Compositions and methods for tendon repair

Abstract

The composition comprises a purified exosome product (PEP) and a pharma- ceutically acceptable carrier comprising a support matrix. The support matrix may comprise a collagen scaffold, a tissue sealant, or a fibrin sealant. The method of repairing damaged tendon tissue typically comprises applying to the damaged tendon tissue a composition comprising the PEP and a pharma-ceutically acceptable carrier. In one or more embodiments, the composition applied to the damaged tendon tissue comprises a support matrix.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 279,839, filed November 16, 2021, which is incorporated by reference in its entirety. Summary of the Invention

[0002] The present disclosure, in one embodiment, describes a composition that generally includes a purified exosome product (PEP) and a pharma- ceutically acceptable carrier that includes a support matrix.

[0003] In one or more embodiments, the PEPs comprise spherical or ellipsoidal exosomes having a diameter of 300 nm or less.

[0004] In one or more embodiments, the PEPs comprise spherical or ellipsoidal exosomes with a mean diameter of 110 nm ± 90 nm. In one or more of these embodiments, the PEPs comprise spherical or ellipsoidal exosomes with a mean diameter of 110 nm ± 50 nm. In one or more of these embodiments, the PEPs comprise spherical or ellipsoidal exosomes with a mean diameter of 110 nm ± 30 nm.

[0005] In one or more embodiments, the PEP comprises 1% to 20% CD63 - Exosomes and 80%-99% CD63 + In one or more of these embodiments, the PEP comprises at least 50% CD63 - Contains exosomes.

[0006] In one or more embodiments, the PEP is 1×10 11 PEP exosomes ~ 1 × 10 13 In one or more of these embodiments, the PEP comprises 1×10 12PEP exosomes ~ 1 × 10 13 Contains PEP exosomes.

[0007] In one or more embodiments, the support matrix comprises a collagen scaffold. In one or more of these embodiments, the collagen scaffold comprises type I fibrillar collagen.

[0008] In another aspect, the present disclosure describes a method of treating damaged tendon tissue comprising applying a composition described herein to the damaged tendon tissue.

[0009] In one or more embodiments, the composition is applied in an amount effective to reduce adhesion incidence compared to injured tendon tissue treated without the composition, hi one or more embodiments, the composition is applied in an amount effective to increase the ratio of type I collagen to type III collagen compared to injured tendon tissue treated without the composition.

[0010] In one or more embodiments, the composition is applied in an amount effective to result in a more organized collagen architecture compared to damaged tendon tissue treated without the composition.

[0011] In one or more embodiments, the damaged tendon tissue comprises a disruption of the tendon. In one or more of these embodiments, the disruption of the tendon comprises a rupture of the tendon. In one or more of these embodiments, the rupture of the tendon comprises a rupture of the Achilles tendon.

[0012] The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly exemplifies illustrative embodiments. Throughout this application, guidance is provided through examples, which examples can be used in various combinations. In each instance, the above list serves only as a representative group and should not be interpreted as an exclusive list. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1A shows the surgical procedure of a rabbit in a prone position with the hind limb prepped and draped. (A) A 2 centimeter (cm) incision was made centered 1.5 cm proximal to the calcaneal tubercle. FIG. 1B shows the surgical procedure of a rabbit in a prone position with the hind limb prepped and draped. (B) The paratendon was incised and the flexor digitorum superficialis (FDS) was identified and isolated. FIG. 1C shows the surgical procedure of a rabbit in a prone position with the hind limb prepped and draped. (C) The Achilles tendon was identified and isolated. FIG. 1D shows the surgical procedure of a rabbit in a prone position with the hind limb prepped and draped. (D) A tenotomy was performed on the Achilles tendon. FIG. 1E shows the surgical procedure of a rabbit in a prone position with the hind limb prepped and draped. (E) A modified Kessler main suture was performed in all groups. Figure 1F shows the surgical procedure with the rabbit in a prone position with pre-operative preparation and draping of the hind limb. (F) In groups 2 and 3, a scaffold was placed at the tenotomy site, followed by final suture fixation. Figure 1G shows the surgical procedure with the rabbit in a prone position with pre-operative preparation and draping of the hind limb. (G) The incision was closed with absorbable sutures. Figure 1H shows the surgical procedure with the rabbit in a prone position with pre-operative preparation and draping of the hind limb. (H) The hind limb was immobilized with a hip spica-like cast at a 150° angle for 3 to 6 weeks. Figure 1I shows the surgical procedure with the rabbit in a prone position with pre-operative preparation and draping of the hind limb. (I) The hind limbs were immobilized in a hip spica cast at a 150° angle for 3–6 weeks. [Diagram 2] Figure 2A: Evaluation of tendon repair. (A) Breaking load at 3 and 6 weeks for each group. Figure 2B: Evaluation of tendon repair. (B) Ultimate tensile strength at 3 and 6 weeks for each group. Figure 2C: Evaluation of tendon repair. (C) Cross-sectional area was reduced by 6 weeks in the PEP treatment group (p=0.04). Figure 2D: Evaluation of tendon repair. (D) Young's modulus was greater in the PEP treatment group (p=0.01) and increased over time (p≦0.03). [Diagram 3]Figure 3. Evaluation of tendon repair. (Left) Young's modulus versus cross-sectional area (labeled "CSA") for each group. (Right) Ultimate tensile strength versus cross-sectional area (labeled "CSA") for each group. There was a significant group interaction for Young's modulus and cross-sectional area (p=0.03), resulting in greater stiffness per cross-sectional area in the PEP-treated group compared to the control group. There was no significant group interaction for ultimate tensile strength and cross-sectional area (p=0.84). [Figure 4-1] FIG. 4A shows trichosome staining of specimens from each group at each endpoint and of normal, contralateral, untreated tendons. [Figure 4-2] Figure 4B shows hematoxylin and eosin (H&E) staining of specimens from each group at each endpoint and from normal contralateral untreated tendons. The images show that in the PEP-treated group, collagen was more organized and denser, with fewer peripheral adhesions, more similar to normal tendons. [Diagram 5] Figure 5A: Evaluation of tendon repair. (A) Macroscopic adhesion grading at 3 and 6 weeks for each group. Figure 5B: Evaluation of tendon repair. (B) Microscopic adhesion grading at 3 and 6 weeks for each group. Group 3 showed significantly less adhesions both macroscopically (p=0.0006) and microscopically (p=0.0062). [Figure 6] Figure 6 shows tendon adhesions after incision at 6 weeks. Macroscopic adhesions were greater in the control group (left) and collagen alone group (center) compared to the PEP + collagen group (right) (p=0.0006). [Figure 7] Figure 7A: Immunohistochemical assessment of tendon repair. (A) Immunohistochemical staining for type I collagen in specimens from each group and normal contralateral untreated tendons. Figure 7B: Immunohistochemical assessment of tendon repair. (B) Immunohistochemical staining for type III collagen in specimens from each group and normal contralateral untreated tendons. Images show increased staining intensity for type I collagen and decreased staining intensity for type III collagen in PEP-treated tendons, similar to staining observed in untreated normal tendons. [Figure 8]Figure 8. Immunohistochemical staining for P-selectin and Ki-67 of PEP-treated tendons. The images show immunoreactivity for Ki-67, but not for P-selectin, indicating that all PEP exosomes were resorbed by adjacent cells. [Figure 9] Figure 9 shows an image of the fixture for MTS testing. The calcaneal end of the tendon was mounted on a grooved plate at the proximal end and the muscle-tendon junction was clamped distally. The distal clamp was frozen with dry ice to increase clamp-tissue friction. The FDS tendon was severed prior to testing because it acts as an internal splint in vivo but would interfere with mechanical property testing in vitro. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present disclosure describes compositions and methods for improving the repair of damaged tendon tissue. Typically, the compositions include a purified exosome product (PEP) that is applied to damaged tendon tissue. Although described herein in the context of an exemplary tendon repair model involving the Achilles tendon, the methods described herein may be implemented to repair and / or treat any damaged tendon in any body location.

