Compositions and methods for repair of a ligament, tendon, cartilage or meniscus

Abstract

This disclosure relates to compositions and methods for repairing tendons, ligaments, cartilage or menisci. In particular the disclosure relates to a barrier comprising a corticosteroid to prevent penetration of synovial fluid and / or blood and aid in the repair of tendons, ligaments, cartilage or menisci.

Description

Docket 88800730-000529COMPOSITIONS AND METHODS FOR REPAIR OF A LIGAMENT, TENDON, CARTILAGE OR MENISCUSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of US Provisional Application Number 63 / 862,377, filed 12 August 2025, and US Provisional Application Number 63 / 746,683 filed 17 January 2025; each of which is incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under W81XWH-20-1-0583 awarded by the Defense Health Agency, Medical Research and Development Branch. The government has certain rights in the invention.TECHNICAL FIELD

[0003] This disclosure relates to compositions and methods for repairing tendons, ligaments, cartilage or menisci. In particular the disclosure relates to a barrier comprising a corticosteroid to prevent penetration of synovial fluid and / or blood and aid in the repair of tendons, ligaments, cartilage or menisci.BACKGROUND

[0004] The anterior cruciate ligament (ACL) is covered by a synovial sheath containing fibroblast-like synoviocytes (FLS) that play a crucial role in regulating the traffic of synovial fluid solutes into the ligament. An ACL injury disrupts this natural barrier. In contrast to periarticular ligaments, intra-articular ligaments exhibit poor healing capacity after injury (e.g, partial or full tear), which has been attributed in part to exposure of the wound to synovial fluid that has been shown to reduce adhesion and proliferation of ACL fibroblasts, potentially compromising their healing role. The latter is critical as studies have determined that glucose permeation from the synovial fluid is 59.7% in the ACL and 18.1% in the MCL, suggesting that nutrients to the extra-articular MCL (which heals) is provided mainly by its vascular supply and synovial fluid permeation is an important transport route for small molecules for the normal ACL.Docket 88800730-000529

[0005] There is a well-established link between ACL injury and development of post traumatic osteoarthritis (PTOA). Once the anterior cruciate ligament (ACL) ruptures, it does not spontaneously repair due to synovial fluid surrounding the joint space and mechanical stress during load-bearing. Even after surgical intervention, the ACL still exhibits a poor healing capacity and a similar incidence of PTOA.

[0006] Blood is known to have a negative impact on articular cartilage, leading to cartilage breakdown. Blood can impede ACL repair, impairing cell health as well as healing capacity, as well as lead to cartilage degeneration. ACL injury is typically accompanied by joint bleeding, but the individual contributions of whole blood components to joint damage are not fully understood. Articular bleeding, known as hemarthrosis, has been shown to trigger joint inflammation and is linked to the deterioration of various joint components, such as synovium and cartilage. Joint inflammation can be induced by pro-inflammatory cytokines as well as blood post injury.

[0007] Therefore, improved compositions and methods for repair of tendons, ligaments, cartilage or menisci would represent a significant advance in the art.SUMMARY

[0008] The present disclosure provides compositions and methods for improved repair of tendons, ligaments, cartilage or menisci using a vitrified collagen wrap (VC-Wrap) membrane wrap that serves as a vehicle for local low-dose (therapeutic) drug delivery of a corticosteroid (for example dexamethasone) in the synovial joint that reduces inflammation and promotes chondroprotection from pro-inflammatory cytokines while stimulating anabolic cell activities that support ligament repair, and enhances primary ACL repair by providing a barrier to synovial fluid solutes (such as hyaluronan) that may inhibit ligament healing, while permitting nutrient access, thereby restoring ligament function and joint stability. This disclosure at least partially addresses at least one of the above inefficiencies. However, this disclosure can prove useful to other technical areas. Therefore, various claims recited below should not be construed as necessarily limited to addressing any of the above inefficiencies.

[0009] The present disclosure provides a method of preventing infiltration of blood and / or synovial fluid to at least one of a ligament, a tendon, a meniscus, or a cartilage, comprising erecting a barrier comprising a vitrified type I collagen gel and at least a first corticosteroid on the at least one of the ligament, the tendon, the meniscus, or the cartilage. In certain embodiments, the barrierDocket 88800730-000529is a sheet or a cuff. In some embodiments, the barrier is selectively permeable. In related embodiments, the barrier is selectively permeable based on size of molecule, charge on molecule, component to be repaired or area of coverage. In other embodiments, the at least a first corticosteroid is comprised in a microsphere. In yet other embodiments, the microsphere is a PLGA microsphere. In additional embodiments, the at least a first corticosteroid is dexamethasone. In further embodiments, the dexamethasone is comprised in PLGA-microspheres. In yet further embodiments, the ligament is an anterior cruciate ligament. In still other embodiments, the barrier provides structural support for the at least one of the ligament, the tendon, the meniscus, or the cartilage.

[0010] The present disclosure also provides a method for repairing an injury to at least one of a ligament, a tendon, a meniscus, or a cartilage, comprising contacting the at least one of the ligament, the tendon, the meniscus, or the cartilage with a barrier comprising a vitrified type I collagen gel and at least a first corticosteroid. In certain embodiments, the ligament is an anterior cruciate ligament.

[0011] Additionally, the present disclosure provides a device comprising a vitrified type I collagen gel sheet and at least a first corticosteroid. In certain embodiments, the vitrified type I collagen gel sheet at least one of blocks or limits a passage of at least one of a blood or a synovial fluid through the device. In some embodiments, the vitrified type I collagen gel sheet, when positioned relative to at least one of a ligament, a tendon, a meniscus, or a cartilage, is configured to at least one of block or limit a passage of at least one of a blood or a synovial fluid to the at least one of the ligament, the tendon, the meniscus, or the cartilage. In other embodiments, the vitrified type I collagen gel sheet is formed into a sleeve or cuff. In yet other embodiments, the sleeve or cuff provides physical support for at least one of a ligament, tendon, meniscus, or cartilage. In still other embodiments, the sleeve or cuff is formed during placement of the device relative to at least one of a ligament, tendon, meniscus, or a cartilage. In further embodiments, the sleeve or cuff is formed prior to placement of the device relative to at least one of a ligament, tendon, meniscus, or a cartilage. In yet further embodiments, the vitrified type I collagen gel sheet is selectively permeable. In still further embodiments, the at least a first corticosteroid is dexamethasone. In additional embodiments, the dexamethasone is comprised in PLGA-microspheres.Docket 88800730-000529

[0012] It should be understood that the various individual aspects and features of this disclosure can be combined with any one or more individual aspect or feature, in any number, to form embodiments of the present invention that are specifically contemplated and encompassed by the present invention.

[0013] This disclosure is embodied in various forms illustrated in a set of accompanying illustrative drawings. Variations are contemplated as being a part of this disclosure, limited only by scope of various claims recited herein.BRIEF DESCRIPTION OF DRAWINGS

[0014] The set of accompanying illustrative drawings shows various exemplary embodiments of this disclosure. Such drawings are not to be construed as necessarily limiting this disclosure. Like numbers and / or similar numbering scheme can refer to like and / or similar elements throughout.

[0015] FIG. 1 shows dexamethasone release characteristics. Panel A. Dexamethasone release rate from VC-Wrap laden microspheres (DEXMS). Panel B. Transport of dextran-FITC of different molecular weight to low density (1 mg / cm2) or high density (3 mg / cm2) VC-Wrap. Permeability decreases with increased collagen concentration (all significant, p<0.05). Panel C. High Density VG allows smaller molecules to approach equilibrium relatively quickly (70% of equilibrium after 24 hours, 100% after 96 hours), while inhibiting equilibrium transport of larger sized molecules (38% of equilibrium after 96 hours).

[0016] FIG. 2 shows dexamethasone release from <5 pm diameter dexamethasone-PLGA microspheres in culture showing sustained release through 25 days.

[0017] FIG. 3A and FIG. 3B show characterization of a VC-Wrap. Average stress-strain at failure (FIG. 3 A) and corresponding modulus (FIG. 3B; n=4). Gels of 2 mg / cm2were about 50-fold weaker.

[0018] FIG. 4A and FIG. 4B show results of the characterization of a VC-Wrap. Release of hyaluronan (HA; FIG. 4A) and collagen (FIG. 4B) into the media monitored over 10 days.

[0019] FIG. 5A and FIG. 5B show biochemical analysis of synovial explants as determined by dry weight (DW) / glycosaminoglycan (GAG) (FIG. 5A) and COL / DW (FIG. 5B). Dark Bars: Synovium alone; Light Bars: Synovium-cartilage co-culture. Data presented as mean and standard deviation (n=10-12 / group). Groups with disparate letters indicate significant differences (p<0.05).Docket 88800730-000529

[0020] FIG. 6 shows analysis of cartilage co-cultured with interleukin- ip (10 ng / mL) ± dexamethasone microsphere-laden VC-Wrap sheets (2.67 mg / mL DEXMS-loaded 3mg / cm2VC-Wrap). Panel A. Co-culture model of synovial joint using Transwell® culture system where dexamethasone-loaded PLGA microspheres in the VC-Wrap sheet were secured to the porous filter with ACL fibroblasts (ACLF) culture atop, and engineered canine cartilage placed in lower chamber. Biochemical measurement of DNA levels (indicative of cell number; Panel B) and GAG (Panel C) content measures showing maintenance of canine (K9) cartilage tissue properties in presence of interleukin- 1 , n=4-6 per group. GAG content correlates with mechanical properties. Panel D. Reciprocal shear loading of cartilage on synovium showing increased metabolic activity.

