedures, and others. In each case, the benefits and actions of the methods disclosed herein include: 1) acute inhibition of oxygen metabolism resulting from CO’s effects on heme-containing enzymes: 2) upregulation of mitochondrial pathways related to heme metabolism directly and healing generally including the Sirtuins, AMP Kinase, and NRF-1; 3) anti-inflammatory effects throughout the joint particular to each tissue type, for example modulation of nitric oxide signaling in macrophages and adipose and inhibition of NFkB in chondrocytes; and / or 4) coordinated, joint-wide upregulation of antioxidants including bilirubin and glutathione as well as associated pathways like NRF-2.

[0053] As used herein, the term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human mammalian subjects.

[0054] As used herein the term “effective amount” or “therapeutically effective amount” refers to the amount or dose of a therapeutic, including but not limited to one or more of the compositions disclosed herein, and such a dose can include a single or multiple dose administration to the subject, which provides the desired effect. An effective amount can be determined by the attending diagnostician, as one skilled in the art, using known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the severity of the wound; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances. As used herein the term ‘‘treating’" refers to reducing, eliminating, or improving a condition or disease, including an intraarticular injury, or lessening the severity of any aspect of a symptom of a condition, disease, or intraarticular injury or damage.

[0055] As used herein, the term “preventative treatment” or “prevent” may be used interchangeably and refers to inhibiting the development of a condition or disease.

[0056] Examples

[0057] Example 1- Activation of Heme Metabolism Promotes Tissue Health After Intraarticular Injury or Surgical Exposure

[0058] This example and study began with the hypothesis that combinations of traumatic injuries and the rigors of intraarticular surgical care applied to repair these injuries might cause similar damage to articular cartilage through well characterized pathways such that patients receiving intraarticular surgery may benefit from therapeutic adjuvants to surgical care in a wide variety of trauma settings. Critical mitochondrial oxidative damage pathways whereby posttraumatic osteoarthritis (PTOA) is initiated after intraarticular fracture, meniscal injury, and a wide variety of in vitro models have been identified. With increasing enthusiasm for translation of mitochondrial strategies in orthopedics, it is proposed that activation of heme metabolism, previously associated with healing in many settings, causes prototypic mitochondrial reprogramming effects in cartilage ideally suited to use perioperatively.

[0059] In this example, carbon monoxide (CO)-containing foam (COF) was employed to stimulate heme metabolism and restore chondrocyte oxygen metabolism in vitro. Heme-oxygenase-1 (HOI), the initiating enzyme of heme metabolism, has anti-inflammatory, antioxidant, and pro- metabolic effects well characterized in other tissues. A cartilage-specific HOI overexpressing transgenic mouse strain was utilized to demonstrate specific features of the mitochondrial reprogramming biology under examination. Intraarticular injection of COF and key redox and safety outcomes in rabbit stifle (knee) joints ex vivo was also demonstrated. It is proposed that activation of heme metabolism is an ideal adjuvant to trauma care that replenishes mitochondrial metabolism and restores redox homeostasis after intracellular insult from trauma.

[0060] Introduction

[0061] Heme metabolism plays a key role in restoring the redox, mitochondrial, and energetic pathways of mammalian cells after injuries or during the resolution of inflammation. Prior generations of CO donors and other means of upregulating the initiating enzyme of heme metabolism, hemeoxygenase-l (HOI), have shown how this pathway provides some degree of protection against inflammatory models of arthritis and a wide variety of non-orthopedic pathologies. Many of these studies have concentrated on the anti-inflammatory features of these agents, but CO and heme metabolism are also know n to improve mitochondrial function. This is observed in throughout the body despite its w ell-known role inhibiting respiration at high doses. Therapeutic doses of CO appear to upregulate mitochondrial electron transport chain (ETC) activity and antioxidant pathways, but relationships between heme metabolism, mitochondria, and redox status in healthy, primary, articular chondrocytes have not been described. Research has focused on early features of posttraumatic osteoarthritis (PTOA), showing how- mitochondrial damage and dysfunction associated with traumatic injury can initiate PTOA. As "‘mitochondrial reprogramming" approaches are increasingly developed and applied, heme metabolism’s coordination of chondrocyte redox and mitochondrial physiology may provide prototypic strategies for cartilage protection and restoration.

[0062] It has been shown how sublethal mechanical injuries to articular cartilage initiate oxidative damage to the mitochondria over the first few hours after injury7that can be prevented by augmenting intracellular glutathione (GSH) or inhibiting mitochondrial electron transport. This results in mitochondrial dysfunction that does not resolve on its own but goes on to contribute to PTOA in multiple preclinical models. Prior studies have also identified damage from intraarticular surgical exposures including air, low osmolarity, unbuffered pH, and iatrogenic injuries. It is proposed that damage from any of these sources renders mitochondria dysfunctional and unable to support cellular metabolism. Activation of heme metabolism with acute delivery of CO to damaged or exposed articular cartilage might mitigate the effects of each these injuries, improve mitochondrial metabolism, and prevent oxidative damage and stress.

[0063] To test this, CO-containing foam (COF) w as applied to tissue culture models, primary7bovine osteochondral explants, and intact lapine joints and murine stifle (knee) joints. To better examine chondrocyte mitochondrial reprogramming by heme metabolism, this was complemented with genetic manipulation of HOI using the TetOn inducible system under a chondrocyte specific promoter. Consistent increases in mitochondrial function and ETC enzyme complement with COF that were associated with increased activity7and decreased oxidation of the GSH pathway were observed. Interestingly, COF increased ATP-linked respiration within 2 h after COF treatment, with marked increases in succinate dehydrogenase and cytochrome oxidase activity persisting 24 h later. These effects were observed in the presence and absence of traumatic injury and were recreated in HOI overexpressing chondrocytes in vivo. COF proved to be well tolerated, demonstrating no toxicity in any species tested. This exciting, novel material may prove a disease modifying adjuvant to surgical care, augmenting the benefits of orthopedic surgical care in any setting where orthopedic trauma, iatrogenic cartilage injury7, or joint exposure is a patient concern.

[0064] Materials and Methods

[0065] Cell and Tissue Culture

[0066] Either primary bovine chondrocytes extracted from healthy cartilage or the human chondrocyte cell line T / C-28a2 (cat# SCC042, Milipore, Temecula, CA) were used for monolayer studies. Primary7chondrocy tes were extracted as previously described (29437147). Cartilage from the explants were digested with collagenase and pronase (0.1 mg / ml; Sigma-Aldrich) overnight. The chondrocytes were then collected after pelleting, resuspended, and plated.

[0067] Cylindrical bovine osteochondral explants were utilized for injury7studies. Intact, fresh bovine stifle (knee) joints obtained from a local abattoir (Bud's Custom Meats, Riverside, IA) were dissected and both medial and lateral femoral condyles were used. Cylindrical explants 8 mm in diameter were obtained from the load bearing region of each femoral condyle. The explants were washed in Hank's balanced salt solution (HBBS) with 50 U / mL penicillin. 50 pg / mL streptomycin, and 2.5 pm / mL amphotericin B, then placed into culture medium. Both explant and monolayer cultures were maintained in cell culture medium with 45% Dulbecco's Modified Eagle's medium, 45% F-12, 10% fetal bovine serum (FBS) (Gibco), 100 U / mL penicillin, 100 pg / mL streptomycin, and 2.5 prn / rnL amphotericin B. All specimens were kept in a tissue culture incubator maintained at 5% 02, 5% CO2 and 37 °C. Explants were equilibrated to culture conditions overnight before any treatment or testing.