[0015] Tendon injuries (e.g., Achilles tendon injuries) can be acute (traumatic) or chronic (degenerative). Tendon healing is typically a slow process because tendon tissue tends to have a low metabolic rate, limited cellularity, and / or poor vascularity compared to tissues such as muscle and bone. Additionally, scars formed after tendon injury and repair are often mechanically inferior to native tendons, which can lead to re-rupture, persistent pain, and / or reduced functional ability, which can delay a patient's return to work or recreational activities.

[0016] Current tendon injury practice includes either non-operative or operative treatment. For example, non-operative treatment of Achilles tendon tears includes functional bracing and casting in a static equinus position with early range of motion and weight-bearing protocols. Operative treatments vary but generally include suture repair followed by 4-6 weeks of immobilization in light plantar flexion followed by various early range of motion and weight-bearing protocols. While non-operative treatments can provide comparable functional outcomes compared to operative treatments, non-operative treatments have been repeatedly shown to be associated with higher re-rupture rates. In both operative and non-operative treatment options, the tendon heals via extrinsic processes that promote adhesion formation, as described in more detail below.

[0017] Tendon healing can be intrinsic, extrinsic, or a combination of the two. Intrinsic healing (healing from within the tendon) results in better outcomes in terms of mechanical strength and has a greater histological similarity to native tissue. Extrinsic healing, by definition, requires migration of cells from the surrounding tendon sheath or soft tissue to the injury site. Tissues undergoing predominantly extrinsic healing have shown higher re-rupture rates due to greater amounts of immature fibers and type III collagen. An increase in the ratio of type III to type II collagen in the newly formed tissue typically increases the risk of re-rupture by decreasing the mechanical strength of the tendon. Current treatments for tendon injuries focus on accelerating extrinsic healing and optimizing early exercise protocols. Although these treatments have been shown to be beneficial, they do not address the underlying problem of insufficient inherent intrinsic healing.

[0018] PEP is a purified exosome product prepared using a cryodesiccation process that results in a product with a structure different from exosomes prepared using conventional methods. For example, PEP typically has a spherical or ellipsoidal structure rather than a crystalline structure. Spherical or ellipsoidal exosome structures usually have a diameter of 300 nanometers (nm) or less. Typically, PEP preparations contain spherical or ellipsoidal exosome structures with a relatively narrow size distribution. In some preparations, PEP contains spherical or ellipsoidal exosome structures with a mean diameter of 110 nm ± 90 nm, with the majority of the exosome structures having a mean diameter of 110 nm ± 50 nm, such as 110 nm ± 30 nm.

[0019] Unmodified PEP preparations (i.e., PEP preparations whose characteristics have not been altered by selection or isolation of the exosome population within the preparation) express CD63 + Exosomes and CD63 - Naturally contains a mixture of exosomes. CD63 - Exosomes can inhibit unlimited cell proliferation, so CD63 + Exosomes and CD63 - Unmodified PEP preparations that naturally contain exosomes can stimulate cell proliferation for wound repair and / or tissue regeneration and limit uncontrolled cell proliferation.

[0020] In addition, CD63 + By sorting exosomes, we were able to identify CD63 from naturally isolated PEP preparations. + After exosome extraction, the desired amount of CD63 + By returning exosomes, CD63 in the PEP product - CD63 on exosomes + The proportion of exosomes can be adjusted. In one or more embodiments, the PEP preparation contains CD63 - May contain only exosomes.