[0021] FIG. 7 shows effect of dexamethasone on inhibiting deleterious effects of pro-inflammatory cytokines. Soluble hyaluronan (ECM product; Panel A), prostaglandin-E2 (Panel B) and NO (Panel C) (inflammatory media markers) in culture media of synovium explants showing elevated levels in response to pro-inflammatory cytokine interleukin- ip that is suppressed by dexamethasone. (Panel A and Panel B: n=4-6 per group, *p<0.05 vs. other groups; Panel C: *p<0.05 between d7 and day 14; #p<0.05 vs. all other groups.

[0022] FIG. 8 shows human ACL fibroblast response to blood. Quantification of ACL fibroblast number (Panel A), lactate dehydrogenase (LDH; Panel B) and NO (Panel C). Cell number decreased with time in culture in a blood concentration-dependent manner; fibroblasts lost their stellate morphology over culture time.

[0023] FIG. 9 shows fluorescent recovery after photobleaching (FRAP) measurements of diffusivity. Panels A, B and C. Experimental fluorescence recovery data, showing FITC (green) and laser bleached region (BR- dark line), inputted into theoretical framework to assess diffusion coefficient (Panel D).

[0024] FIG. 10A and FIG. 10B show measurement of pro-inflammatory markers. FIG. 10A - lactate dehydrogenase (LDH). FIG. 10B -NO.

[0025] FIG. 11 shows results of wound assay of confluent human ACL.

[0026] FIG. 12 A, FIG. 12B, FIG. 12C and FIG. 12D show gene expression of human ACL fibroblasts cultures with varying blood concentrations at day 2 and day 4.

[0027] FIG. 13 shows the shear stress of cultured ACL fibroblasts cultured under conditions of 10% FBS-DMEM or with high molecular weight hyaluronan (HMWHA).Docket 88800730-000529

[0028] FIG. 14 shows results of human ACL fibroblast-collagen gel assay showing control gel on Day 0 and experimental groups on Day 4.

[0029] FIG. 15 shows healthy hACLF [Ca2+], response towards lysed blood exposure *p<0.05, **p<0.01, ***p<0.00I and ****p<0.0001.

[0030] FIG. 16 shows results of effect of pro-inflammatory cytokines. Panel A. Normalized NO data for cultured chondrocytes and fibroblast-like synoviocytes (FLS) subject to control (CTL), and interleukin- ip (1 ng / mL or 50 ng / mL). Panel B. Corresponding raw DNA data.

[0031] FIG. 17 shows conditioned media from the Day 3 Chondrocytes and Day 6 FLS co-cultured with VC-Wrap for up to 16 weeks. The VC-Wrap lost ~40 percent collagen weight at 1 week and plateaued out to 16 weeks.

[0032] FIG. 18 shows the biomechanical performance of wraps (barriers) incorporating microspheres. Panel A. Representative stress vs. strain curve for MS-embedded VC-Wrap. Panel B. Energy derived from area under the curve (AUC). Panel C. Young’s modulus derived from linear portion of stress-strain curve.

[0033] FIG. 19 shows canine ACL transection with dexamethasone microsphere-laden VC-Wrap. Panel A. Arthroscopy images of canine ACL transection model with internal brace (IB) placement. Panel B. Schematic of dexamethasone-laden PLGA microspheres in collagen VC-Wrap that is used to cover sutured ligament ends at transection (t) site. Panel C. Images of DEX-VC-Wrap delivery and placement over adjoined tissue transection site.

[0034] FIG. 20 shows canine ACL transection with dexamethasone microsphere-laden VC-Wrap over time. Panel A. Schematic of how FiberTape (suture bracing) is placed in parallel with primary anterior cruciate ligament (ACL) repair (suturing of surgically cut ligament ends together) where DEX-Wrap has been positioned over the adjoined tissue. Panel B. 3-month arthroscopy image of control, primary repair without DEX-wrap. Panel C. Experimental group with DEX-wrap. Grossly, slight fibrillation of cartilage is evident particularly in the lateral surfaces of the canine stifle (knee) joint. LFC: left femoral condyle, PFJ: Patellofemoral Joint, IB: internal bracing.

[0035] FIG. 21 A, FIG. 2 IB and FIG. 21 C show results of in vivo studies with DEX-wrap in a primary ACL (CCL) repair. FIG. 21A. Clinical measures of dogs at the 3-month terminal time point. Lower scores / measures are “better” except for CROM and Function. FIG. 21B.Docket 88800730-000529Scoring of arthroscopic images of stifle joint compartments accessing synovium and ACL status (cut ends being intact, partially intact, or not intact). FIG. 21C. and cartilage. Non-Surg: contralateral limbs; Surg: ACL repair ± DEX-wrap.

[0036] FIG. 22A shows dexamethasone release characteristics from VC-Wrap laden microspheres (DEXMS) over a ten week period.

[0037] FIG. 22B shows diffusion coefficients of 3 solutes in a bovine ACL sheath vs. VC-wrap.

[0038] FIG. 23 A shows representative arthroscopic images of the CCL 12-weeks after surgery with and without a VC-wrap, the inset showing the VC-wrap alone.

[0039] FIG. 23B shows clinical assessments of anterior drawer, CROM, Pain, Xray, OA, Cartilage integrity, and ACL Status. Assessments were made on the visual analog (VAS) scale according to OARSI scoring. Data reported as mean plus / minus standard deviation and analyzed via t-test or Mann-Whitney test (a = 0.05), *p < 0.05, **p < 0.01.

[0040] FIG. 24A shows a schematic of 2D wound closure assay.

[0041] FIG. 24B shows the percent closure of ACLF wounds with FLS plated on the bottom or top side of a Transwell in co-culture. The ACLF co-culture control group was run simultaneously.

[0042] FIG. 24C shows HA concentration assayed from GM in the bottom compartment of the Transwell.

[0043] FIG. 24D shows analysis of agarose gel electrophoresis demonstrated the distribution of HA in the supernatants of the co-culture experiment. Bars with different letters are significantly different from each other.

[0044] FIG. 25 A shows dose-dependent effect of HMWHA on ACLF wound healing.

[0045] FIG. 25B shows mean adhesion strength of ACLF pretreated + HMWHA.

[0046] FIG. 25C shows average number of focal adhesions per cell after pretreatment with HMWHA or FAK-I. Bars with different letters are significantly different from each other.

[0047] FIG. 25D shows representative images of focal adhesions and actin cytoskeleton of ACLF pretreated + HMWHA. Scale bar = 10 pm.

[0048] FIG. 26 A shows dose-dependent effect of HMWHA on ACLF gels.Docket 88800730-000529

[0049] FIG, 26B shows viability of ACLF gels following 48 hours of treatment. The dashed line represents the initial seeding amount. Bars with different letters are significantly different from each other.

[0050] FIG. 27A shows representative images of human collagen gels at 12 hours of treatment.

[0051] FIG. 27B shows gel contraction measured at 12 and 24 hours demonstrated a dosedependent effect on 3D gel contraction.

[0052] FIG. 27C shows average number of cells measured during the treatment timeline. Bars with different letters are significantly different from each other.DETAILED DESCRIPTION

[0053] The present disclosure provides a barrier, which in certain embodiments is a selectively permeable barrier, that is used to augment primary ACL repair by protecting the torn ends of the ACL from negative factors in the surrounding synovial fluid, such as large molecular weight hyaluronan (HA), while maintaining the ability of critical nutrients from the synovial fluid to diffuse into the repair site. The barrier can be acellular or cellular, and have incorporated drugs, growth factors or other desired agents that may impact ligament healing as well as the synovial joint.

[0054] One embodiment of the present disclosure is an acellular barrier composed of vitrified type I collagen gel (VC-Wrap). This VC-Wrap barrier can include a corticosteroid, for example dexamethasone-PLGA microspheres, to facilitate sustained delivery of the corticosteroid within the synovial joint. The present disclosure is also useful for tendon, cartilage, and meniscus repair as well repair of other ligamentous tissues.

[0055] EXAMPLE 1 - Characterization of synovial wrap

[0056] This example provides the characterization of dexamethasone release from wraps over a range of microsphere concentrations and collagen (VC-Wrap) wrap formulations for up to 10 weeks, and characterization of the VC-Wrap biomechanical properties and suture pull-out strength.

[0057] Characterization of Dexamethasone Release

[0058] To monitor dexamethasone release characteristics from high density VC-Wraps (1 mg / cm2) including varying concentrations of PLGA dexamethasone-laden microspheres,Docket 88800730-000529samples were incubated in PBS and dexamethasone in the supernatant quantified spectrophotometrically over 70 days. As anticipated, as the concentration of microspheres (mg / ml) increases, the percent release of (total) dexamethasone released as a function of time was observed to decrease (FIG. 1, Panel A). A control group of 2.75 mg / mL soluble dexamethasone in the media adsorbed by the VC-Wrap was performed and reaches equilibrium immediately (i.e., no sustained release) as reflected by the flat percent yield curve (top line). Another control, bottom line, confirms that the VC-Wrap itself does not release dexamethasone (or lead to changes in spectrophotometer readings). A VC-Wrap concentration of 3 mg / cm2(2.5 mg / mL) dexamethasone microspheres was selected. This formulation unexpectedly yielded twice the release rate compared to lower density gel (1 mg / cm2). This gel concentration permits good suturability and transport characteristics (FIG. 1, Panel B and Panel C) with respect to preventing permeability of large MW solutes (similar size to HA molecules that impede ligament healing). The drug release profiles and concentration ranges are similar to those previously reported for agarose hydrogel and used successfully in vitro and in vivo to decrease interleukin- 1 -induced catabolic breakdown of cartilage. High Density VC-Wrap allows smaller molecules to approach equilibrium relatively quickly (70% of equilibrium after 24 hours, 100% after 96 hours), while inhibiting equilibrium transport of larger sized molecules (38% of equilibrium after 96 hours; FIG.1, Panel C).