[0068] Mechanical Injury

[0069] After overnight equilibration to culture conditions, the mechanical impact injury was delivered via drop tower as previously described [44, 10, 12], As described, 2 J of impact on a 0.6 cm2contact surface area. The explants were leveled and adhered to a 3 x 3 cm stainless steel plate with polycaprolactone prior to impact. Release of the drop tower was secured to ensure a single impact. The explants were then rinsed with HBSS before being returned to normal cell culture medium. Gas Delivery via Gas Entrapping Foam

[0070] A novel material developed by By me et al.

[0031] was applied with various entrapped gases including room air (RAF), nitrogen (NF), and CO. The material includes 0.5% wt xanthan gum (Modernist Pantry), 0.8% wt methylcellulose (Modernist Pantry) in phosphate buffered saline (PBS). The gas entrapping foam was prepared and gassed. Dishes were rinsed with clean media and aspirated then each treatment was foamed and applied to fill dishes. Foams were applied for 30 min for monolayer cell cultures or 1 h for explants.

[0071] Ex vivo Stifle Culture

[0072] Intact but immediately post-mortem animals from other studies were used to explore acute effects of COF and RAF within the joint. New Zealand White rabbits, female aged 8 to 9 months (Charles River) and C57B6J mice, both sexes, aged 8 to 15 weeks (Jackson) were utilized. After euthanasia, foam was introduced into the stifle joint via injection immediately medial of the patellar tendon and the presence of foam within the joint was confirmed via fluoroscopy and inclusion of 20% Isovue-370 in the foam material. Stifles were extended and flexed gently and then left for 30 minutes. Lapine injections were 0.5 ml foam and murine injections were 0.05 ml foam.

[0073] Western Blot

[0074] Protein extracts were denatured and reduced by addition of LDS sample buffer (Invitrogen) and DTT reducing agent (Invitrogen). heated at 70°C by 10 min. A total of 20 ug of protein was loaded per well and electrophoresed through a 10% Bis-Tris NuPage acrylamide gel (Invitrogen), at 120 Volts constant by 2h with MES-SDS running buffer (Invitrogen). After the electrophoresis, the gel proteins were transferred to a 0.2 uM PVDF membrane (BioRad), at 300 mAmp constant by 2h with 20% methanol 0.1% SDS transfer buffer (Invitrogen). The blotted membrane was stained using Ponceau S, as a loading and protein quality control. The membrane was blocked by 30 min with Bovine serum albumin (BSA) 5% solution. The primary antibodies mitofusin-1 (Abeam), sirtuin 1 (Cell Signaling), mitochondrial electron transport chain complexes I-V (cocktail. Abeam?), p65 (Abeam), OPA-1 (Cell Signaling) and MFF (Cell Signaling) were diluted 1: 1000 in BSA 2.5% and incubated overnight at 4°C in a rotator shaker. After TBST rinsing, HRP -linked secondary abs (Cell Signaling) diluted 1 : 2000 in BSA 2.5% were incubated by Ih RT. Once TBST rinsed, the protein signal was developed by Super Signal West Femto (Thermo 34095) and visualized with Amercham Hyperfilm ECL system (GE Healthcare 28906839) film. Extracellular Flux Analyses

[0075] To evaluate mitochondrial activity by live chondrocytes as rapidly after COF treatment as possible, 20,000 primary bovine chondrocytes per well were plated on XF96 Extracellular Flux Analyzer (Seahorse Bioscience, Agilent) plates. After 3 days to allow attachment, wells were exposed to either RAF or COF then media was changed and standard mitochondrial stress test measures were conducted as previously described []. Briefly, the following reagents were successively injected into each well (final concentration in the well shown): oligomycin (2 pM), carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (250 nM). antimycin A (5 pM), rotenone (2 pM). After completion of the assay, the number of cells was counted. The oxygen consumption rates (OCR) were then normalized to the number of cells. Basal respiration is reported as basal OCR minus OCR after rotenone and antimycin A injection. ATP-linked respiration is reported as basal OCR minus OCR after oligomycin injection. Proton leakage is OCR after oligomycin injection minus OCR after rotenone and antimycin A injection, normalized to each specimen’s basal respiration.

[0076] ETC Complex Activity Assays

[0077] Electron transport chain (ETC) activities were measured as previously described [Birch- Machin et al., Biochem Med Metab Biol 51, 35-42 (1994); Tao et al.. Molecular cell. 2010 December 22;40(6): 893-904. PMID:21172655. PMCID: PMC3266626; and Owens et al., Free radical biology & medicine. 2012 January 1;52(1): 160-6. PMID:22041456. PMCID: PMC3249516], Briefly, Complex II samples were incubated with or without succinate in 25 mM potassium phosphate buffer, 5 mM magnesium chloride, 2 mM potassium cyanide, and BSA (2.5 mg / ml) for 10 min at 30°C. After 10 min, antimycin A (200 pg / ml), rotenone (200 pg / ml), 5 mM 2,6-dichloroindophenol (Sigma-Aldrich), and 7.5 mM coenzyme QI were added. The activity was calculated as difference of the absorption at 600 nm. divided by 19.1, and this number was normalized by the protein content. Complex IV samples were assayed in 25 mM potassium phosphate buffer, 0.5 mM n-dodecyl |3-maltoside, and 1.5 mM reduced cytochrome c. The activity was calculated as difference of the absorption at 550 nm, divided by 19.6, and then normalized to each sample’s protein content.

[0078] Cartilage-specific HOI overexpression

[0079] Cartilage-specific HOI overexpression was achieved using a doxycycline-inducible overexpression system implemented in two strains of transgenic mice that, when crossed, provide HOI overexpression upon introduction of doxycycline. We created a Col2Al- TetOn+ / - strain to enable the necessary cartilage specific expression of TetOn, and a Tet response element (TRE)-mHMOXl. TRE-mHMOXl+ / - strain to provide doxycyclineresponsive HOI overexpression. Both strains were created via knock-in on a B6 background with the help of the Genome Editing Facility at the University of Iowa, which performed all cloning, embryo injection, and genetic characterization of this strain. Briefly, Col2Al-TetOn mice have a knock-in. optimized, second-generation reverse tetracycline transactivator (rtTA*M2) gene [Gossen et al. Science. 1995;268(5218): 1766-1769] under a promoter taken from a portion of Col2Al [Zhou et al., J Cell Sci. 1995;108 (Pt 12):3677-3684. doi: 10.1242 / jcs.l08.12.3677] that provides chondrocyte specific expression in utero [Zhou et al.] up to the 10-15 week age groups shown in this study. Inserts are shown in FIGS. 8A and 8B. Briefly, the inserted Col2Al-TetOn transgene contains an SV40 polyadenylation signal, the previously characterized 2 x 182 bp doublet taken from the Col2Al promoter sequence [Zhou et al.], a minimal woodchuck hepatitis vims post-transcriptional regulatory element, the TetOn gene [Gossen et al ], a Factor Xa site and a polyadenylation signal. TRE-mHMOXl mice have a different gene knocked-in containing the murine HM0X1 under the Tet Response Element (TRE) promoter [Gossen et al.]. Similar to the Col2Al-TetOn mouse, but with distinct promotion and payload, the inserted TRE-mHMOXl transgene contains an SV40 polyadenylation signal, the tet response element critical for rtTA*M2 binding (7 repeats of TCCCTATCAGTGATAGAGA separated by spacer sequences), a minimal cytomegalovirus promoter, the Kozak sequence and mHMOXl, a P-globin intron and a polyadenylation signal. Double transgenic Col2Al -TetOn+ / -, TRE-mHMOXl + / - mice were confirmed with genotyping and both strains are maintained separately as heterozygotes. Mice aged 8-15 weeks were fed between 1-5 days of doxycy cline-containing chow (TD.01306 at 625 mg / kg, Teklad). Stifles for histological studies and specimens for Western blots were prepared as previously described [Hines et al.. Free Radic Biol Med. 2022;188: 175-184],

[0080] Immunohistochemistry (IHC)

[0081] Slides were deparaffinized and rehydrated via a series of washes: 100% xylene. 100% ethanol, 95% ethanol, 80% ethanol, and distilled water. Slides were then placed in 0. 1 M sodium citrate, pH 6, overnight, up to 16 h, at 55 °C. The samples were rinsed, and peroxidases were quenched. Samples were blocked using normal goat serum blocking solution. The primary anti -HOI antibody was incubated overnight (1: 150) at 4 °C. The secondary antibody (1:200) was incubated 30-40 min at room temperature. Samples were then incubated with ABC reagent Elite Standard (Vector laboratories) for 30 min. The samples were developed using DAB reagent (Vector laboratories). No counterstain was used.