[0021] In one or more embodiments, the PEP preparation comprises CD63+ Exosomes and CD63 - Exosomes may contain both CD63 and CD63. - CD63 on exosomes + The proportion of exosomes may vary, at least in part, depending on the amount of cell expansion desired in a particular application. + / CD63 - Exosome rate is CD63 + Exosomes induce desired cell proliferation and CD63 expression via cell contact inhibition - In certain scenarios, such as tissues with non-adherent cells (e.g., blood-derived components), this ratio may be adjusted to provide the right balance of cell proliferation and cell inhibition for the tissue being treated. For example, in tissues with non-adherent cells, CD63 may be used to avoid uncontrolled cell proliferation due to the lack of cell-cell contact triggers. + Conversely, if it is desired to expand a clonal cell population, such as for autologous cell-based therapy or immunotherapy, it may be desirable to use CD63 clonal antibodies so that a large cell population can be obtained from a very scarce source. + The ratio of exosomes should be increased.

[0022] Thus, in one or more embodiments, CD63 in the PEP preparation - CD63 on exosomes + The ratio of exosomes may be at least 1:1, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 11:1, at least 12:1, at least 13:1, at least 14:1, at least 15:1, or at least 16:1. - CD63 on exosomes +The ratio of exosomes may be at most 15:1, at most 16:1, at most 17:1, at most 18:1, at most 19:1, at most 20:1, at most 25:1, or at most 30:1. For example, CD63 - CD63 on exosomes + The ratio of exosomes may be 1:1 to 30:1, 2:1 to 20:1, 4:1 to 15:1, or 8:1 to 10:1. In one or more particular embodiments, the PEP product is + Exosomes:CD63 - In one or more specific embodiments, the exosomes are formulated to contain a 9:1 ratio of exosomes. - CD63 on exosomes + In some cases, PEP with unaltered exosome ratios may be used.

[0023] The production of purified exosome product (PEP) involves separating plasma from blood by filtration and centrifugation and isolating exosome fluid from the separated plasma. PEP has been well characterized, and methods for preparing PEP are described in International Patent Application No. PCT / US2018 / 065627 (published as WO 2019 / 118817), U.S. Patent Publication No. 2021 / 0169812A1, and U.S. Patent No. 10,596,123, each of which is incorporated herein by reference in its entirety.

[0024] In vivo experiments The compositions and methods described herein may be used to measure efficacy in tendon repair using any suitable animal model. As discussed herein, the compositions and methods have been shown to be effective in treating a rabbit Achilles tenotomy model. However, any suitable animal model may be used, such as mice, rats, horses, pigs, or primates. Furthermore, the tendon repair model is not limited to Achilles tenotomy. Any suitable tendon rupture model may be used.

[0025] surgical technique The rabbits were divided into three groups. In group 1, an Achilles tenotomy was performed followed by a standard suture repair. In group 2, an Achilles tenotomy was performed followed by a standard suture repair and a type I collagen scaffold was applied to the repair site. In group 3, an Achilles tenotomy was performed followed by a standard suture repair and a type I collagen scaffold supplemented with 20% PEP was applied to the repair site.

[0026] Forty-four (98%) rabbits survived to their respective endpoints. One rabbit was euthanized on postoperative day 8 due to pain but was found to have a contralateral patellar luxation at necropsy. Eighteen (40%) rabbits required cast revision (12 for misalignment and 6 for toe swelling). Four rabbits from group 2 were found to have postoperative hematuria on postoperative day 1 but recovered spontaneously. These four rabbits were part of a group of nine rabbits in group 2 operated on the same day. Mean weight loss was 0.21 ± 0.14 grams and was not significantly different between treatment groups (p = 0.49).

[0027] Mechanical Property Testing Repair of a ruptured tendon may be measured by changes to mechanical properties of the tendon, such as failure load, tensile strength, stiffness, and Young's modulus. In one or more embodiments, mechanical property testing may be used to compare the progress of tendon repair in animals treated with different compositions, for example, comparing PEP-treated animals with non-PEP-treated animals. In one or more embodiments, compositions and methods described herein that include PEP may improve the mechanical properties of a ruptured tendon more quickly and / or more completely compared to compositions and methods that do not include PEP.

[0028] The breaking load and ultimate tensile strength were found to be similar across all groups (p≧0.15), but the tensile strength at 6 weeks was significantly higher than at 3 weeks in the collagen and collagen+PEP groups (p<0.05) (Table 2, Figure 2A, Figure 2B). The cross-sectional area measured before MTS testing was found to be smaller by 6 weeks in Group 3 specimens compared to Groups 1 or 2 (p=0.04) (Table 1, Figure 2C). Young's modulus increased with time in all groups (p≦0.03) (Table 1, Figure 2D). There was a significant group interaction for Young's modulus and cross-sectional area (p=0.03), resulting in higher stiffness per cross-sectional area in the PEP-treated group compared to the control group. There was no significant group interaction for ultimate tensile strength and cross-sectional area (p=0.84). The most common failure mode was at the repair site (65%, n=17) (Table 2). Other failure modes included calcaneal avulsion (n=6) and slippage at the distal toothed clamp (n=3).

[0029] Histological analysis The repair of ruptured tendons may be measured by histological analysis of the tendons. Histological characteristics that may be measured include collagen fiber density, collagen fiber organization, and microscopic and macroscopic adhesion grade assessment. In one or more embodiments, tissue analysis may be used to compare the progress of tendon repair in animals treated with different compositions, for example, to compare PEP-treated animals with non-PEP-treated animals. In one or more embodiments, the compositions and methods described herein that include PEP may improve histological measures of ruptured tendons compared to compositions and methods that do not include PEP.

[0030] Six specimens from each group were subjected to histological analysis by both hematoxylin-eosin and Masson's trichrome staining. PEP-treated tendons contained dense collagen fibers with a parallel organization (Figure 4), which more closely resembled normal tendons compared to the disorganized structure commonly seen in groups 1 and 2. Over time, the PEP-treated group showed mature (flattened) nuclei (Figure 4), approaching the acellular nature of normal tendons.