[0059] To counter the phenomena where concentration of microspheres increases, counterintuitively, the release of dexamethasone was observed to decrease, techniques to fabricate smaller PLGA microspheres were developed that may be able to be mixed with the original sized microspheres to modulate the drug release profde. To monitor dexamethasone release characteristics from PLGA DEX-laden microspheres, samples were incubated in PBS and dexamethasone in the supernatant quantified spectrophotometrically over 33 days (FIG. 2).

[0060] VC-Wrap Mechanical Properties and Suture Retention

[0061] The VC-Wrap sheet system was developed with well-defined transport characteristics that would cover the anterior cruciate ligament (ACL) wound site with a thickened peripheral edge region extending beyond the wound for suture placement. A VC-Wrap (3 mg / cm2) is formed and then superimposed with a concentric collagen ring (20 mm outer diameter and 10 mm inner diameter) which is combined by the vitrification process. VC-Wraps with edges were re-hydrated, sutured with a 5-0 polypropylene suture, and underwent tensile testing at a strain rateDocket 88800730-000529of 0.01 mm / s (FIG. 3A and FIG. 3B). All VC-Wraps failed at the clamping site rather than the suture site, indicating favorable suturability properties. VC-Wrap exhibited collagen orientation as evident via second harmonic generation (SHG) Imaging. Suture technique for VC-Wraps was also assessed in culture using living bovine knee ligament explants that were transected and then “repaired” by placing the ends in juxtaposition.

[0062] Additional studies were performed on a VC-Wrap with well-defined transport characteristics that would cover the anterior cruciate ligament (ACL) wound site with a thickened peripheral edge region extending beyond the wound for suture placement. In addition to suture pull out tests, a protocol was developed to determine equibiaxial mechanical properties of the VC-Wrap sheet using an inflation test and digital image correlation. The properties are relatively similar to that of native synovium tissue (which is the natural lining of the synovial joint and outer surface of the ACL).

[0063] EXAMPLE 2 - Analysis of biological function of dexamethasone wrap

[0064] This example provides the assessment of the biological function of DEX-wrap on mitigating chondrocyte death / cartilage breakdown and ligament repair in a novel in vitro model of PTOA.

[0065] Engineered Ligament Optimization: Studies were performed to produce more biofidelic engineered ligament constructs for evaluation of the VC-Wrap using co-culture studies. Engineered ligament constructs were fabricated using 250,000 fourth passaged ligament fibroblasts in fibrinogen and varying numbers of porous titanium anchors. Constructs were fed every other day with 2 mL 10% aMEM supplemented with 50 pg / mL vitamin C, 5.75 mg / mL L-proline, and 5 ng / mL FGF2. Cultures were initially spiked with 1 ng / mL TGFpi to stimulate concentration. Media release of hyaluronan (FIG. 4A) and collagen (FIG. 4B), as measured by ELISA and OHP assay, respectively, was performed over 10 days.

[0066] Cartilage-Synovium Interactions in Co-culture: Toward development of a tri-culture model system of the synovial joint with ACL, cartilage and synovium, the co-culture interaction of cartilage and synovium tissues was assessed to establish a baseline. Synovium was co-cultured in the Transwell® system with cartilage in media supplemented with and without proinflammatory cytokines. This system allows co-cultures of cartilage explants with tissue synovium, a key component being the two separate fluid reservoirs separated by the engineered synovium. This co-culture model has also been used to study the interaction of mechanically-Docket 88800730-000529injured cartilage and synovium, as well as influence of triamcinolone (steroid). The preliminary data shows cross-talk between the synovial tissues and that dexamethasone, a synthetic corticosteroid, can prevent cytokine-induced changes in synovium collagen composition (FIG. 5A and FIG. 5B). Transport of FITC-labeled dextran (or dexamethasone) was performed to demonstrate selective permeability of VC-Wrap by solute molecular weight. Initial studies have not shown significant change to VC-Wrap transport behavior with proinflammatory cytokines in the culture media.

[0067] To better understand the impact of dexamethasone on inhibiting deleterious effects of pro-inflammatory cytokines, cartilage was co-cultured with interleukin- 1 P (10 ng / mL) ± dexamethasone microsphere-laden VC-Wrap sheets (2.67 mg / mL DEXMS-loaded 3mg / cm2VC-Wrap) using a Transwell® culturewell system. The study provided paracrine (non-contact) communication between anterior cruciate ligament (ACL) fibroblasts and articular chondrocytes. Representative data is shown in FIG. 6, Panels A, B and C. The studies demonstrate chondroprotection from interleukin, established previously to lead to tissue breakdown by elevation of degradative enzymes, by dexamethasone and supports the strategy to use the VC-Wrap system to suppress the pro-inflammatory cytokine environment post joint injury. In other studies, the biological response of physiologic shear loading of cartilage and synovium was assessed using an MTT colorimetric assay (FIG. 6, Panel D). The latter is one component of the in vitro model system to evaluate the efficacy of the VC-Wrap system.

[0068] To better understand the impact of dexamethasone on inhibiting deleterious effects of pro-inflammatory cytokines, synovial explants were co-incubated with interleukin- 1 ± dexamethasone. As observed in the past for articular cartilage, the corticosteroid was able to protect synovium as determined by maintenance of control levels of hyaluronan (HA) and prostaglandin-E2 (PGE2) and nitric oxide (NO) production (FIG. 7, Panels A, B and C, respectively). These results support the efforts to provide sustained delivery of dexamethasone within the synovial joint to mitigate development of post traumatic OA (PTOA) associated with ACL injury.

[0069] Atomic Force Microscopy (AFM) was used to measure the Young’s modulus of synovium tissue as a more sensitive measure of tissue property changes (than inflation method described previously). For determining the spatially-varying modulus of the synovium, atomic force microscopy (AFM, Asylum MFP 3D Bio Infinity) can be used as performed in preliminaryDocket 88800730-000529tests on synovium samples where 5 different “local test regions” (100 pm xlOO pm) were subjected to 5 elastic modulus indentation tests at each area. The mean modulus for human synovium from osteoarthritic knees, which are subject to pro-inflammatory cytokines in situ, was 10.29 ± 3.8 kPa, about half that of healthy synovium. Note the much greater variability in modulus for human OA native tissues versus engineered. The engineered synovium serves as a cellularized version of the VC-Wrap that is being tested in parallel with the acellular VC-Wrap.

[0070] Joint inflammation can be induced by pro-inflammatory cytokines as well as blood post injury. In this regard, immune cells are thought to facilitate lysis of red blood cells (RBC, or erythrocytes) causing their release of iron rich hemoglobin that can induce ferroptosis and cell death. Studies were performed looking at co-culture effects of blood with cartilage, synovium and ACL fibroblasts. Hemoglobin has a molecular weight of 65 kDa (similar to the lower molecular weight FITC-dextran adopted herein). As such, efforts are made to create a VC-Wrap with synovium-like transport properties, able to exclude large molecular weight (~2 MDa) molecules, analogous to larger hyaluronan molecules that can inhibit tissue repair, and modulate entry of hemoglobin and its associated iron. Dexamethasone is an anti-inflammatory corticosteroid that may therefore serve to mitigate PTOA (e.g, cartilage degeneration) as well as blood effects contributing to poor ACL repair.

[0071] Whole human blood was purchased from the New York Blood Center and separated into its individual components via Ficoll-Paque separation technique. Reported studies have shown that a minimum concentration of 10% v / v blood for 2 days induces long-lasting detrimental changes to cartilage in vitro (i.e., inhibition of proteoglycan synthesis) while concentrations exceeding 50% v / v blood has been clinically shown in traumatic joint bleeds. In addition, some clinical practices anticipate a blood volume fraction of 100% due to the negligible synovial fluid volume following acute injury, and has been confirmed in in vivo rat and rabbit models. Blood was co-cultured with human anterior cruciate ligament (ACL) fibroblasts that were isolated from ACL using enzymatic digestion. Media was analyzed for markers of cell damage (lactate dehydrogenase, LDH) and pro-inflammation (NO). The studies indicate that blood has a negative impact on ACL fibroblast health in a dose-dependent manner, FIG. 8, Panels A, B and C. A wound assay was performed by culturing a confluent monolayer of human ACL fibroblasts and scratching a cruciform wound (“cross”) using a pipet tip. Cultures were monitored over time to assess the impact of blood (whole and lysed) on wound closure time. Control cultures exhibited closure byDocket 88800730-00052972 hours, whereas the presence of blood inhibited closure indicating an adverse blood impact on wound healing capacity.