[0082] The GSH content was evaluated using a mouse antibody (1:50) () and goat anti-mouse secondary (1 :200) (Thermofisher). Immunofluorescence of 3-NT was done using a rabbit anti- nitrotyrosine antibody (Millipore, 1 : 150) and goat anti-rabbit Cy5 fluorescent secondary (Roche, 760-238) on a Discovery Ultra (Roche, Switzerland).

[0083] Redox Status Visualization via Live Cell Imaging

[0084] In order to assess the redox status, cell viability, and mitochondria of chondrocytes in situ, we applied live cell stains to cultured tissue and visualized cross sections of the cartilage using confocal microscopy. First, the explants were cut with a precision saw' (IsoMet 1000, Buehler) and a clean cross section was revealed via removal of a 0.25 mm deep portion of the face of the cartilage with a scalpel. Monochlorobimane (MCB) (M1381MP, Invitrogen) was applied to reveal the reduced (as opposed to oxidized) thiols as Cook et al. (1868449), alongside calcein AM (C3099, Invitrogen), to assess cellular viability. MCB reacts with endogenous glutathione- S-transferase and reduced thiols (predominantly GSH) to create a blue fluorescent complex. This blue fluorescence thus indicates the presence of reduced thiols and decreases in that blue staining would indicate oxidation. Both explants and cells were stained in 20 pM MCB and 1 pM calcein AM. All staining and imaging occurred in DMEM / F-12 media without phenol red (Gibco). The samples were then observed with the confocal microscopy with using excitations at 405 nm and 488 nm for MCB and calcein AM, respectively.

[0085] For mitochondrial staining, the explants or cells were stained with MitoTracker Deep Red (MTDR) (Invitrogen) at 1 pM and co-stained with 1 pM of calcein AM, for cell viability. The samples were observed with the confocal microscopy with excitation at 633 nm for MTDR and 488 nm for calcein AM. Cartilage images were exported, manually segmented according to appropriate depths or areas, and analyzed with a previously published, custom MATLABbased algorithm []. This algorithm uses calcein AM to identify live cells only and then determines intracellular MCB or MTDR fluorescent intensity in each live cell.

[0086] Biochemical Measurement of GSH and GSSG

[0087] Griffith's GSH / GSSG assay as previously described [Griffith OW, Anal Biochem. 1980 Jul 15; 106(1 ):207- 12. PMID: 7416462] was deployed to confirm MCB results. Cartilage samples cut from explants were minced into 5% sulfosalicylic acid, and subjected to three freeze-thaw cycles to lyse the cells. Tissue and cell lysates were then mixed with buffer solution containing dithionitrobenzoic acid (DTNB). GSH reductase, and NADPH (all from Sigma- Aldrich). GSH reacts with DTNB to form a yellow product, while the GSH reductase recycles the GSH. As a result, the absorbance rate change at 412 nm as measured via spectrophotometer is proportional to GSH concentration. All sample concentrations were determined via comparison to appropriate standard curves. For GSSG determination, 20% sample volume of 2-vinylpyridine (Sigma- Aldrich) was added to sample aliquots for 1 h before analysis to quench GSH and prevent the reaction of reduced GSH with DTNB.

[0088] Image Analysis & Statistics

[0089] The averaged MCB or MTDR intensity will be statistically compared in zones and injury / noninjury group with one-way ANOVA analysis.

[0090] Results

[0091] CO Rapidly Induces HOI and Increases Mitochondrial Function

[0092] We first wanted to demonstrate that COF induces HOI in articular cartilage, as shown in other tissues [Ryter et al., Physiol Rev. 2006 Apr;86(2):583-650. PMID: 16601269; Chi et al., Mediators Inflamm. 2014:2014:279171. Epub 2014 Jan 29. PMID: 24616552; PMCID: PMC3927740; Desmard et al., Antioxid Redox Signal. 2007 Dec;9(12):2139-55. PMID: 17854278; Chin et al., Proc Natl Acad Sci U S A. 2007 Mar 20;104(12):5109-14. PMID: 17360382; PMCID: PMC1820823; and Lancel et al., J Pharmacol Exp Ther. 2009 May;329(2):641-8. PMID: 19190234], Following 30 min exposure to either CO or RAF, cartilage samples were harvested between 0 and 12 h after exposure for western blotting of explant cartilage. Results showed that CO induces HOI as soon as 1 h after foam exposure. This elevation of HOI lasted for at least 12 h after the exposure, FIG. 1A. Increases in mitofusin-1, a marker of mitochondrial health previously associated w ith CO exposure in other tissues,was also observed (FIG. IB). Based on prior reports [Haab et al.. Expert entia. 1990 Dec 1 :46(11-12): 1202-6. PMID: 2174793; Lavitrano et al., FASEB J. 2004 Jul;18(10): 1093-5. PMID: 15132974; Tsui et al., Shock. 2007 Feb;27(2): 165-71PMID: 17224791; Lancel et al., J Pharmacol Exp Ther. 2009 May;329(2):641-8. PMID: 19190234; Kapetanaki et al., Biochemistry. 2009 Feb 24;48(7): 1613-9. PMID: 19183042: Hull et al., JC1 Insight. 2016;l(2):e85817. PMID: 27110594; PMCID: PMC4838906; and Piantadosi et al., Circ Res. 2008 Nov 21;103(l 1): 1232-40. PMID: 18845810; PMCID: PMC2694963], we hypothesized that CO exposure improves the mitochondrial respiratory' function of chondrocytes. This hypothesis was tested using primary chondrocytes extracted from bovine cartilage subjected to mitochondrial stress tests as rapidly after foam treatment as possible. This measurement was approximately 1.5 h after initial exposure, extracellular flux equilibration, and early portions of the mitochondrial stress test. The result showed COF-treated chondrocytes had higher oxygen consumption rates (OCR) in the basal and ATP-linked portions of the mitochondrial stress test, as shown in FIG. 1C and ID. These studies demonstrated that COF increased primary’ chondrocytes' HOI content and augmented their mitochondrial function.