[0031] The tendons treated with PEP were found to have lower adhesion grades (p≦0.006) both macroscopically and microscopically compared to groups 1 and 2 (Table 3, Figure 5, Figure 6). Microscopic adhesion grade assessment was performed by three physicians and showed a mean interrater coefficient of variation of -0.16.

[0032] immunohistochemistry Repair of a ruptured tendon may be measured by immunohistochemical analysis of the tendon. Immunohistochemistry may be used to detect the expression of specific proteins or genes associated with tendon repair. Proteins associated with tendon repair include, but are not limited to, collagen type I, collagen type III, or Ki-67. In one or more embodiments, immunohistochemical analysis may be used to compare the progression of tendon repair in animals treated with different compositions, for example, comparing PEP-treated animals with non-PEP-treated animals. In one or more embodiments, compositions and methods described herein that include PEP may improve immunohistochemical assessment of ruptured tendons during healing compared to compositions and methods that do not include PEP.

[0033] In the case of immunohistochemical analysis combining multiple antibodies, six specimens from each group were analyzed. Analysis under a fluorescent microscope showed that the PEP-treated tendons stained more similarly to normal tendons in terms of the ratio of collagen types I and III compared to groups 1 and 2 (Figure 7). Although there was significant cell proliferation in all groups as indicated by the Ki-67 marker, the antibody marker for PEP (P-selectin) was not visualized at any time point in group 3, indicating that exosomes had been resorbed by the 3-week time point (Figure 8).

[0034] That is, the present disclosure describes the treatment of injured tendons with a support matrix (e.g., a collagen scaffold) loaded with PEP. When compared to tendons treated with collagen or treated with suture repair alone, the treatment that included PEP resulted in a greater degree of endogenous healing compared to the other groups. This finding was supported by both mechanical and histological findings.

[0035] Native tendons have high Young's modulus and ultimate tensile strength. Specifically, human Achilles tendons can have an ultimate tensile strength of 100-110 MPa. Scar tissue formed by extrinsic healing has been shown to exhibit inferior material properties, such as significant reductions in load to failure, ultimate tensile strength, and Young's modulus, in rabbit Achilles tendons treated with primary suture repair, both 3 and 6 weeks after surgery, compared to normal tendons. In this study, the load to failure and ultimate tensile strength were similar across all groups, but the cross-sectional diameter of the PEP-treated tendons was smaller, suggesting increased stiffness in the PEP-treated tendons. In addition, a significant group interaction was found for Young's modulus and cross-sectional area (p=0.03), resulting in higher stiffness per cross-sectional area in the PEP-treated group, and fewer adhesions in the PEP-treated group. All these findings suggest that PEP-treated tendon repairs tend to favor intrinsic healing versus extrinsic healing.

[0036] Histologically, native tendons consist of 80% collagen type I, up to 5% collagen type III, 2% elastin, minimal cellularity, and minimal vascularity. A higher proportion of collagen type III, a greater proportion of fibroblasts, and disorganized collagen structure are observed throughout each phase of extrinsic healing compared to native tendons. While collagen type III is present in higher proportions in scar tissue, tendons undergoing intrinsic healing have a higher proportion of collagen type I. Tendons undergoing intrinsic healing have upregulated collagen type I and downregulated collagen type III. In this study, the structure and staining of PEP-treated tendons were more similar to native tendons when compared to collagen-only and suture-only controls, further supporting the hypothesis that PEP may promote intrinsic healing.

[0037] Extrinsic tendon healing is prone to adhesion formation. Adhesion supports tendon repair but can lead to complications including limited motion, reduced tendon gliding, and / or pain. Prevention of adhesions after tendon repair has been an active area of ​​research for decades. Several treatments have been used to help prevent and / or treat adhesions, including nonsteroidal anti-inflammatory drugs (NSAIDs), 5-fluorouracil (5-FU), and barrier sheaths, but none have been able to completely prevent adhesion formation. In this study, tendons treated with PEP were shown to have fewer peripheral adhesions under macroscopic and microscopic observations. This finding supports the hypothesis that PEP promotes intrinsic healing.

[0038] This disclosure provides evidence that PEP promotes intrinsic healing of tendons by reducing adhesion development, increasing the ratio of type I to type III collagen, and showing a more organized collagen architecture while maintaining comparable breaking load and ultimate tensile strength. PEP is an acellular, off-the-shelf product that can promote tendon regeneration, providing physicians and patients with a viable solution to reduce pain, improve functional ability, and thereby promote return to work and / or recreational activities. Given the lack of solutions for patients with tendon-related injuries, the results herein support the translation of this technology into the clinic in patients with chronic disabling tendon diseases.

[0039] Thus, the present disclosure describes compositions and methods for improving tendon tissue repair. Typically, the compositions include PEP and a pharma- ceutically acceptable carrier. In a surgical setting, the PEP may be combined with a carrier suitable for application to tendon tissue, such as, for example, a surgical adhesive, a tissue adhesive, and / or a support matrix (e.g., a collagen scaffold). As used herein, "collagen scaffold" refers to a three-dimensional network that includes collagen, such as a hydrogel.

[0040] Combining PEP with collagen can increase the rate at which reconstituted PEP forms a gel at 37°C. Indeed, the rate at which reconstituted PEP gels in the presence of collagen is at least partially influenced by collagen concentration. Increased gelation rates can be achieved by using higher concentrations of collagen, with a maximum concentration of 10mg / ml. In some embodiments, PEP is used in combination with collagen at a collagen concentration of 5mg / mL. Combination with other gelling agents including thrombin glue (e.g., TISSEEL, Baxter Healthcare, Deerfield, IL), hyaluronic acid, polyvinyl alcohol (PVA), polylactic-co-glycolic acid (PLGA), etc. When PEP is formulated as a gel + collagen, PEP exosomes can attach to collagen fibrils, resulting in a "beads and beads" appearance.