[0072] Transport Behavior of DEX-FITC from VC-Wrap in the Presence of Proinflammatory Cytokines

[0073] Fluorescence Recovery After Photobleaching (FRAP) was utilized to assess transport properties of VC-Wrap, with the goal of achieving wrap properties similar to that of native synovium tissue (FIG. 9, Panels A, B, C and D). FRAP was performed in 3 areas of 4 regions each using 488 nm laser using a method previously described. The protocol consisted of 5 cycles monitored at 2% laser power for baseline and 60 cycles photobleached at 100% laser power in a 30 pm circle. 600 cycles monitored at 2% laser power for recovery. The transport properties of VC-Wrap compare favorably with that of native synovium tissue, which has similar properties to the ACL surface sheath.

[0074] RBC and ACL Fibroblast Culture: Whole human blood (O+, New York Blood Center) was separated via the Ficoll-Paque separation technique. Healthy human ACL was obtained from the Musculoskeletal Transplant Foundation (Edison, NJ). Cells were expanded until confluency in 10% DMEM supplemented with 5 ng / ml FGF-2 and cultured with various blood concentrations for 4 days. Previous dose response experiments have shown that 20% v / v is the minimum blood concentration to induce significant cell toxicity. Therefore, lysed blood and osmolarity-controlled lysates were added to culture media at 20% v / v. To study mechanotransduction, human ACLF were plated in silicone isolators on type 1 collagen-coated glass slides at a cell density of 4 x 104cells / cm2. Cells were pre-conditioned in 20% v / v lysed blood and osmolarity-controlled lysed blood, with parallel untreated controls. To track changes in intracellular calcium ([Ca2+]), hACLF were stained using 5 pM Fura Red. Fluid flow-induced shear stress was applied in a parallel plate flow chamber at 0.1 Pa. Unidirectional flow experiments were conducted using Hank’ s Buffered Salt Solution (HBSS) supplemented with 0.1% fetal bovine serum (FBS). Fluid shear data were collected for 100 cells per slide, pooled across four slides per group to yield a representative response. Fluorescence intensity versus time measurements were taken for individual cells, where increasing ([Ca2+]) resulted in decreased fluorescence. Cells were considered to be responders if relative ([Ca2+]), increased 20% above baseline equilibrium measurements. The total percentage of responding cells was determined using a custom Matlab (MathWorks) code. GPX4 activity was measured in cell culture media after experimentalDocket 88800730-000529treatment normalized to control using the 2-AACTmethod. Target genes were normalized to GAPDH and control values.

[0075] Human blood (0+) was separated via the Ficoll-Paque separation technique. Healthy human ACL obtained from MTFBiologics (Edison, NJ). ACL fibroblasts were isolated via enzymatic digestion and cultured with various blood concentrations for 4 days. Isolated intact RBC were added with chondrogenic media (CM) at subphysiologic (20% v / v), physiologic (40% v / v), and supraphysiologic (60% v / v) blood concentrations.

[0076] Media and Gene Expression Analysis: The media from each treatment condition was analyzed for NO release, a pro-inflammatory marker, and lactate dehydrogenase (LDH), a marker of cell damage. GPX4, STEAP3, TFRC, and TP53 activity were measured in cell culture media after experimental treatment normalized to control. GPX4 converts peroxidized lipids into lipid alcohols. TRFC is associated with iron metabolism, and STEAP3 and TP53 modulate the ferroptosis response.

[0077] Directional Cell Migration: Human ACL fibroblasts were cultured at 5 x 104cells per well used on a tissue culture treated 12-well plate until confluent. A cruciform scratch wound was made in each well using a 20 / d pipette tip before adding experimental treatment groups. Images were collected at 0, 8, 24, and 72 hours following injury. Data was evaluated using two-way ANOVA with Tukey HSD post-hoc tests (a=0.05). Values are presented as mean ± standard deviation

[0078] Statistical analysis: Biochemical content and gene expression were analyzed with ANOVA on GraphPad Prism 9 with a Tukey post-hoc for multiple comparisons, and significance was determined at p < 0.05.

[0079] Results

[0080] Pro-Inflammatory Markers: Relative LDH activity and NO concentration increased significantly with blood concentration (FIG. lOAandFIG. 10B). Significant differences were observed between each treatment group and timepoint.

[0081] In vitro Scratch Assay: 60% blood treatment significantly inhibited wound closure rate (FIG. 11). Wounds in the control culture fully closed (100%) by the 72-hour timepoint.

[0082] Effect of Blood on Ferroptosis Markers: A significant decrease was seen in GPX4, TRFC and TP53 activity with blood treatment (FIG. 12A, FIG. 12C and FIG. 12D). No statistical differences observed in STEAP3 with treatment (FIG. 12B).Docket 88800730-000529

[0083] Analysis of Large Molecular Weight Hyaluronan (LMWHA): Another study analyzed how controlling the access of large molecular weight hyaluronan (LMWHA) present in synovial fluids could impact ACL wound healing. HMWHA should impact ACL fibroblast ability to attach and migrate when the outer sheath is compromised. ACL fibroblasts, passage 1-4 were cultured in 10% DMEM + 5 ng / mL FGF2. Cells were lifted, diluted, and kept in suspension before treatments: 1 mg / mL HMWHA suspension for 3 hours, 1 mg / mL HMWHA adsorption for 1.5 hours and 10% FBS-DMEM suspension. HMWHA reduced the adhesion strength of ACL fibroblasts more than 3-fold, suggesting a mechanism for impaired ACL healing upon injury (FIG. 13). These findings support the approach to include a selectively permeable VC-Wrap to exclude HMWHA from the site of primary ACL repair.

[0084] Impact of Blood on Human ACL Fibroblast Would Healing: Impact of blood on human ACL fibroblast wound healing response in 3D as measured in a collagen gel contraction assay. Cells were seeded at a concentration of 8 x 106cells / mL in a 2 mg / mL type I collagen solution based on manufacturer’s instructions. Cell-seeded collagen gels were cultured for 96 h in serum-free DMEM (CM) and imaged at various time points to analyze changes in gel surface area over the course of treatment (FIG. 14). The presence of blood (20% V / V lysed red blood cells (RBC) or intact RBCs, and whole blood) significantly decreased gel contraction, where intact RBCs and whole blood suppressed gel contraction maximally. These findings indicate that blood and blood products can significantly impair ACL fibroblast contractile behavior and suggest deleterious effects of joint bleeding on ACL repair strategies. These studies are complementary to the 2D wound healing studies performed above.

[0085] Intracellular Calcium Response. The healthy hACLF [Ca2+] response was significantly greater in comparison to the blood treatment groups. FBS was flowed (N=100 cells / slide, 3 slides / group). Fluorescence intensity over time was measured for individual cells and normalized as relative [Ca2+]i. The lysed blood and osmolarity-controlled group responded similarly for hACLF, suggesting no osmolarity effects (FIG. 15).

[0086] Gene Expression: Downregulation of glutathione peroxidase 4 (GPX4), known for converting peroxidized lipids into lipid alcohols, shows lysed blood exposure activates the ferroptosis pathway. Additionally, immunocytochemistry staining indicated that GPX4 expression was also decreased after blood exposure. Findings of decreased expression of GPX4 with blood exposure is consistent with ferroptosis as a modulator of the ACLF response to blood.Docket 88800730-000529

[0087] Discussion

[0088] Calcium signaling was significantly decreased in lysed blood treatment groups in comparison to control. The significant decrease in cell responders indicates that exposure to lysed blood significantly affects mechanotransduction and highlights a mechanism that leads to changes in hACLF [Ca2+]i responses. Downregulation of GPX4, known for converting peroxidized lipids into lipid alcohols, shows lysed blood exposure activates the ferroptosis pathway. Additionally, immunocytochemistry staining indicated that GPX4 expression was also decreased after blood exposure. These studies provide new insights on ACL biology and biomechanics, and optimize clinical intervention and / or therapeutic targets to mitigate or reverse the negative effects of intraarticular bleeding in ACL treatment. In vivo studies will further characterize blood interactions in the joint.

[0089] EXAMPLE 3 - Studies of RBC crosstalk with human ACL fibroblasts

[0090] This example provides the results of studies to assess the degradative effects of RBC crosstalk with human ACL fibroblasts in the context of biochemical content, media analysis, and viability staining.

[0091] Methods and Experimental Design

[0092] Cell Culture: ACL fibroblasts were enzymatically isolated from healthy human ACL (MTFBiologics). Isolated intact RBC and lysed blood (NYBC) were added to chemically-defined media (CM) at 20% v / v.

[0093] 3D Collagen Gels: Human ACLF were cultured at 3520 cells / cm2until confluency in 10% DMEM supplemented with 5 ng / ml FGF-2. To cast collagen gel constructs, 5X DMEM, rat tail type I collagen, INNaOH, and cell suspension were combined until homogeneously mixed. Solid gels matured at 37°C for 4 days in 24 well plates before being transferred to a 12-well plate for direct blood co-culture or to a 12-well plate with Transwell® (blood apical compartment) for indirect RBC exposure. Images were collected at day 4 following treatment exposure.

[0094] Media Analysis: Supernatants from each treatment condition were collected at day 4 and analyzed for lactate dehydrogenase (LDH), a marker of cell damage and NO, a pro-inflammatory marker.

[0095] LEGENDplex: Human inflammation target analytes: IL1 ?, IFN-cr2, IFN-y, TNF-a, CCL2 (MCP1), IL-6, IL- 10, IL- 12, IL-17A, IL- 18, IL-23, and IL-33 concentrations were measured in cell culture media.Docket 88800730-000529

[0096] Biochemistry: At day 4, collagen constructs were assessed for biochemical content (DNA and GAG), with ELISA for human collagen 1 alpha 1. Media supernatants and 3D construct homogenates for collagen were normalized to DNA.