[0093] HO-1 Overexpression in Articular Chondrocytes In Vivo Increases Mitochondrial Metabolism and Decreases Basal p65 Expression

[0094] Because culturing primary chondrocytes is associated with a variety of well-known mitochondrial artifacts, to better examine the effects of increasing HOI and activating heme metabolism in primary’ chondrocytes in vivo we designed Col2Al-TetOn+ / -, TRE- mHMOXH- / - mice to overexpress HOI in articular chondrocytes in the presence of doxycycline. We fed mice doxycycline containing chow for 1-5 days and examined both immunohistochemical staining for HOI and western blots for HOI. Our result showed that by 2 days of Dox Chow feeding, increased HO-1 was seen in cartilage but not in meniscus, FIG. 2A (right panel). Wider views of the joint demonstrate no staining outside the articular cartilage, FIG. 2A (left panel). To examine ETC content with HO-1 overexpression, western blots for complexes I, II, III, IV, and V were run on cartilage from murine hind foot preparations. FIG. 2B. These samples also showed elevation of the pro-mitochondrial sirtuin 1 (SirTl) and decreased basal p65 staining after doxycycline feeding, FIG. 2C. These data demonstrate in vivo that the activation of heme metabolism triggers key molecular effects in articular chondrocytes corresponding to the effects of COF.

[0095] COF Augments Articular Chondrocyte GSH Metabolism and Protects Against Injury

[0096] Prior reports of antioxidant benefits from CO as w ell as the pro-mitochondrial effects of COF in cartilage suggested that COF might be protective against mitochondrial damage and dysfunction we have previously shown mediate PTOA at the earliest stages after injury’. We hypothesized that COF could prevent GSH oxidation from a defined model of traumatic injury and tested this hypothesis by applying COF for 1 h before a 2 J / cm2 impact injury known to oxidize GSH in situ. To provide detailed view s of GSH status at the articular surface, w e utilized the live cell stain MCB. This dye fluoresces blue in the presence of reduced GSH (vs oxidized) and this intensity decreases in the presence of decreased reduced GSH (i.e. dimmer blue = greater oxidation). To demonstrate these specificities in articular cartilage we applied either buthionine sulfoxamine (BSO) for 24 h prior to staining to inhibit glutathione synthesis or ethacrynic acid (ETA) immediately prior to staining to inhibit glutathione-S-transferases that contribute to MCB activation by GSH. Both inhibitors decreased MCB by greater than 90%, FIG. 3A.

[0097] After exposure to either RAF, NF, or COF, explants were given impacts or sham impacts, and returned to normal grow th media for 24 h. MCB of cross sections of the articular surface showed that RAF and NF decreased MCB fluorescence intensity to a comparable degree to mechanical injury whereas COF maintained MCB staining in either setting, FIG. 3B. Oxidation in the absence of mechanical injury not observed in sham groups suggest that extended exposure to RAF and NF are injurious to a similar extent to mechanical injury, potentially because these foams are constructed with hypo-osmolar PBS relative to growth media. We observed significant damage in the NF treated explants including some cell death, suggesting important distinctions between NF and COF despite both being constructed without oxygen gas. This demonstrates that CO’s specific biochemical activity is important to its benefits.

[0098] Intraarticular CO is Non-Toxic and Improves Cartilage Health In Situ

[0099] Beyond the data above, COF provides ideal physical characteristics for intraarticular injection that also confer lubrication and may prove especially useful by rapidly delivering CO to the entirety of the intraarticular j oint. However, prior to clinical implementation, it is also crucial to demonstrate the safety and efficacy of this material. To first demonstrate that intraarticular COF rapidly filled the joint space after injection, we gave 2-4 ml injections of COF containing contrast agent to the medial compartments of the stifles of New' Zealand White rabbits immediately post-mortem. Observation via fluoroscopy shows how COF rapidly fills the intraarticular space, including the far distal and proximal portions of the joint, around the patella, and in the opposite compartment. This result was obtained with both rabbit fluoroscopic images and munne CT imaging after injection of the same contrast agent containing COF. FIG. 4A. An example image of an injection into a rabbit is shown in FIG. 4F.

[0100] To interrogate whether COF has similar protective effects to our in vitro model, MCB staining of these rabbit stifles ex vivo 24 h after exposure was utilized. Rabbits were given RAF in one stifle and COF in the contralateral stifle, then 30 min later the limb was harvested, the articular surface was cut from the rest of the bone, and this was cultured for 24 h. This was intended to examine whether foam delivered while the joint was intact could provide similar significant increases in MCB intensity as was observed with osteochondral explants. We hypothesized that COF increased reduced GSH signal indicated by MCB in situ. COF treated cartilage showed significantly higher MCB fluorescent intensity than cartilage from pairwise contralateral joints exposed to comparable RAF doses, FIG. 4B. To support this result, Griffith’s GSH recycling assay was applied to assess total GSH and oxidized GSH (GSSG). COF decreased %GSSG, FIG. 4C, compared to RAF, suggesting that COF provides antioxidant support to the cartilage when injected intraarticularly. FIG. 5D shows that 1 week and 4 weeks after injection of COF or vehicle (RAF) there is no decrease in viable cells in the cartilage. The data in FIG. 4D was generated from a 20X objective lens and incorporates automated analysis of the entire volume scanned confocally. The data in FIG. 5E was generated from a 10X objective lens and is analyzed without volumetric analysis.

[0101] To demonstrate the safety' of this material, live rabbits were given either RAF or COF injections to the medial compartment of their left stifle. Animals were sacrificed either 1 week or 4 weeks after injection and articular surfaces of the tibia and femur were imaged for cell viability while synovia were fixed and prepared for histological staining with hematoxylin and eosin. This experiment demonstrated no decreases in articular chondrocyte viability. We next analyzed synovia from these same animals given material and then sacrificed 1 week and 4 weeks after injection to assess whether the material caused any damage or inflammation. This experiment demonstrated no apparent changes in synovial cell morphology or thickness, FIG. 5 A, and no indications of thickening or increased presence of immune cells within the synovium in any of the rabbits given intraarticular injections of RAF or COF, FIGS. 5B and 5C. These data demonstrated that 1 week after injection and 4 weeks after injection of COF the tissues of the joint appear completely normal, viable, and undamaged by the material. FIG. 5D shows that 1 week and 4 weeks after injection of COF or vehicle (RAF) there is no decrease in viable cells in the cartilage. The data in FIG. 5D was generated from a 20X objective lens and incorporates automated analysis of the entire volume scanned confocally. The data in FIG. 5E was generated from a 10X objective lens and is analyzed without volumetric analysis.

[0102] Activation of HOI in Articular Chondrocytes Prevents PTOA

[0103] Mice given destabilization of the medial meniscus (DMM) reliably develop PTOA by 8-10 weeks. TgHOl mice given doxycycline containing chow (625 mg / kg) for 1 week during the fourth week after injury show decreased average (FIG. 6B) and maximum (FIG. 6C) OARSI scores, suggesting HOI prevents PTOA. N = 4, p = 0.0857 by Kruskal Wallis. Discussion

[0104] Early articular cartilage damage contributes to PTOA via oxidation and mitochondrial dysfunction. There are also mitochondrial abnormalities in chondrocytes later in disease progression. This has led to enthusiasm in the orthopedic community for improving mitochondrial function and mitigating redox stress via ‘'mitochondrial reprogramming"-based approaches that might prevent PTOA. Despite a history as a “silent killer”, scientific literature has shown therapeutic benefits of CO in line with these types of approaches. We propose that activation of heme metabolism by CO causes prototypic mitochondrial reprogramming not only of great value to translational efforts, but to understanding cartilage healing processes as well. In this study, we have shown how this gas provides an ideal adjuvant to intraarticular injuries that may be of value for patients with recently injured joints or joints that have begun to progress to PTOA. COF rapidly induced HOI and increased healthy mitochondrial respiratory function in large animal tissue explants. COF also protected against GSH oxidation in vitro and ex vivo both in the presence and absence of mechanical injury without incurring any toxicity or inflammation on its own. Increases in mitochondrial respiration with HO1- induction and upregulation of heme metabolism in explant cartilage were reproduced in vivo using a cartilage-specific overexpressor of HOI. These data demonstrated acute, protective effects from COF to articular cartilage that may be efficacious for protecting against the stresses of intraarticular procedures when applied immediately pre-operatively.