[0041] In one or more embodiments, the support matrix comprises least one extracellular matrix component. Suitable extracellular matrix components include, but are not limited to, proteins such as collagen, elastin, fibronectin, and laminin, proteoglycans, and hyaluronic acid. In embodiments in which the composition comprises collagen, the collagen may be provided as procollagen, fibrillar collagen such as collagen type I, collagen type III, or combinations thereof. In embodiments in which the composition comprises collagen, the collagen may be provided as a collagen scaffold. In one or more other embodiments, the extracellular matrix component may be provided in any suitable form, such as purified recombinant protein. In one or more embodiments, the composition may comprise PEP and one or more support matrix components (e.g., collagen) in a ratio of 1:20 to 1:5 (5% v / v to 20% v / v). In one or more alternative embodiments, the ratio of PEP to support matrix components may be 1:100, 1:500, 1:1000, 1:10, 1:5, 1:2, or 1:1 by volume. Any medically suitable form of collagen may be included in the composition, such as collagen type I, collagen type II, or collagen type III. The collagen may be from a mammalian source, such as a bovine source or a human source. The structure of the collagen fibrils may be native, atelocollagenous, hydrolyzed, or a combination of several types. Typically, 10% or less collagen in the collagen scaffold exhibits faster than alpha characteristics using gel electrophoresis.

[0042] That is, the method includes administering an effective amount of the composition to tendon tissue in need of repair. In this embodiment, an "effective amount" is an amount effective to reduce adhesion occurrence, increase the ratio of collagen type I to collagen type III, and / or provide a more organized collagen architecture compared to untreated tendon tissue or tendon tissue treated with support matrix alone (without PEP). The method may improve at least one histological measure of the ruptured tendon. Exemplary histological measurements include, but are not limited to, increased fiber continuity, increased parallel fiber orientation, increased collagen fiber density, decreased vascularity, or decreased cellularity compared to tendons treated without PEP.

[0043] As used herein, a "subject" can be a human or any non-human animal. Exemplary non-human animal subjects include, but are not limited to, livestock animals or companion animals. Exemplary non-human animal subjects include hominoid animals (including, for example, chimpanzees, gorillas, or orangutans), bovine animals (including, for example, cows), caprine animals (including, for example, goats), ovine animals (including, for example, sheep), porcinoid animals (including, for example, pigs), equine animals (including, for example, horses), members of the cervid family (including, for example, deer, elk, moose, caribou, reindeer, etc.), bison (including, for example, reptiles ... Examples of the animal include, but are not limited to, members of the order Acanthurus (e.g., bison), felines (e.g., domestic cats, tigers, lions, etc.), canines (e.g., domestic dogs, wolves, etc.), birds (e.g., turkeys, chickens, ducks, geese, etc.), rodents (including, for example, mice, rats, etc.), members of the order Leporidae (e.g., rabbits or hares), members of the family Mustelidae (e.g., ferrets), or members of the order Chiroptera (e.g., bats).

[0044] PEP may be combined with a pharma- ceutically acceptable carrier to form a pharmaceutical composition. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, hydrogel, carrier solution, carrier suspension, carrier colloid, water, and the like. The use of such media and / or agents for pharma- ceutically active substances is well known in the art. Any conventional media or agent is contemplated for use in therapeutic compositions unless it is incompatible with the active ingredient. Additional active ingredients may also be incorporated into the composition. As used herein, "pharma-ceutically acceptable" refers to a substance that is not biologically or otherwise undesirable, i.e., the substance may be administered to an individual together with PEP without causing undesirable biological effects and without adversely interacting with any of the other components of the pharmaceutical composition containing the substance. As mentioned above, in surgical settings, exemplary suitable carriers include surgical adhesives, tissue adhesives, or support matrices (eg, collagen scaffolds).

[0045] Pharmaceutical compositions containing PEP may be formulated into various forms adapted to preferred administration routes. That is, pharmaceutical compositions can be administered via known routes, such as oral, parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., application to tendon tissue exposed during surgery, intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transdermal, rectal, etc.). Pharmaceutical compositions can be administered to mucosal surfaces, such as by administration (e.g., by spray or aerosol) to nasal or respiratory mucosa. Pharmaceutical compositions can also be administered via sustained or delayed release.

[0046] That is, the pharmaceutical composition may be provided in any suitable form, including but not limited to, solution, suspension, emulsion, spray, aerosol, or any mixture form.The pharmaceutical composition may be delivered in combination with any pharma- ceutically acceptable excipient, carrier, or vehicle.For example, the formulation may be delivered in a conventional topical dosage form, such as cream, ointment, aerosol formulation, non-aerosol spray, gel, lotion, etc.The formulation may further include one or more additives, such as adjuvants, skin penetration enhancers, colorants, fragrances, flavorings, moisturizers, thickeners, etc.

[0047] Suitable excipients include, for example, human or bovine collagen, hyaluronic acid-based compounds, human fibrinogen, or human thrombin.

[0048] The lyophilized composition containing PEP may be combined with additional excipients, which may be further lyophilized. The components of the lyophilized composition may be co-packaged or provided separately and mixed prior to use to prepare the PEP-loaded biocompatible scaffold. The lyophilized excipients may be, for example, lyophilized human or bovine collagen, hyaluronic acid-based compounds, human fibrinogen, human thrombin, or other lyophilized powders that form biocompatible gels upon contact with bodily fluids (e.g., blood or interstitial fluid).

[0049] In one or more embodiments, the compositions described herein are administered via injection into / onto the tendon during arthroscopic or open surgical repair. The compositions may be administered alone or in addition to traditional surgical repair methods such as suturing or stapling. The products may also be used to enhance the biocompatibility and therapeutic efficacy of tendon sutures, anchors, patches, or other devices used to repair tendon injuries.