[0097] Statistical analysis: ANOVA with a Tukey post-hoc for multiple comparisons, and significance was determined at p < 0.05.

[0098] Results

[0099] Live / dead staining showed that most cell death in constructs treated with RSL-3, a ferroptosis-inducing agent group, with a significant amount of cell death observed in constructs directly exposed to lysed RBC and intact RBC. The majority of cell death was observed on the periphery of the construct with less cell death seen towards the center. Intact RBC treatment significantly increased NO concentrations and relative lactate dehydrogenase levels (p<0.001 for day 4). Analysis of collagen content in media supernatant revealed that there was significantly higher collagen content in the intact RBC group in comparison to the control (p<0.05 for day 4). Collagen content in tissue homogenates showed a relative decrease in collagen content following intact RBC exposure. Interestingly, there was no significant changes in DNA content of collagen constructs following 4-day blood exposure. A significant decrease in GAG content was observed in the intact RBC group in comparison to the control (p<0.01 for day 4). All inflammatory analytes exhibited a significant increase following direct exposure with intact RBC (Table 1, p<0.05). For indirect exposure, CCL2 concentration was significantly increased following intact RBC exposure (Table 1, p<0.05).Table 1Docket 88800730-000529

[0100] Discussion

[0101] Under the conditions of the current study, hACLF-seeded collagen constructs directly exposed to RBCs exhibited significant levels of inflammatory cytokines and cytotoxic markers compared to control. Greater cell death in the RSL-3 treatment group indicates that induction of the ferroptotic pathway significantly induces ACLF cytotoxicity and cell death observed with intact and lysed RBC groups showcases the harmful effects of blood exposure. After 4 days, the direct exposure of intact RBC led to an upregulation of all inflammatory cytokines. In contrast, Transwell® exposure of intact RBC physically separated from ACL cells only significantly increased CCL2. CCL2, also known as MCP1, is an important chemotactic protein that regulates the macrophage immune response. Together this study shows that direct contact and / or close proximity of RBC to ACL fibroblasts induce cell death pathways and construct matrix breakdown.

[0102] EXAMPLE 4 - Modification of VC-Wrap

[0103] This example provides the results of modification of the VC-Wrap. During preparing for the in vivo canine studies, an unanticipated issue arose with the integrity of the VC-Wrap (e. ., tearing) due to the surgical manipulation required to apply the collagen wrap over the ACL ligament transection site. After limited success using the VC-Wrap with troubleshooting, it was decided to transition away from a sheet-based approach to one where the VC-Wrap is delivered as a prefabricated cylindrical (hollow) sleeve or cuff that can be pulled over the ACL. A trial was performed using the VC-Wrap as a sleeve using a method that is used clinically for a femoral avulsion, which comprises almost all repairs. The procedural steps: 1) Transected at femoral origin, 2) Drill tibial and femoral tunnels, 3) Place VC-Wrap cuff and place a mattress suture to secure to ACL and 4) Placed transarticular-transACL FiberTape internal brace and secure.Docket 88800730-000529

[0104] Studies were performed to look at ability of the vitrigel wrap to survive an in vitro pro-inflammatory cytokine environment that reflects the synovial joint milieu post injury. Healthy human FLS and chondrocytes were digested from human tissue provided by MTFBiologics. Passage 2 cells were plated in a 12-well plate and allowed to expand until confluence. ILip was applied to the monolayers for 6 days. Media samples were taken at 3 and 6 days for downstream applications, including for NO (FIG. 16, Panel A), and for conditioned media studies (FIG. 16, Panel B), where VC-Wraps were co-incubated for up to 16 weeks (FIG. 17). These results suggest that the VC-Wrap can withstand the potentially harsh post-implantation chemical environment.

[0105] EXAMPLE 5 - Fabricate microspheres and VC-Wrap for in vivo implantation

[0106] This example provides the results of fabrication of microspheres and VC-Wrap (barrier) for implantation.

[0107] The VC-Wrap was fabricated using 6 mg / mL rat tail collagen type I (Corning) combined with 10X PBS, NaOH, and water, pipetted into a rectangular rubber mold (total volume = 1 mL, surface area = 2 cm2for a final density of 3 mg / cm2), gelled for 2 hours in the cell culture incubator, transferred to a flat rubber platen, vitrified for 16 hours at room temperature and humidity in the biosafety cabinet to maintain sterility, then rehydrated in IX PBS for 5 min prior to mechanical testing. In order to make the wraps, the rehydrated rectangular sheet was wrapped around an 8 mm tube (the part of the serological pipette that's inserted into the pipette gun works well for this purpose and it's sterile), then the sheet is permitted to dry out and vitrify again. Mechanical characterization of fabricated VC-Wraps showed that the incorporation of microspheres did not significantly modify the biomechanical performance of the wraps (FIG. 18, Panel A, B and C).

[0108] For the in vivo canine studies, the efficacy of a dexamethasone microsphere-laden VC-Wrap was tested using a method that is used clinically for a femoral avulsion, which comprises almost all repairs. The procedural steps: 1) Transected at femoral origin, 2) Drill tibial and femoral tunnels, 3) Place transarticular-trans ACL FiberTape (Arthrex) internal brace and secure, and 4) Place VC-Wrap and place a mattress suture to secure to ACL (FIG. 19, Panel A, B and C, FIG.20, Panel A, B and C). The experiments were carried out for 3 months where degenerative changes of the synovial joint resulting from ACL transection, which remains unhealed, are well-established in the literature (and serve as historical un-repaired controls).Docket 88800730-000529

[0109] The clinically relevant efficacy of aDEX-eluting collagen VC-Wrap in modulating development and progression of OA in a primary ACL (CCL) repair performed on a validated preclinical large animal (canine) model of ACL transection and repair was studied. Adult purpose-bred research hounds (>20kg) (n=12; 12 knees) were be used. Dogs were divided up into two groups: ACL transection + ACL repair (ACLR) with DEX-laden VC-Wrap (N=6 knees), ACL transection + ACLR only (N=6 knees) - control group, where Contralateral knees (N=12) -unoperated controls.

[0110] Primary outcome measures

[0111] Clinical assessments: (lameness grade, VAS function, comfortable range of motion): Day 0 and 12-wk post-operatively.

[0112] Arthroscopy: (2nd look): Day 0 and 12-wk on RIGHT (surgery) knees.

[0113] Knee radiographs: Day 0 (pre-op) and 12-wk on RIGHT (surgery) knees. Secondary measures on saved specimens: Gross exam (with India ink; determine the effects of articular defects on apposing articular cartilage): 12-wk on RIGHT (surgery) knee. Histology (OARSI): 12-wk on RIGHT (surgery) knee.

[0114] After 3 months, clinical, arthroscopy and radiograph measures indicated that canine knees where ACL repair was augmented with a VC-Wrap had improved ACL repair, with more normal synovium and cartilage presentation (FIG. 21A, FIG. 21B and FIG. 21C). The ACL status (not intact:0, partially intact:!, intact:2) was significantly correlated with CROM (Spearman r=0.79, p=0.004), Function (Spearman 4= 0.94, p<0.0001), and negatively correlate with lameness (Spearman r= -0.64, p=0.283), pain (Spearman r= -0.94, p<0.0001), Drawer (Spearman r= -0.68, p=0.022), and internal rotation (Spearman r= -0.62, p=0.035).

[0115] Together with insights gained from in vitro studies, the in vivo studies found that the DEX-wrap is able to facilitate ACL primary repair as well as to mitigate development of degenerative change to articular cartilage consistent with PTOA (FIG. 21C). The cartilage properties may have been directly impacted by sustained release of the corticosteroid DEX from the wrap into the joint space, known to be chondroprotective and to modulate the inflammatory environment (FIG. 2 IB). Alternatively, cartilage maintenance may be the beneficiary of more stable biomechanics and normal knee function afforded by enhanced ACL repair with wrap augmentation as noted by decreased joint laxity on clinical measures from the anterior draw test and internal rotation (FIG. 21A).Docket 88800730-000529

[0116] Further in vivo canine studies were performed. DEXMS VC-Wraps were shipped dry, and rehydrated in IX PBS for 5 min before use. They tend to shrivel a bit during the drying process so it may be challenging to fit them over the ligament if they aren't hydrated. Six subjects: ACLT, suture tape bracing, primary repair with DEX-Wrap; 6 Dogs: ACLT, suture tape bracing, primary repair. ACLT, ± bracing historical control. Arthroscopy showed better cartilage protection (i.e., lateral side) with DEXMS-Laden VC-Wraps. XR PTOA severity scores approach significance, p=0.08; DEX: 5 + 2; No-DEX: 7.9 + 3, Scale: 0-20, 20 most severe. For reference, ACL-transection (ACLT) without brace scores are a little higher, i.e., 8-12, at 3 months but not a lot higher at that time point and importantly most braces without DEX were loose or broken which is major finding in this study.

[0117] EXAMPLE 6 - Fabricate microspheres and ligament wrap for in vivo implantation

[0118] This example provides the results of fabrication of microspheres and ligament wrap (barrier) for implantation.