[0105] Various strengths were included in the design of this study to increase the rigor and reproducibility7of the articular cartilage biology under study. In vitro large animal explant models, ex vivo models of rabbit and mouse stifle joints, and in vivo animal models w ere included with consistent effects upon heme metabolism, mitochondria and GSH. The protective effects of CO were consistent across multiple species, with each demonstrating increased mitochondrial readouts as well as improved GSH status. GSH w as assessed using multiple methods, each with detailed controls or standard curves as appropriate, to provide a more comprehensive understanding of the redox status of chondrocytes in situ. Similarly, multiple approaches were adopted for investigating mitochondria, including both expression and activities of respiratory complexes as well as a variety of associated pro-mitochondrial pathways.

[0106] Several challenges and limitations to this study became clear during experimentation. It w ould be valuable to measure the oxygen consumption rate while CO is present and as CO is replaced with normal atmosphere; however, given the technical details of the mitochondrial stress test setup, it is unclear whether the foam can be rinsed effectively from extracellular flux analyzer wells to provide reliable, immediate quantitation. In lieu of this, we have chosen to manually confirm foam removal and then initiate mitochondrial stress tests, yielding the data provided. We note that the paradox that a molecule known to inhibit respiration would augment mitochondrial activity after removal has been observed in other tissues. More detailed, depthand diffusion-focused research could provide interesting kinetic views of how CO’s chemical activity alters short term chondrocyte metabolic behavior. Because the delivery of CO from COF relies on diffusion, depth- and time-specific effects may be important to future preclinical and translational efforts. This model system may prove especially useful in future studies describing time-dependent and dose-dependent features of gasosignaling intraarticular biology.

[0107] The paradox that CO inhibits heme-containing proteins has already been noted, including the mitochondrial complexes, yet an increase of mitochondrial respiration and expression with CO or HO-1 overexpression was observed. This was observed as soon as 1 h after COF and persisted to 24 h after administration. Increases in reduced GSH also persisted to 24 h. In this way, we suggest that activation of heme metabolism in articular chondrocytes induces an endogenous mitochondrial reprogramming that can provide lasting benefits to articular cartilage. By stimulating mitochondrial biogenesis, supporting antioxidant activities through GSH, and thereby coordinating a return to healthy oxygen metabolism after exposures to COF, this material might be applied immediately prior to any procedure where low osmolarity', low temperature, high oxygen saline and the other rigors of intraarticular surgery might present a threat to cartilage health. The direct effects of COF (healthier, more functional mitochondria and robust antioxidant defenses) and, by extension, the activation of heme metabolism are ideally suited to this setting.

[0108] This study has concentrated on specific protective effects of COF to bolster application of COF within the context of preventive care for recently injured but otherwise healthy cartilage; however, similar benefits are expected in the other tissues of the articular joint or for cartilage already along the continuum of PTOA and studies are currently underway to delineate these benefits. Long term, this material and local activation of heme metabolism are expected to provide protection against PTOA not only by coordinating chondrocyte intracellular metabolism, but also by coordinating metabolic function and related pro-inflammatory / anti - inflammatory signaling throughout the joint. Coordinated protection and restoration of cartilage metabolism coincident with stimulation of the synovium by COF, specifically upregulation of anti -infl ammatory macrophages, may represent not only an ideal system in which to examine intraarticular gasosignaling and cartilage-synovium crosstalk, but may also provide revolutionary therapeutic benefits. The COF shown here providing these benefits consists of simple, GRAS materials which confer meaningful, lasting safety and biocompatibility. COF is easily injectable into rodent joints, thus represents a realistic solution for human intraarticular injection. Thus, COF is an ideal adjuvant to orthopedic surgical care in any context where damage to the cartilage is a concern.

[0109] Example 2 Gas-containing foam treatment increases mitochondrial content and decreases cellular oxidative stress

[0110] PTOA is a debilitating joint disease that can develop after a joint injury. Injuries to articular cartilage are known to initiate oxidative damage to resident articular chondrocyte mitochondria over the first few hours after injury [1,2], Similar damage to chondrocytes can result from intraarticular surgical exposures, whether from exposure to air [3,4,5], low osmolarity [6,7], unbuffered pH [8], or iatrogenic injuries [9], This led to our working preclinical hypothesis that applying protective strategies prior to intra-articular surgery might improve the efficacy or success of those surgeries. We have shown protection from cartilage injuries, with N- acetylcysteine-mediated increases in intracellular glutathione (GSH) or the inhibition of mitochondrial electron transport, but these non-specific approaches only provided partial protection from PTOA after intra-articular fracture [10,11,12], This study investigates a new approach to protecting articular cartilage through the coordinated manipulation of chondrocyte mitochondrial and GSH pathways.

[0111] When considering small molecules that might provide the necessary mitochondrial benefits while supporting GSH, we became interested in exploring the effects of carbon monoxide (CO). This endogenously produced gasotransmitter broadly binds to heme-containing proteins, inhibiting them and upregulating heme oxygenase-1 (HOI) [12,13,14,15,16,17,18.19], This leads to the restoration of redox balance, mitochondrial function, and energetic pathways in mammalian cells following injury [20,21,22,23,24,25], CO donors [16,26] and the transgenic upregulation of HOI have demonstrated some degree of protection against inflammatory arthritis and decrease inflammatory readouts in arthritic cartilage [27,28,29] as wel 1 as a wide variety of non-orthopedic pathologies [30,31,32.33,34,35,36]; however, the direct mitochondrial and redox effects of CO on normal cartilage and chondrocytes have not been described and prior examinations relied upon CO donors rather than the direct administration of CO gas. The response of otherwise healthy tissues is an important consideration for intraarticular delivery’, therapeutic outcomes, and safety. Here, we hypothesized that CO would improve mitochondrial metabolism while decreasing oxidation in chondrocytes after cartilage injuiy’ without demonstrating any adverse effects in vitro or in vivo.

[0112] To examine the potential benefits of CO, we opted to use CO foam (COF)

[0031] which is capable of being injected intra-articularly. We investigated the application of COF to chondrocytes in monolayer culture, primary bovine osteochondral explants, and rabbit and mouse stifle (quadruped knee) joints. We demonstrate that, after cartilage injury, CO can improve GSH and increase mitochondrial activity in chondrocytes in vitro, and that the intra-articular application of COF replicates these effects in situ.

[0113] Results

[0114] Increased mitochondrial activity was demonstrated in live osteochondral explants. Explants from bovine articulate cartilage were stained for ATP (red) and mitochondrial content (blue). COF increased ATP, and demonstrated that COF treatment increased ATP. Positive staining dyes in live cell for ATP (red) and mitotracker (blue) colocalization (magenta) (FIG. 9A) Quantitation of ATP staining demonstrated COF staining w as significantly increased compared to paired RAF controls (FIG. 9B). These data demonstrate that CO increases HOI as well as mitochondrial metabolism in articular chondrocytes rapidly after exposure.