[0050] The formulations may conveniently be provided in unit dosage form and may be prepared by methods well known in the art of pharmacy. Methods for preparing compositions containing pharmaceutically acceptable carriers include the step of bringing the PEP into association with the carrier, which constitutes one or more accessory ingredients. In general, the formulations can be prepared by uniformly and / or intimately bringing the PEP into association with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired formulation.

[0051] The dosage of PEP may vary depending on various factors, including but not limited to the content and / or source of PEP administered, the weight, physical condition, and / or age of the subject, and / or the route of administration. That is, the absolute weight of PEP contained in a given unit dosage form may vary widely and depends on factors such as the species, age, weight, and physical condition of the subject, and / or the method of administration. Therefore, it is not practical to generalize the amount that corresponds to the effective amount of PEP for every possible application. However, a person skilled in the art can easily determine the appropriate amount by fully considering such factors.

[0052] In one or more embodiments, a single dose of PEP may be measured in terms of PEP exosomes delivered per dose. That is, in one or more embodiments, the method may include, for example, administering a single dose of 1×10 6 PEP exosomes ~ 1 × 10 15 This may include administering to a subject sufficient PEP to provide a single dose of PEP-exosomes, although in one or more embodiments, the method may be practiced by administering a single dose of PEP outside this range.

[0053] In one or more embodiments, the method thus comprises the step of: 6 PEP exosomes, at least 1 x 10 7 PEP exosomes, at least 1 x 10 8 PEP exosomes, at least 1 x 10 9PEP exosomes, at least 1 x 10 10 PEP exosomes, at least 1 x 10 11 PEP exosomes, at least 2 × 10 11 PEP exosomes, at least 3 × 10 11 PEP exosomes, at least 4 × 10 11 PEP exosomes, at least 5 × 10 11 PEP exosomes, at least 6 × 10 11 PEP exosomes, at least 7 × 10 11 PEP exosomes, at least 8 × 10 11 PEP exosomes, at least 9 × 10 11 PEP exosomes, at least 1 x 10 12 PEP exosomes, 2 × 10 12 PEP exosomes, at least 3 × 10 12 PEP exosomes, at least 4 × 10 12 PEP exosomes or at least 5 × 10 12 PEP exosomes, at least 1 x 10 13 PEP exosomes, or at least 1 × 10 14 This may include administering sufficient PEP to provide a minimum single dose of PEP-exosomes.

[0054] In one or more embodiments, the method comprises: 15 PEP exosomes, 1×10 14 PEP exosomes, 1×10 13 PEP exosomes, 1×10 12 PEP exosomes, 1×10 11 PEP-exosomes or less than 1 × 10 10 This may include administering sufficient PEP to provide a maximum single dose of no more than 100 PEP exosomes.

[0055] In one or more embodiments, the method may include administering sufficient PEP to provide a dose characterized by a range defined by any minimum dose above and any maximum dose above the minimum dose, including the endpoints. For example, in one or more embodiments, the method may include administering 1×10 11 ~1×10 13 A single dose of PEP exosomes, e.g., 1 x 10 11 ~5×10 12 A single dose of 1×10 PEP exosomes 12 ~1×10 13 5 x 10 PEP exosomes per dose 12 ~1×10 13 In one or more particular embodiments, the method may include administering sufficient PEP to provide a single dose equal to any of the minimum single doses or any of the maximum single doses described above. That is, for example, the method may include administering sufficient PEP to provide a single dose equal to 1×10 10 PEP exosomes, 1 × 10 11 PEP exosomes, 5 × 10 11 PEP exosomes, 1 × 10 12 PEP exosomes, 5 × 10 12 PEP exosomes, 1 × 10 13 PEP exosomes, or 1 × 10 14 This may include administering a single dose of PEP exosomes.

[0056] Alternatively, a single dose of PEP may be measured in terms of the concentration of PEP upon reconstitution from a lyophilized state. That is, in one or more embodiments, the method may involve administering PEP to a subject, for example, to the subject in a single dose of between a 0.01% solution and a 100% solution, although in one or more embodiments, the method may be practiced by administering a single dose of PEP outside of this range. As used herein, a 100% PEP solution refers to one vial of PEP (2×10 11exosomes (i.e., 75 mg) solubilized in 1 ml of a liquid or gel carrier (e.g., water, phosphate buffered saline, serum-free medium, surgical glue, tissue adhesive, etc.). By way of comparison, a single dose of 0.01% PEP corresponds roughly to a standard dose of exosomes prepared using conventional methods for obtaining exosomes, such as exosome isolation from cells in vitro using standard cell-conditioned medium.

[0057] In one or more embodiments, the method may thus include administering sufficient PEP to provide a minimum bolus dose of at least 0.01%, at least 0.05%, at least 0.1%, at least 0.25%, at least 0.5%, at least 1.0%, at least 2.0%, at least 3.0%, at least 4.0%, at least 5.0%, at least 6.0%, at least 7.0%, at least 8.0%, at least 9.0%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, or at least 70%.

[0058] In one or more embodiments, the methods may include administering sufficient PEP to provide a maximum bolus of 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 9.0% or less, 8.0% or less, 7.0% or less, 6.0% or less, 5.0% or less, 4.0% or less, 3.0% or less, 2.0% or less, 1.0% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less.

[0059] In one or more embodiments, the method may include administering sufficient PEP to provide a dosage characterized by a range defined by any minimum dosage above and any maximum dosage above the minimum dosage, including the endpoints. For example, in one or more embodiments, the method may include administering sufficient PEP to provide 1%-50% of the dosage, such as 5%-20% of the dosage. In some embodiments, the method may include administering sufficient PEP to provide a dosage equal to any minimum dosage or any maximum dosage above. That is, for example, the method may involve administering 0.05%, 0.25%, 1.0%, 2.0%, 5.0%, 20%, 25%, 50%, 80%, or 100% of the dosage.