[0119] Fabrication of Biomaterials: DEXMS were made by encapsulating 100 mg DEX in 75:25 poly(lactic-co-glycolic acid). The DEXMS were then mixed into a collagen type I solution with NaOH, phosphate buffered saline (PBS), and ddFLO. The solution was allowed to gel in a mold at 37°C for 2hr, then vitrified at 25°C overnight to create a flat sheet (final collagen concentration = 3mg / cm2). The collagen sheet was rehydrated in PBS, then wrapped to create a hollow, cylindrical sleeve (0 = 8mm). The vitrified collagen wrap (VC-Wrap) was used for all downstream testing and applications. Initial mechanics of the biomaterial were measured using tensile testing and fluorescent recovery after photobleaching (FRAP) to determine diffusion coefficient. To contextualize outcomes, FRAP measurements were also made of juvenile bovine ACLs collected from a local abattoir. Samples were saturated in FITC-dextrans of multiple sizes (20, 70, and 2000 kDa) prior to photobleaching. DEX release was evaluated over 10 weeks by culturing VC-Wraps in PBS and assaying the absorbance (242nm).

[0120] In Vivo Surgeries: A pre-clinical canine model of injury was used to assess functionality and durability of the VC-Wrap (IACUC: 42907). Adult, purpose-bred research hounds (>20kg, female) underwent aseptic surgery of the right stifle. The native CCL was debrided of the ligament sheath, transected at the midpoint, then secured at the femoral anteromedial and tibial anterolateral portals with the braided FiberTape ± VC-Wrap (n = 6). 12 weeks after surgeryDocket 88800730-000529arthroscopic images and X-rays of the right stifles were captured for gross analysis and OA scoring. Clinical assessments of anterior drawer, comfortable range of motion (CROM), pain, and XRay OA were measured by two board-certified veterinary surgeons. Subjects were then euthanized with an intravenous injection. Stifles were dissected to isolate CCLs and synovia. Tissues were fixed in 10% formalin, embedded in paraffin, sectioned, and stained for blinded OARSI histological scoring.

[0121] Statistics: A post-hoc analysis determined the power to be (1 - ) = 0.407. Data was reported as mean ± standard deviation. One-way ANOVA with Tukey’s HSD post-hoc test (a = 0.05) was used for comparing differences in VC-Wrap formulation. A t-test or Mann-Whitney test (a = 0.05) was used to compare all other differences.

[0122] Results: Functionality of the VC-Wrap: After 10 weeks it was determined that 2.5mg / mL DEXMS embedded into the VC-Wrap would deliver the critical dosage of 0.9ng DEX / day after an initial burst release (FIG. 22A). This concentration of DEXMS was used for all further downstream tests and applications. The diffusion coefficients of all dextrans were not significantly different between the VC-Wrap and the ACL sheath (FIG. 22B). The presence of DEXMS in the VC-Wrap did not significantly weaken the material properties of the collagen wrap compared to a MS-free control. Young’s Modulus was measured to be 1.90 ± 1.88 MPa vs. 1.16 ± 0.69 MPa and Ultimate Tensile Strength was measured to be 0.39 ± 0.50 MPa vs. 0.070 ± 0.04MPa for the control vs. VC-Wrap, respectively. Surgical Outcomes: All surgical sites healed fully and no subjects developed any infection or other diseases during the duration of this study. Subjects repaired with the VC-Wrap appeared to have less degradation of the FT than subjects without the augmented repair (FIG. 23 A). Significant improvements were seen in anterior drawer, CROM, pain, XRay OA, and cartilage integrity scores, where most areas showed no signs of damage. Furthermore, evaluation of histology revealed the CCLs were more often fully intact after augmented repair than FT-alone (FIG. 23B). Histology of synovia showed similarly fibrotic ECM in both groups. There were greater overall OARSI scores on the medial side of the stifle compared to the lateral side. Neither CCL nor synovia scores were significantly different between surgical groups.

[0123] The testing was undertaken to determine whether a DEX-eluting VC-Wrap could improve primary ACL repair outcomes in a canine model. The VC-Wrap group showed reduced anterior laxity, pain, and lameness, along with improved CCL tissue integrity. Histological andDocket 88800730-000529arthroscopic assessments revealed the wrap maintained a healing-conducive microenvironment while fully degrading by 12 weeks. The VC-Wrap appeared to act as a surrogate synovial sheath, protecting the repair site and supporting ECM deposition and cellular infiltration along the periphery of the CCL. Localized DEX delivery likely improved outcomes by attenuating inflammation and reducing the risk of PTOA. Cartilage across the joint was better preserved in DEX-treated subjects, supporting DEXMS as an effective disease modifying OA drug. This experiment demonstrates that a DEX-eluting biomaterial wrap can significantly improve ACL healing by reducing inflammation, preserving tissue integrity, and preventing early joint degeneration.

[0124] EXAMPLE 7 - Negative impact of high molecular weight hyaluronan on anterior cruciate ligament wound repair

[0125] The average annual incidence of anterior cruciate ligament (ACL) injuries in the United States has been reported to be as high as 68.6 per 100,000 people, with disproportionately elevated incidences in active individuals under the age of 25 years old. As intra-articular ligaments like the ACL have an inferior healing capacity compared to that of extra-articular ligaments, such as the medial collateral ligament (MCL), reconstructive surgery is the gold-standard treatment for patients to restore stability and function. Despite initially successful surgical reconstructions, failures in terms of re-tears still occur in 6 to 20% of patients, and a significant proportion of patients develop posttraumatic osteoarthritis (PTOA) within a decade. PTOA is likely due to the combination of an inability to restore native biomechanics of the joint through tendon autograft or allograft reconstruction and an upregulation in pro-inflammatory and catabolic factors in the synovial fluid stemming from the injury and subsequent surgical intervention.

[0126] However, recent studies have shown that the ACL has an innate healing response marked by myofibroblasts and the formation of new blood vessels, as indicated in patient populations where the ruptured ACL is attached to the posterior cruciate ligament. Primary repair of the torn native ACL has been considered a “holy grail” when compared to reconstruction, but long-term clinical studies indicate that the majority of patients do not achieve acceptable tissue material properties or morphology, such that instability or failure were common. Importantly, an intact synovial sheath around the ACL is associated with lower repair failure rates resulting in re-tears or laxity than those with partial or full sheath tears. ACL repair may therefore be facilitatedDocket 88800730-000529by rapid recapitulation of the physical and physiological barrier separating the tissue from its surrounding synovial fluid environment after injury.

[0127] The synovium that envelops the mobile joint is comprised of fibroblast-like synoviocytes (FLS) that produce synovial fluid, which contains hyaluronic acid (HA). HA plays an integral role in the joint as a lubricating molecule. Synovial fluid in the intact knee is known to be beneficial both chemically and physically due to its role in joint lubrication and cellular signaling, but it is possible that it plays a negative role in wound healing of ligament fibroblasts. In fact, supplementation of cell culture models with synovial fluid restricts ACL fibroblast (ACLF) proliferation. It was also demonstrated that high molecular weight HA (HMWHA) of 1.6 and 3.6 MDa inhibited tendon fibroblast proliferation in a dose-dependent manner. The inventors reasoned that in situ, the violated sheath allows HMWHA to enter the wounded area and inhibit healing of the ACL. Accordingly, in vitro studies were performed to test whether HMWHA is detrimental to ACLF healing capacity in experiments investigating: (1) transwell co-culture of ACLF with HMWHA or FLS, and (2) the impact of HMWHA pretreatment of ACLF on contraction of 3D collagen constructs.

[0128] Methods

[0129] Isolation of Cells

[0130] FLS were isolated as previously described (Estell et al., J. Biomech. 60:91-99, 2017). Briefly, synovial tissue explants were harvested from juvenile bovine joints (Green Village Packing) and digested in collagenase type II (Worthington Biochemical) for 6 hours with shaking at 37°C. Bovine tissue was chosen due to its similarities to native human ACL and availability. Cells were then passed through a 40 pm nylon mesh and expanded in growth media (GM) until passage 2 to 4. GM was comprised of aMEM (Fisher Scientific) supplemented with 10% fetal bovine serum (R&D Systems), 1% antibiotic-antimycotic (Gibco), and 5 ng / mL human fibroblast growth factor-2 (Fisher Scientific). ACLF was recovered from the same joints using an identical digestion protocol. Bovine ACLs were degloved of the synovial sheath by pulling taught with foreceps and cutting away with a scalpel prior to tissue digestion.

[0131] Transwell Co-Culture

[0132] ACLF were plated onto 24-well plates and expanded until confluent. Then, ACLF and FLS were seeded onto either the top or bottom of 0.4 pm pore Transwells (Coming; FIG. 24A) at a density of 10,000 cells / filter. Cells were allowed to attach overnight before the filters wereDocket 88800730-000529placed into co-culture with the ACLF monolayers. Following co-culture studies, GM was assayed for HMWHA concentration via the cetyltrimethylammonium bromide turbidimetric method. Agarose (0.5% w / v) gel electrophoresis was used to determine the relative sizes of HMWHA present in both compartments of the cell culture chamber. Supernatants and HA ladders (HAWorks) were supplemented with 0.005% w / v Stains-All (Sigma-Aldrich) in 50% ethanol in order to visualize the HA proteins following electrophoresis. Relative mobility of HA in the supernatants was assessed in ImageJ and compared against the known protein ladders.

[0133] Wound Closure Assay

[0134] Cruciform scratch wounds were created on ACLF monolayers in 24-well plates using a 2 pL pipette tip. Cell monolayers were rinsed with IX phosphate-buffered saline (PBS) before GM ± HMWHA (1.8 MDa, Sigma— Aldrich) was added. Images of the monolayers were taken immediately after scratching and every 2 hours until the wound had closed. Image analysis was completed with a custom ImageJ tracing tool based on the extension previously created (Suarez -Arnedo et al., PLoS One 15(07):e0232565, 2020).