[0115] Further to demonstrating that CO provides support to the GSH pathway in articular chondrocytes above, mitochondrial content was evaluated, and considered in relation to prior studies showing loss of mitochondria after injury. CO pretreatment is shown to be mitochondrial protective and decreases oxidative damage after injury. Immunofluorescent staining was applied for mitochondrial marker TOMM20 in fixed tissues and the intensity’ in the superficial zone quantified. COF treatment prior to impact significantly increased the TOMM20 staining compared to the RAF + impact process, but this was not different compared to COF controls, as shown in FIGs. 10A and 10B. To measure oxidation in the superficial zone of these tissues, IF of 3NT-modified proteins was applied to fixed tissues and the intensity' of the staining per area quantified. The application of COF prior to impact reduced 3NT formation relative to RAF controls, as shown in FIGS. IOC and 10D. These data show that CO is stimulating the GSH pathway, protecting chondrocyte mitochondria and decreasing oxidation after injury.

[0116] The benefits of COF treatment shown in FIGs. 10A-10D are dependent upon molecular iron released as a direct result of biochemical activity of the treatment, direct biochemical catalyzation of molecular iron release by the CO treatment. Adding DFO (deferoxamine) abrogates mitochondrial protection, as viewed by staining mitochondria of the articular surface and imaging via confocal microscope in FIGs. 11A-11B. CO stimulates heme degradation, which by definition releases iron stored in those hemes, making that iron available for the cells’ needs. Concurrently treating the cells with 1 mM DFO prevented released iron from being used by the cell during CO treatment, demonstrating that iron is critical to the mechanism of action. Treatment stimulates the cells to utilize their iron through their endogenous metabolism (heme metabolism).

[0117] Successful delivery’ and retention of CO gas in the joint space was confirmed in stifles. Murine stifles were examined for CO concentrations using mass spectrometry 24 h and 72 h after injection. Significant CO was observed remaining in the joint 24 h after injection that did not remain 3 days after injection (FIG. 12). This demonstrates that significant amounts of CO are retained for at least 24 h after injection.

[0118] Materials and Methods

[0119] 2.1 Application of CO

[0120] Intact bovine stifle (knee) joints obtained from a local abattoir (Bud’s Custom Meats, Riverside, IA, USA) were dissected approximately 2 h after death. Fresh, healthy cylindrical bovine osteochondral explants 10 mm in diameter were obtained from the load-bearing region of both the medial and lateral femoral condyles. Explants were washed in Hank’s balanced salt solution (HBBS) with 50 U / mL penicillin, 50 pg / mL streptomycin, and 2.5 pm / mL amphotericin B. They were then placed into culture medium (45% Dulbecco’s Modified Eagle’s medium, 45% F-12, 10% fetal bovine serum (FBS) (all Gibco, Waltham, MA. USA), 100 U / mL penicillin. 100 pg / mL streptomycin, and 2.5 pm / mL amphotericin B). All specimens were kept in a humidified tissue culture incubator maintained at 5% O2, 5% CO2, and 37 °C. Explants were equilibrated to culture conditions overnight before any treatment or testing.

[0121] To generate the COF and control foams (room air (RAF), nitrogen (NF), and 5% O2), we dissolved xanthan gum (0.5 weight (wt) %, Modernist Pantry. Eliot, ME, USA) and methylcellulose (0.8 wt %, Modernist Pantry, Eliot, ME. USA) in phosphate-buffered saline (PBS) to form a pre-foam solution. The pre-foam solution was then inserted into a custom- made whipping siphon and pressurized to 200 PSI with each gas of interest according to Byrne et al. [31, 35], Foams containing each gas were filled into 15 rnL conical tubes, and the explants submerged in the foam for 60 min. An explant cultured in media as normal and not exposed to any foam, was used as a no-foam or sham control.

[0122] 2.2. Induction of HOI by CO

[0123] For this experiment, we utilized COF as well as a control foam containing 5% oxygen-like incubation conditions to minimally disturb control cartilage oxygen tension during the determination of HOI responses. After exposure to the foam, the explants were rinsed with culture media equilibrated to incubator conditions. For Western blotting, the explants were harvested 0, 1, 3, 6, or 12 h after the initial 60 min exposure to either foam. The cartilage was minced and mixed with RIPA buffer (Invitrogen, Waltham, MA, USA) and placed into -80 °C freezer. Protein extracts from explants specimens were denatured and reduced by the addition of LDS sample buffer (Invitrogen, Waltham, MA, USA) and DTT reducing agent (Invitrogen, Waltham, MA, USA), heated at 70 °C for 10 min. A total of 20 ug of protein was loaded per well and electrophoresed through a 10% Bis-Tris NuPage acrylamide gel (Invitrogen, Waltham, MA. USA) at a 120-volt constant for 2 h with MES-SDS running buffer (Invitrogen, Waltham, MA, USA). After electrophoresis, the gel proteins were transferred to a 0.2 uM PVDF membrane (BioRad, Hercules, CA, USA) at 300 mAmp constant for 2 h with 20% methanol 0.1% SDS transfer buffer (Invitrogen, Waltham, MA, USA). The blotted membrane was stained using Ponceau S. as a loading and protein quality control. The membrane was blocked for 30 min with bovine serum albumin (BSA) 5% solution. The primary antibodies HOI (Cell Signaling, Danvers, MA, USA) and mitofusin-1 (Abeam, Cambridge, UK) were diluted 1: 1000 in BSA 2.5% and incubated overnight at 4 °C in a rotator shaker. After TBST rinsing, HRP-linked secondary antibody (Cell Signaling, Danvers, MA, USA), diluted 1:2000 in BSA 2.5%, was incubated by 1 h RT. After TBST rinsing, the protein signal was developed by Super Signal West Femto (Thermo, Waltham, MA, USA) and visualized with Amersham Hyperfilm ECL system (GE Healthcare, Chicago, IL, USA) film. Densitometry was performed in ImageJ Fiji 2. 16.0 using the actin staining as a standardized control and represents the mean of n = 3.

[0124] 2.3. Chondrocyte Extracellular Flux Analyses

[0125] Primary bovine chondrocytes were extracted from articular cartilage and used to evaluate mitochondrial activity in live chondrocytes immediately after COF or room air-containing foam (RAF) exposure. Briefly, cartilage from the explants was digested with collagenase and pronase (0. 1 mg / mL; Sigma-Aldrich, St. Louis, MO, USA) overnight. Chondrocytes were then pelleted, resuspended, and plated. Chondrocytes were cultured as described above for explants. Then, 20,000 primary bovine chondrocytes per well were plated on XF96 Extracellular Flux Analyzer (Seahorse Bioscience, Agilent, Santa Clara, CA, USA) plates. After allowing 3 days for attachment, the wells were exposed to either RAF or COF for 30 min. Media were then changed and a mitochondrial stress test was conducted as soon as possible after foam treatment, as previously described

[0010] , Briefly, we used the following successive injections into each well using concentrations previously determined to be effective and non-lethal in primary chondrocytes (final concentration in the well is shown): oligomycin (2 pM), carbonyl cyanide- 4-(trifluoromethoxy) phenylhydrazone (FCCP) (250 nM), antimycin A (5 pM), and rotenone (2 pM). Mitochondrial stress test measurements were obtained approximately 1.5 h after initial exposure, following extracellular flux equilibration and early portions of the mitochondrial stress test. After completing the assay, the cells were trypsinized in 100 pL of trypsin. After cells were lifted from plates, 150 pL of media was used to neutralize the trypsin and cells were counted using a hemocytometer. Oxygen consumption rates (OCRs) were normalized to the number of cells. Basal respiration is reported as basal OCR minus OCR after rotenone and antimycin A injection. ATP-linked respiration is reported as the basal OCR minus OCR after oligomycin injection. Proton leakage is the OCR after oligomycin injection minus the OCR after rotenone and antimycin A injection, normalized to each specimen’s basal respiration. The mitochondrial data were statistically compared with a Student's t test. Statistical significance was defined as a p- value of less than 0.05.