[0060] A dose may be administered all at once, continuously over a period of time, or in multiple doses. When multiple doses are used, each dose may be the same or different. For example, a given daily dose may be administered as a single dose, continuously over 24 hours, or as two doses that may be equal or unequal. When multiple doses are used to deliver a dose, the interval between doses may be the same or different. In one or more specific embodiments, PEP may be administered from a single dose, for example during a surgical procedure.

[0061] In one or more particular embodiments in which multiple doses of the PEP composition are administered to the subject, the PEP composition may be administered as needed to regenerate tendon tissue to the desired extent. Alternatively, the PEP composition may be administered 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 times. The interval between doses may be a minimum of at least 1 day, such as at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, or at least 21 days. The interval between doses may be a maximum of 6 months or less, such as 3 months or less, 2 months or less, 1 month or less, 21 days or less, or 14 days or less.

[0062] In one or more embodiments, the method may include multiple administrations of PEP to a subject at an interval (in the case of two administrations) or multiple intervals (in the case of three or more administrations), characterized by a range including the endpoints defined by any minimum intervals as above and any maximum intervals greater than the minimum intervals. For example, in one or more embodiments, the method may include multiple administrations of PEP at one or more intervals between 1 day and 6 months, such as between 3 days and 10 days. In one or more particular embodiments, the method may include multiple administrations of PEP at intervals equal to any minimum interval or any maximum interval as above. That is, for example, the method may involve multiple administrations of PEP at intervals of 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, 1 month, 2 months, 3 months, or 6 months.

[0063] In one or more embodiments, the methods may include administering a cocktail of PEPs prepared from different cell types, each with its own unique tendon-supporting profile (e.g., protein composition and / or gene expression). In this manner, the PEP composition may provide a broader range of tendon-supporting activity than if the PEP composition was prepared from a single cell type.

[0064] In the foregoing description and in the claims that follow, the term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements; the terms "comprises", "comprising", and variations thereof are to be construed as open ended, i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, "a", "an", "the", and "at least one" are used interchangeably and mean one or more than one; the recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0065] In the foregoing description, specific embodiments may be described in isolation for clarity. A particular embodiment may include any combination of compatible features described herein in connection with one or more embodiments, unless a feature of a particular embodiment is expressly stated to be incompatible with a feature of another embodiment.

[0066] In any method disclosed herein that includes separate steps, those methods may be carried out in any order practicable, and, where appropriate, any combination of two or more steps may be carried out simultaneously.

[0067] In this specification, when the term "one embodiment," "an embodiment," "a particular embodiment," "one or more embodiments," or "some embodiments" is used, it means that a particular feature, configuration, composition, or characteristic described in connection with an embodiment is included in at least one embodiment of the present disclosure. That is, the appearance of such a phrase in various places in this specification does not necessarily refer to the same embodiment of the present disclosure. Furthermore, particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Furthermore, particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. That is, a feature described in the context of one embodiment may be combined with a feature described in the context of a different embodiment, unless those features are necessarily mutually exclusive.

[0068] As used herein, the words "preferred" and "preferably" refer to embodiments of the invention that may offer certain advantages, under particular circumstances. Other embodiments, however, may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, or is intended to exclude other embodiments from the scope of the invention.

[0069] Preferred Embodiments In embodiment 1, Purified exosome product (PEP), and a pharma- ceutically acceptable carrier comprising a support matrix; The composition comprises:

[0070] Embodiment 2 is the composition of embodiment 1, wherein the PEP comprises spherical or ellipsoidal exosomes having a diameter of 300 nm or less.

[0071] Embodiment 3 is the composition of embodiment 1, wherein the PEP comprises spherical or ellipsoidal exosomes having an average diameter of 110 nm ± 90 nm.

[0072] Embodiment 4 is the composition of embodiment 3, wherein the PEP comprises spherical or ellipsoidal exosomes having an average diameter of 110 nm±50 nm.

[0073] Embodiment 5 is the composition of embodiment 4, wherein the PEP comprises spherical or ellipsoidal exosomes having an average diameter of 110 nm±30 nm.

[0074] Embodiment 6 is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: 1%–20% CD63 - Exosomes, and 80%-99% CD63 + Exosomes, The composition according to any one of embodiments 1 to 5, comprising:

[0075] In embodiment 7, the PEP is at least 50% CD63 - The composition according to any one of embodiments 1 to 5, which comprises exosomes.

[0076] In embodiment 8, the PEP is 1×10 11 PEP exosomes ~ 1 × 10 13 The composition according to any one of embodiments 1 to 7, comprising PEP exosomes.

[0077] In embodiment 9, the PEP is 1×10 12 PEP exosomes ~ 1 × 10 13 The composition according to any one of embodiments 1 to 8, comprising PEP exosomes.

[0078] Embodiment 10 is the composition of any one of embodiments 1-9, wherein the support matrix comprises a collagen scaffold.

[0079] Embodiment 11 is the composition of embodiment 10, wherein the collagen scaffold comprises type I fibrillar collagen.

[0080] Embodiment 12 is a method of treating damaged tendon tissue comprising applying a composition according to any one of embodiments 1-11 to the damaged tendon tissue.

[0081] Embodiment 13 is a method according to embodiment 12, wherein the composition is applied in an amount effective to reduce adhesion incidence compared to injured tendon tissue treated without the composition.

[0082] Embodiment 14 is the method of embodiment 12, wherein the composition is applied in an amount effective to increase the ratio of type I collagen to type III collagen compared to damaged tendon tissue treated without the composition.

[0083] Embodiment 15 is a method according to embodiment 12, wherein the composition is applied in an amount effective to result in a more organized collagen architecture compared to damaged tendon tissue treated without the composition.