[0135] Adhesion Strength Assay

[0136] ACLF in suspension was preincubated in GM ± HMWHA for 3 hours prior to plating 30,000 cells / slide onto glass slides. Focal adhesion kinase inhibitor (FAK-I) was included as a negative control group at a concentration of 10 pM. Slides were then loaded into a custom-built parallel plate flow chamber and subjected to increasingly greater flow rates of Hank’s Buffered Salt Solution (Sigma- Aldrich), calculated at the wall of the chamber by T =6 pQ / h2w, where p is viscosity of the fluid, Q is the volumetric flow rate, h is the height of the chamber, and w is the width of the chamber. The shear rate was increased step-wise every 30 seconds from 0 to 250 dyne / cm2while a time-lapse of cells was captured. In order to visualize the morphology and focal adhesions of the ACLF, cells were simultaneously preincubated and plated onto glass-bottom dishes. Monolayers were fixed in 4% w / v paraformaldehyde and stained with DAPI (Fisher Scientific), phalloidin (Abeam), and vinculin (EMD Millipore) for nuclear and cytoskeleton visualization. Quantification of the focal adhesions was performed with ImageJ.

[0137] Three-Dimensional Collagen Contraction Assays

[0138] Bovine ACLF were cast into 3D collagen constructs at a concentration of 250,000 cells / construct. Cells were first mixed with 5X DMEM (Fisher Scientific) before adding acid-solubilized collagen type I (Corning) to a final concentration of 2 mg / mL. The mixture wasDocket 88800730-000529neutralized with IN NaOH and mixed thoroughly to ensure even distribution of cells and collagen. A total of 1 mL gels were made by pipetting the mixture into a sterile 24-well plate and allowing the liquid to gel at 37°C. Following gelation, GM ± HMWHA was added to each well, and constructs were cultured for 24 hours. A total of 10 pM FAK-I was included as a negative control, and 10 ng / mL transforming growth factor-Pl (TGFP1) was included as a positive control for contraction. The diameter of each gel was measured every 6 hours to calculate the contracted area in the XY plane relative to the starting size. At the conclusion of the study, the constructs were digested in 1 mg / mL Proteinase K (MP Biomedicals) and assessed for cytotoxicity and proliferation using a PicoGreen dsDNA Assay (ThermoFisher Scientific).

[0139] Human-derived ACLF were likewise cast into 3D collagen constructs. Human ACLFs were isolated from non-OA intact- ACL donors. Collagenase type II was used to digest ACL from n =4 unique donors (all male, < 30 years old) and then pooled into one population. The pooled donors were expanded out to passage 2 prior to casting into engineered collagen constructs at a concentration of 150,000 cells / construct. GM ± HMWHA was added to each well, and constructs were cultured for 72 hours. FAK-I and TGFP1 treatments were run simultaneously. The diameter of each gel was measured every 12 hours to calculate the contracted area in the XY plane relative to the starting size.

[0140] Statistical Analysis

[0141] Data are presented as mean ± standard deviation with outliers removed via the ROUT method and analyzed in Prism 10 (Graph-Pad). Two-way ANOVA with Tukey’s post hoc test (a = 0.05) was used to analyze all data unless otherwise noted.

[0142] Results

[0143] FLS Inhibits ACLF Migration

[0144] When FLS were seeded on the bottom side of the Transwell filter, the wound healing was significantly attenuated after 10- and 12-hours compared to controls (p = 0.0145 and 0.001, respectively). However, when the FLS were plated on the top side of the 0.4 pm filter, there was no difference (FIG. 24B). All wounds closed by 24 hours regardless of treatment. The inventors reasoned that the FLS were secreting HA of various MW in both scenarios, but were physically separated from the ACLF monolayers when the cells were on the top side of the Transwell filter. GM was assayed from the supernatant of ACLF monolayers with FLS on both the top side and bottom side of the filter. There was a significantly greater concentration of HA inDocket 88800730-000529the supernatant of cells where the shared media did not first pass through a 0.4 pm Transwell filter (p= 0.0072; FIG. 24C). Gel electrophoresis was used to assess the supernatants for HA content following co-culture. Wells with FLS seeded on the top side of the filter demonstrated a lower distribution of HMWHA than those with FLS seeded on the bottom side, indicating that the filter was able to exclude much of the larger-sized HA during the study (FIG. 24D).

[0145] HMWHA Inhibits ACLF Migration and Adhesion

[0146] In order to assess the direct effects of HMWHA on ACLF, various concentrations of exogenous HA were dissolved in GM and plated over wounded ACLF monolayers. The ACLF wounds healed more slowly in a dose-dependent manner over a 12-hour period. At both 6- and 12-hours, the highest concentration of HMWHA was significantly inhibitory to the ACLF (FIG. 25A). A linear correlation coefficient was calculated between HMWHA concentration and percent closure to be r = -0.65 (p < 0.001). Again, all ACLF wounds closed after 24 hours regardless of HMWHA dosage.

[0147] ACLF in suspension was pretreated with HMWHA prior to plating on glass slides to assess its ability to form focal adhesions. Cells that were exposed to HMWHA formed significantly weaker focal adhesions compared to those suspended in GM alone (all p < 0.05; FIG. 25B). This trend was observed in a dose-dependent manner, where the highest concentration of HMWHA showed similar adhesion strength to FAK-I. A strong negative correlation was found between the mean adhesion strength of ACLF and HMWHA concentrations (r =-0.9247, p = 0.0245). Simultaneously treated ACLF were fixed and stained for visualization of the actin cytoskeleton and focal adhesions. Untreated cells demonstrated more intense, widespread staining of both actin and adhesion markers compared to HMWHA-treated cells. In fact, the pretreatment of ACLF with FAK-I prevented all cells from adhering to the culture dish (FIG. 25C). Untreated cells demonstrated more intense, widespread staining of both actin and adhesion markers compared to HMWHA (FIG. 25D).

[0148] HMWHA Prevents ACLF Contraction in 3D

[0149] Contraction of the bovine collagen constructs was significantly impacted by HA pretreatment of the ACLF in a dose-dependent manner. A total of 100 pg / mL HA treatment was the most inhibitory concentration, where constructs were only able to contract by 59.12% (p = 0.0243; FIG. 26A). FAK-I-treated constructs were significantly less contracted after 6- and 12-hours, while TGFpi-treated constructs demonstrated maximal contraction at both timepoints.Docket 88800730-000529At the conclusion of the study, a PicoGreen DNA assay revealed that none of the treatments impacted the number of ACLF in each group (FIG.26B), meaning all significant differences cannot be attributed to differences in cell viability.

[0150] Human ACLF-seeded collagen gels exhibited similar contraction patterns to the constructs made from bovine cells (FIG. 27A). TGFpi treatment immediately caused significant contraction within each construct. A dose-dependent inhibition of contraction was observed after 12 hours of treatment, where 100 pg / mL HA was significantly inhibitory to the cells’ ability to contract within the gel. However, by 24 hours, the level of contraction had begun to plateau (FIG 27B). There was no significant difference in average cell number observed over the treatment timeline, indicating the human cells remained viable throughout the study (FIG. 27C).

[0151] Discussion

[0152] Based on the current 2D and 3D cell culture findings, the results show that HMWHA is detrimental to ACLF healing capacity. Immediately following injury, the damaged ACL is exposed to a high concentration (1-4 mg / mL) of HA in synovial fluid produced by FLS in the synovial lining. When ACLF were cultured in the same chamber as FLS, the secreted HMWHA had an inhibitory impact on wound healing, suggestive of a role for synovium-ACL chemical crosstalk via synovial fluid that may mediate poor ligament healing in vivo. However, ACLF cultured in a separate chamber from FLS were not exposed to a high concentration or size of HA, leading to improved wound healing.

[0153] This effect was mirrored by the culture of ACLF with exogenous HA. A total of 100 pg / mL of HMWHA significantly impedes wound healing, which can reflect weakening ligament fibroblast focal adhesions in vitro. One explanation underlying this phenomenon is the ability of HA to bind fibronectin in vitro and within the ECM. The binding of HA to fibronectin from the GM could occur with a higher affinity than that of integrins to fibronectin, preventing adequate assembly of focal adhesions by the cells. It has been shown that the expression of vinculin in rat tendons was significantly decreased after treatment with HA, similar to the results presented herein. The latter is indicative of the mechanism underlying the application of exogenous HA injections to prevent scar formation in tendon healing following reconstruction / repair. HA-mediated inhibition of ACLF healing was translated to 3D, where HMWHA significantly impeded contraction of the construct in a dose-dependent manner. HA treatment did not affect cell viability, indicating that the sole effect occurred through inhibition ofDocket 88800730-000529focal adhesions within the construct. The juvenile bovine ACLF responded to the treatments along a shorter time course than the human ACLF.

[0154] HA-mediated inhibition of scar formation during the wound healing process presents a novel therapeutic target. The rapid restoration of the synovial barrier via repair, wraps, or a scaffold is one solution. In vivo porcine studies have shown that primary ACL repair augmented with a collagen scaffold could reduce postsurgical complications or even exceed the mechanical properties of suture repair alone up to 4 weeks postoperatively. These studies have resulted in a renewed interest in ACL repair over graft reconstruction. Successful repair preserves the proprioception and native biomechanics of the ACL, leading to a faster return to function. It has also been shown that ACL repair where the torn ends are protected from HMWHA from the synovial fluid, although indirectly, can mitigate the development of PTOA. A pilot study has demonstrated that patients may prefer ACL repair compared to reconstruction, where both surgical interventions were able to restore similar functionality to the knee, likely due to the pain and swelling that accompanies donor-site morbidity in reconstruction. A combination therapeutic that directly addresses the damaging infiltration of HMWHA into the wound site of the ruptured ACL while repairing the native tissue provides a solution for the aforementioned complications.