[0126] 2.4. Live Cell Evaluation of ATP and Mitochondrial Content After Gaseous Manipultaion

[0127] To determine the effect of gaseous manipulations on articular chondrocyte ATP abundance, osteochondral explants were exposed to RAF or COF for 1 h then returned to 5% O2 pre-gassed media and cultured for 24 h. Live cell stains were prepared in DMEM / F-12 media without phenol red (Gibco, Waltham, MA, USA). BioTracker ATP-Red Live Cell Dye (Sigma, SCT045, St. Louis. MO, USA) was diluted to 5 pM and co-stained BioTracker 405 Blue Mitochondria Dye (Sigma, #SCT135, St. Louis, MO. USA) at a concentration of 200 nM for 15 min. Images were gathered using a confocal microscope (Olympus / Evident, Tokyo, Japan) with a 20 x immersion objective using wavelengths (450 nm, DAPI, respectively). Three images were taken from each sample for analysis. Images were analyzed in the red channel using ImageJ Fiji 2.16.0 to determine the mean intensity’ and the area of the image. All quantitative measurements were graphed, and statistical analysis was performed in Graphpad Prism 9. 2.5. Mechanical Injury and Evaluation of Thiol Redox Status

[0128] The effect of CO on chondrocytes receiving direct mechanical injury7was assessed by applying foams and subjecting explants to well-characterized, energy-controlled impact injuries similar to prior studies [1,10.11,12], This model creates a simple and highly reproducible impact injury with cell biological features comparable to those of in vivo traumatic joint injuries

[0012] , After overnight equilibration to culture conditions, explants were treated with either sham, RAF, COF, or NF for 1 h. Following treatment, the explants were rinsed with media. Explants w ere adhered to a 3 x 3 cm stainless steel plate with polycaprolactone. The cartilage was then impacted with a flat, stainless steel impermeable platen via a drop tower that delivered an energy-controlled, gravity -driven 2 J / cm2[1,10,11,12], The explants were rinsed with HBSS before being returned to the normal cell culture medium for 24 h. Four explants were used in each group unless otherwise noted.

[0129] To assess the redox status and cell viability of chondrocytes in situ, we used confocal microscopy of monochlorobimane (MCB) (Invitrogen. Waltham, MA, USA) to provide detailed view's of intracellular thiol status throughout a cross-section of the articular surface. First, explants were cut through the center of the impacted region (or equivalent in nonimpacted control samples) with a precision saw (IsoMet 1000, Buehler, Lake Bluff, IL, USA). Next, a scalpel was used to cut away a full-thickness cross-section, i.e., from the articular surface to the subchondral bone, excising a 0.25 mm layer of cartilage to remove the tissue abraded by the saw; thus exposing an undamaged surface. Explants w ere stained in DMEM / F- 12 media without phenol red (Gibco, Waltham, MA, USA) containing MCB (20 pM) and Calcein AM (1 pM, Invitrogen, Waltham, MA, USA), to assess cellular viability. MCB reacts with endogenous glutathione-S-transferase and reduced thiols (predominantly GSH) to create a blue, fluorescent complex. This signal is not entirely specific to GSH but is dominated by GSH, as shown previously [37,38] . Thus, blue fluorescence indicates the presence of reduced thiols and decreases in blue fluorescence w ould indicate oxidation. We previously show ed that GSH is a critically important mediator of chondrocyte injury by using Griffith’s biochemical GSH / GSSG assay on whole cartilage

[0039] , but MCB allows the visualization of GSH status in space on a per cell basis

[0040] , This is important for testing the application of a diffusible, gaseous therapy like CO to a thick, architecturally complex tissue like cartilage. The samples were then visualized with a confocal microscopy (Olympus / Evident. Tokyo, Japan), using excitations at 405 nm and 488 nm for MCB and Calcein AM. respectively. Three micrographs of each impacted area from each specimen were exported and manually segmented into regions of interest. Here, we report data for the superficial zone, where the effects of each foam and impact are clearly apparent. MCB intensify was analyzed with a previously published, custom MATLAB (version 2024)-based algorithm

[0040] , This algorithm uses a watershed-based algorithm to identify individual calcein AM-stained live chondrocytes (several hundred per micrograph), and then only quantifies intracellular MCB fluorescent intensify in each live cell while discarding data from any dead cells in the image. The average intensify of intracellular MCB staining was compared between foam-treated and injury and non-injury groups then analyzed using two-way ANOVA with Sidak’s multiple comparisons test. Normality was confirmed using a Shapiro-Wilk test, yielding a / value of 0.4673.

[0130] To support that MCB measurements are related to GSH specifically, we also utilized Griffith’s GSH / GSSG assay

[0039] , Cartilage samples cut from explants were minced into 5% sulfosalicylic acid and subjected to three freeze-thaw cycles to lyse the cells. Tissue and cell lysates were then mixed with buffer solution containing dithionitrobenzoic acid (DTNB), GSH reductase, and NADPH (all from Sigma-Aldrich, St. Louis, MO, USA). GSH reacts with DTNB to form a yellow product, while the GSH reductase recycles the GSH. As a result, the absorbance rate change at 412 nm as measured via a spectrophotometer is proportional to GSH concentration. All sample concentrations were determined via comparison to appropriate standard curves. For GSSG determination, a 20% sample volume of 2-vinylpyridine (Sigma- Aldrich, St. Louis, MO, USA) was added to sample aliquots for 1 h before analysis to quench GSH and prevent the reaction of reduced GSH with DTNB.

[0131] 2.6. Immunofluorescent Evaluation of Mitochondira and Oxidative Damage

[0132] To evaluate mitochondrial content and oxidative damage in tissue after gaseous manipulation and to assess impact, articular cartilage was evaluated for the mitochondrial marker translocase of outer mitochondrial membrane (TOMM20) and for oxidative damage marker 3-nitrofyrosine (3NT). Following the foam and impact assessment and the bisection of the osteochondral explants, one of the halves from each group was placed 10% neutral-buffered formalin with 0.03% Safranin-0 (Saf-O). In brief, the samples were decalcified, processed, paraffin- embedded. and sectioned to 5 pm onto super frost slides. The slides were stained on the Discovery Ultra (Roche, Basel, Switzerland) using either rabbit primary anti-TOMM20 (1 :50 Cell Signaling, #42406) or anti-nitrofyrsoine (1: 150, Millipore, St. Louis, MO, USA). The fluorescent secondary antibody was goat anti-rabbit cy5 (Roche, 760-238. Basel, Switzerland). The slides were imaged using the Olympus VS200. Images were analyzed by selecting 3 sites on the edges and center of the impact site or similar regions on unimpacted samples. Analysis was manually segmented to cover the superficial and transitional zone of cartilage (the first 200 pm from the articular surface). The mean intensify and area were measured for each image in ImageJ Fiji 2.16.0. The average mean intensity / area was calculated and graphed and statistical analysis was performed in Graphpad Prism 9.

[0133] 2.7. Intra-Articular COF and Evaluation In Vivo

[0134] All animal studies were conducted under the review and approval of the Institutional Animal Care and Use Committee at the Universify of Iowa [Approval #3062034, approved 19 July 2023] and all associated regulations were followed. To first demonstrate that the intra-articular injection of COF could effectively and efficiently fill the joint space, we administered up to 4 mL of COF containing the contrast agent Isovue-370 into the medial compartments of the stifles joints of 8- to 9-month-old female New Zealand White rabbits (Charles River, Wilmington, MA, USA) immediately post-mortem. The COF was injected through the stifle joint via a 27 Ga needle placed immediately medial to the patellar tendon. During injections, we observed no backflow out of injection sites, supporting reliable intra-articular delivery’. The presence of foam within the joint was confirmed via the incorporation of 20% Isovue-370 in the foam solution followed by fluoroscopic imaging. The same contrast agent-containing foam was also used to assess the retention of COF in murine stifle joints.

[0135] To investigate whether the intra-articular injection of COF has similar effects to in vitro experimentation showing increased intracellular GSH or reduced thiols, we utilized the RAF and COF stifle joints, as described above. The stifle joints were then disarticulated, rinsed in culture media, and equilibrated in the same media and conditions overnight as explants for comparison. This was followed by MCB staining of the articular surfaces 24 h later. The lateral femoral condyles were imaged at the center of the articular surface for live cell imaging using MCB and calcein AM, as described above. The MCB intensify was compared with paired t tests.

[0136] To determine if COF increased HOI expression in situ, post-mortem bilateral injections of either COF or RAF. The stifles were dissected and incubated for 24 h, fixed, decalcified, processed, paraffin-embedded, and sectioned at 5 pm. HOI was evaluated using immunofluorescence. In brief, slides were placed in citrate-buffered antigen retrieval at 55 °C overnight, blocked with normal goat serum (88.5% PBS, 10% normal goat serum, 1% bovine serum albumin, 1% cold water fish gelatin 9% in PBS, and 0.05% Tween 20, followed by overnight primary antibody rabbit anti-HOl (1:150, Cell Signaling, Danvers, MA, USA) incubation at 4 °C. The samples were rinsed followed by 30 min incubation of the goat antirabbit Alexa Cy5 (1 :500, Abeam, AB656, Cambridge, UK) fluorescent antibody, followed by DAPI mounting media and cover slipping. The slides were images using a confocal microscope (Olympus / Evident, Tokyo, Japan) at 20x wavelengths (Cy5, and DAPI).

[0137] To investigate the safety and toxicity of COF for normal synovium and cartilage, NZW rabbits were randomly distributed for 1-week and 4-week observation, with n = 10 (5 male, 5 female) in each observation period. We compared stifle joints from rabbits with no injection (Normal, n = 2) to stifles from rabbits that received a 4 mL injection of RAF in one stifle and COF in the opposing stifle, alternating on the left and right for both injections in individual animals. At harvest times of 1 week or 4 weeks after injection, articular surfaces were placed into culture media to assess chondrocyte viability, described below, while synovia were dissected from the posterior of the stifles and then pinned to cork sheets for fixation (10% NBF) and processed. After dehydration, synovia were bisected at the midline and paraffin-embedded with the cross-sectional facedown to provide full-thickness views of the synovia. Crosssections of the synovium were stained with hematoxylin and eosin (H&E) and then synovial thickness was measured using Olympus Desktop. We next estimated the cellularity of synovia as the number of cells thick the tissue presented in these cross-sections, counting 5 random sites per synovium and then averaging these counts. To assess chondrocyte viability, we stained the tibial articular surfaces with Calcein AM (Life Technologies, Waltham, MA, USA), imaged via confocal microscope (Olympus / Evident, Tokyo, Japan) using a 10x objective to view the cartilage from above, and then counted viable cells per visual field with watershed-based ImageJ Fiji 2.16.0 macros. The cell counts were compared among groups with two-way ANOVA.

[0138] To assess CO concentrations after intra-articular injection, we utilized C57B6J mice of both sexes, aged 8 to 15 weeks (Jackson, Bar Harbor, ME, USA) Six mice were randomly assigned to each group (3 males and 3 females) and were given injections of 0.05 mL RAF or COF under anesthesia with a 31 Ga needle; they were then returned to their cages to move freely. Stifles were harvested 24 h later and frozen. For analysis of CO content, stifles were dissected down to the joint capsule, rinsed free of blood with PBS, and placed in 2 mL bead mill tubes with stainless steel beads (2.4 mm) and diluted 10-fold with ice cold water. Samples were then homogenized on a bead-mill homogenizer (VWR, Radnor, PA. USA), and placed in an ultrasonic bath (Branson / Emersen, St. Louis, MO, USA) for 5 min. Using a gas-tight syringe (Hamilton, Bonaduz, Switzerland), 20 pL of homogenate was then added through a septum to a CO-free amber borosilicate glass vial that contained 20 pL of sulfosalicylic acid (20%). The CO released into the headspace of the vial was then flushed through a gas chromatograph with a reducing compound photometer (Peak Labs, Mountain View, CA, USA; Peak Performer 1) and quantified using a standard curve, created daily using different volumes of 1 ppm CO gas (AirGas, Radnor, PA, USA).

[0139] In the foregoing description, it will be readily apparent to one skilled in the art that vary ing substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably7may be practiced in the absence of any7element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and / or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0140] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0141] Citations to a number of patent and non-patent references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification. References

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Claims

CLAIMSWhat is claimed is:

1. A method of treatment or preventative treatment of intraarticular inj ury in a subj ect, comprising: applying a gas-entrapped composition to cartilage and / or a joint in a subject.

2. The method of claim 1, wherein the composition comprises a carrier composition and a gas.

3. The method of claim 2. wherein the gas comprises carbon monoxide.

4. The method of claim 2, wherein the gas is present as bubbles in the carrier composition.

5. The method of any one of claims 2-4, wherein the carrier composition comprises one or more thickeners and / or foaming agents that are Generally Recognized As Safe (GRAS).

6. The method of claim 5, wherein the one or more thickeners and / or foaming agents are selected from the group consisting of hyaluronic acid, sodium alginate, methyl cellulose, xanthan gum, guar gum, gellan gum, carrageenin gum, polyethylene glycol, decyl glucoside, coco glucoside, hydroxypropyl methylcellulose, hydroxylpropyl cellulose, ethyl hydroxyethyl cellulose, cellulose, ethylcellulose, gelatin, lecithin, and locust bean.

7. The method of claim 6, wherein the one or more thickeners and / or foaming agents comprises methylcellulose, xanthan gum, and / or hyaluronic acid.

8. The method of any one of claims 3-7, wherein the carbon monoxide is not conjugated to a heavy metal and / or wherein the gas-entrapped composition does not include a heavy metal.

9. The method of any one of claims 1-8, wherein the gas releases from the carrier composition over a period about 15 minutes or more, about 30 minutes or more, about 1 hour or more, about 6 hours or more, about 12 hours or more, about 24 hours or more, about 2 days or more, about 3 days or more, about 4 days or more, about 5 days or more, about 6 days or more, about 7 days or more, about 15 minutes to about 7 days, about 15 minutes to about 5 days, about 15 minutes to about 3 days, about 15 minutes to about 2 days, about 15 minutes to about 36 hours, about 15 minutes to about 24 hours, or about 15 minutes to about 16 hours.

10. The method of any one of claims 1-9, wherein the gas-entrapped composition is a foam or a hydrogel.

11. The method of any one of claims 1-10, wherein the gas-entrapped composition is applied via injection.

12. The method of any one of claims 1-11, wherein the gas-entrapped composition is applied to at least a portion of a knee, ankle, elbow, shoulder, wrist, hip, metatarsals, metacarpals, temporomandibular joint, or a spinal disc.

13. The method of any one of claims 1-12, wherein the gas-entrapped composition is applied to one or more of articular cartilage, hyaline cartilage, fibrocartilage, or osteophytes.

14. The method of any one of claims 1-13, wherein the subject is diagnosed with or at risk of developing rheumatoid arthritis and / or osteoarthritis.

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