[0155] This example provides more complete information regarding how synovial fluid constituents, specifically large MWHA, serve as a key regulatory factor in ACLF behavior. Evidence is provided that restoration of native ACL solute transport properties, to exclude damaging HA, after injury, is a promising strategy to promote ACL healing.

[0156] This disclosure can be embodied in many different forms and should not be construed as necessarily being limited to the exemplary embodiments disclosed herein. Rather, the exemplary embodiments are provided so that this disclosure is thorough and complete, and fully conveys various concepts of this disclosure to those skilled in a relevant art.

[0157] Features described with respect to certain exemplary embodiments can be combined and sub-combined in and / or with features described with respect to various other exemplary embodiments, even if such combinations and / or sub-combinations are not specifically described herein. Also, different aspects and / or elements of exemplary embodiments, as disclosed herein, can be combined and sub-combined in a similar manner as well. Further, some exemplary embodiments, whether individually and / or collectively, can be components of a larger system, wherein other procedures can take precedence over and / or otherwise modify their application.Docket 88800730-000529Additionally, a number of steps can be required before, after, and / or concurrently with exemplary embodiments, as disclosed herein. Note that any and / or all methods and / or processes, at least as disclosed herein, can be at least partially performed via at least one entity in any manner.

[0158] Various terminology used herein can imply direct or indirect, full or partial, temporary or permanent, action or inaction. For example, when an element is referred to as being "on," "connected" or "coupled" to another element, then the element can be directly on, connected or coupled to the other element and / or intervening elements can be present, including indirect and / or direct variants. In contrast, when an element is referred to as being "directly connected" or "directly coupled" to another element, there are no intervening elements present.

[0159] Although the terms first, second, etc. can be used herein to describe various elements, components, regions, layers and / or sections, these elements, components, regions, layers and / or sections should not necessarily be limited by such terms. These terms are used to distinguish one element, component, region, layer or section from another element, component, region, layer or section. Thus, a first element, component, region, layer, or section discussed below could be termed a second element, component, region, layer, or section without departing from various teachings of this disclosure.

[0160] Various terminology used herein is for describing particular exemplary embodiments and is not intended to be necessarily limiting of this disclosure. As used herein, various singular forms "a," "an" and "the" are intended to include various plural forms as well, unless a context clearly indicates otherwise. Various terms "comprises," “includes,” "comprising" and / or “including” when used in this specification specify a presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence and / or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0161] As used herein, a term "or" is intended to mean an inclusive "or" rather than an exclusive "or." That is, unless specified otherwise, or clear from context, "X employs A or B" is intended to mean any of a set of natural inclusive permutations. That is, if X employs A; X employs B; or X employs both A and B, then "X employs A or B" is satisfied under any of the foregoing instances.

[0162] Exemplary embodiments of this disclosure are described herein with reference to illustrations of idealized embodiments (and intermediate structures) of this disclosure. As such,Docket 88800730-000529variations from various illustrated shapes as a result, for example, of manufacturing techniques and / or tolerances, are to be expected. Thus, various exemplary embodiments of this disclosure should not be construed as necessarily limited to various particular shapes of regions illustrated herein, but are to include deviations in shapes that result, for example, from manufacturing.

[0163] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in an art to which this disclosure belongs. Various terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with a meaning in a context of a relevant art and should not be interpreted in an idealized and / or overly formal sense unless expressly so defined herein.

[0164] Furthermore, relative terms such as "below," "lower," "above," and "upper" can be used herein to describe one element's relationship to another element as illustrated in the set of accompanying illustrative drawings. Such relative terms are intended to encompass different orientations of illustrated technologies in addition to an orientation depicted in the set of accompanying illustrative drawings. For example, if a device in the set of accompanying illustrative drawings were turned over, then various elements described as being on a "lower" side of other elements would then be oriented on "upper" sides of other elements. Similarly, if a device in one of illustrative figures were turned over, then various elements described as "below" or "beneath" other elements would then be oriented "above" other elements. Therefore, various example terms "below" and "lower" can encompass both an orientation of above and below.

[0165] As used herein, a term "about" and / or "substantially" refers to a + / - 10% variation from a nominal value / term. Such variation is always included in any given value / term provided herein, whether or not such variation is specifically referred thereto.

[0166] If any disclosures are incorporated herein by reference and such disclosures conflict in part and / or in whole with this disclosure, then to an extent of a conflict, if any, and / or a broader disclosure, and / or broader definition of terms, this disclosure controls. If such disclosures conflict in part and / or in whole with one another, then to an extent of a conflict, if any, a later-dated disclosure controls.

[0167] In some embodiments, various functions or acts can take place at a given location and / or in connection with the operation of one or more apparatuses or systems. In some embodiments, a portion of a given function or act can be performed at a first device or location,Docket 88800730-000529and a remainder of the function or act can be performed at one or more additional devices or locations.

[0168] The corresponding structures, materials, acts, and equivalents of all means or step plus function elements in the claims below are intended to include any structure, material, or act for performing the function in combination with other claimed elements as specifically claimed. The embodiments were chosen and described in order to best explain the principles of the disclosure and the practical application, and to enable others of ordinary skill in the art to understand the disclosure for various embodiments with various modifications as are suited to the particular use contemplated.

[0169] The diagrams, images and drawings depicted herein are illustrative. There can be many variations to the diagram or the steps (or operations) described therein without departing from the spirit of the disclosure . For instance, the steps can be performed in a differing order or steps can be added, deleted or modified. All of these variations are considered a part of the disclosure. It will be understood that those skilled in the art, both now and in the future, can make various improvements and enhancements which fall within the scope of the claims which follow.

[0170] The description of this disclosure has been presented for purposes of illustration and description, but is not intended to be fully exhaustive and / or limited to the disclosure in the form disclosed. Many modifications and variations in techniques and structures will be apparent to those of ordinary skill in an art without departing from a scope and spirit of this disclosure as set forth in the claims that follow. Accordingly, such modifications and variations are contemplated as being a part of this disclosure. A scope of this disclosure is defined by various claims, which include known equivalents and unforeseeable equivalents at a time of filing of this disclosure.

Claims

Docket 88800730-000529CLAIMSWhat is claimed is:

1. A method of preventing infiltration of blood and / or synovial fluid to at least one of a ligament, a tendon, a meniscus, or a cartilage, comprising erecting a barrier comprising a vitrified type I collagen gel and at least a first corticosteroid on the at least one of the ligament, the tendon, the meniscus, or the cartilage.

2. The method of claim 1 , wherein the barrier is a sheet or a cuff.

3. The method of claim 1, wherein the barrier is selectively permeable.

4. The method of claim 3, wherein the barrier is selectively permeable based on size, charge, component to be repaired or area of coverage.

5. The method of claim 1, wherein the at least a first corticosteroid in comprised in a microsphere.

6. The method of claim 5, wherein the microsphere is a PLGA microsphere.

7. The method of claim 1, wherein the at least a first corticosteroid is dexamethasone.

8. The method of claim 7, wherein the dexamethasone is comprised in PLGA-microspheres.

9. The method of claim 1, wherein the ligament is an anterior cruciate ligament.

10. The method of claim 1, wherein the barrier provides structural support for the at least one of the ligament, the tendon, the meniscus, or the cartilage.

11. A method for repairing an injury to at least one of a ligament, a tendon, a meniscus, or a cartilage, comprising contacting the at least one of the ligament, the tendon, the meniscus, or the cartilage with a barrier comprising a vitrified type I collagen gel and at least a first corticosteroid.Docket 88800730-00052912. The method of claim 11, wherein the ligament is an anterior cruciate ligament.

13. A device comprising a vitrified type I collagen gel sheet and at least a first corticosteroid.

14. The device of claim 13, wherein the vitrified type I collagen gel sheet at least one of blocks or limits a passage of at least one of a blood or a synovial fluid through the device.

15. The device of claim 13, wherein the vitrified type I collagen gel sheet, when positioned relative to at least one of a ligament, a tendon, a meniscus, or a cartilage, is configured to at least one of block or limit a passage of at least one of a blood or a synovial fluid to the at least one of the ligament, the tendon, the meniscus, or the cartilage.

16. The device of claim 13, wherein the vitrified type I collagen gel sheet is formed into a sleeve or cuff.

17. The device of claim 15, wherein the sleeve or cuff provides physical support for at least one of a ligament, tendon, meniscus, or cartilage.

18. The device of claim 17, wherein the sleeve or cuff is formed during placement of the device relative to at least one of a ligament, tendon, meniscus, or a cartilage.

19. The device of claim 18, wherein the sleeve or cuff is formed prior to placement of the device relative to at least one of a ligament, tendon, meniscus, or a cartilage.

20. The device of claim 13, wherein the vitrified type I collagen gel sheet is selectively permeable.

21. The device of claim 13, wherein the at least a first corticosteroid is dexamethasone.

22. The device of claim 21, wherein the dexamethasone is comprised in PLGA-microspheres.

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