[0084] Embodiment 16 is the method of any one of embodiments 12 to 15, wherein the damaged tendon tissue comprises a disrupted tendon.

[0085] Embodiment 17 is the method of embodiment 16, wherein the disruption of the tendon comprises a rupture of the tendon.

[0086] Embodiment 18 is the method of embodiment 17, wherein the tendon rupture comprises an Achilles tendon rupture. EXAMPLES

[0087] The present invention is illustrated by the following examples, it being understood that the specific examples, materials, contents, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as described herein.

[0088] Example 1 In this example, the effect of collagen or collagen + PEP on tendon repair was investigated using a rabbit Achilles tenotomy model.

[0089] Study design The study was conducted in young adult (10-12 weeks old) female New Zealand White rabbits with body weights ranging from 2.7 kg to 3.0 kg. The study design included three groups of 15 rabbits, totaling 45 rabbits. Endpoints were 3 and 6 weeks after surgery, based on histological differences between these time points shown in predicate studies. The transition from type III to type I collagen does not begin until the remodeling phase (6-10 weeks), supporting the time points chosen.

[0090] Group 1 was the control group and underwent standard suture repair after Achilles tenotomy. Group 2 underwent standard suture repair after Achilles tenotomy and in addition, type I collagen scaffold was applied to the repair site. Group 3 underwent standard suture repair after Achilles tenotomy and in addition, type I collagen scaffold supplemented with 20% PEP was applied to the repair site. Only one Achilles tendon was operated on in each rabbit.

[0091] Three weeks after surgery, six rabbits from each group were sedated and sacrificed by intravenous injection of Fatal Plus (Vortec Pharmaceuticals, Dearborn, MI). Three specimens from each group were used for histological examination and the remaining three specimens were used for mechanical property testing. The unoperated contralateral Achilles tendons were also collected for comparative analysis. Six weeks after surgery, the remaining nine rabbits from each group were sacrificed. Three specimens from each group were used for histological examination and the remaining six specimens were used for mechanical property testing. All rabbits that survived to the assigned time point (3 or 6 weeks) were included in the data analysis.

[0092] Scaffold preparation The scaffolds used in groups 2 and 3 were made from research grade type I fibrillar pH neutral collagen (50 mg / mL, Collagen Solutions, Eden Prairie, MN). Group 2 scaffolds consisted of collagen only, while group 3 scaffolds contained 1 × 10 12The exosomes were combined with PEP (Lyon, Rochester, MN) to achieve a PEP exosome concentration of 10000 exosomes / mL. PEP has been shown to contain fibroblast growth factor 2 (FGF-2), platelet-derived growth factor BB (PDGF-BB), insulin-like growth factor 1 (IGF-1), and transforming growth factor beta (TGF-β). The PEP used in these experiments was unmodified PEP, which is a marker for CD63 - CD63 on exosomes + This means that the ratio of exosomes was not altered.

[0093] The scaffold preparation method was designed to be performed via sterile technique during surgery to mimic the expected clinical scenario. In group 2, scaffolds were fabricated using type I fibrillar collagen (Collagen Solutions, Inc., Eden Prairie, MN) and saline in an 80:20 ratio. In group 3, type I fibrillar collagen (Collagen Solutions, Inc., Eden Prairie, MN) was mixed with PEP in an 80:20 ratio. This resulted in the scaffold being in a paste form that could be applied over the repaired tenotomy site.

[0094] Surgical procedure Approval for this study was obtained from the Institutional Animal Care and Use Committee (IACUC). Rabbits were divided into three groups. In group 1, Achilles tenotomy was performed followed by a standard suture repair. In group 2, Achilles tenotomy was performed followed by a standard suture repair and a type I collagen scaffold was applied to the repair site. In group 3, Achilles tenotomy was performed followed by a standard suture repair and a type I collagen scaffold supplemented with 20% PEP was applied to the repair site.

[0095] On the day of surgery, rabbits were given antibiotics and analgesics before surgery. Rabbits were anesthetized with isoflurane gas inhalation, and gas was continued throughout the procedure. Achilles tendon dissection was performed with a 2 cm longitudinal incision starting 0.5 cm proximal to the calcaneal tubercle (Figure 1A). Dissection was performed down to the flexor digitorum superficialis (FDS). The paratenon connective tissue around the FDS was dissected. The FDS was isolated and retracted laterally to expose the Achilles tendon (Figure 1B). The Achilles tendon bundle was isolated and tenotomized 1.5 cm proximal to the calcaneal tubercle (Figure 1C, Figure 1D). Care was taken not to cut the FDS, as it acts as an internal splint for the repair site. Both ends of the tendon were repaired in 150° plantar flexion using a modified Kessler main suture (Figure 1E). The purpose was to evaluate the effect of the scaffold, not the strength of the suture repair, but a minimal suture repair was desirable, as sutures would help prevent early gap formation. The suture repair was performed similarly across all groups with 5-0 polydioxanone sutures (PDS, Ethicon, Raritan, NJ) to allow for fair tissue specimen evaluation. In groups 2 and 3, 0.2 mL of the scaffold was placed topically at the tenotomy site, followed by final tightening of the suture repair (Figure 1F). The solution gelled rapidly after application. The paratendinous connective tissue did not repair. The skin was closed with 3-0 absorbable Vicryl sutures (Ethicon, Raritan, NJ) (Figure 1G). The hind limb was immobilized with a hip spica-like cast from the toes to the high groin, and the ankle was molded to 150 degrees of plantar flexion (Figure 1H). Next, a VetWrap was wrapped over the cast, from the toes up the leg and in a figure-of-eight pattern around the abdomen (Figure 1I).

[0096] Forty-four (98%) rabbits survived to their respective endpoints. One rabbit was euthanized on postoperative day 8 due to pain and was found to have a contralateral patellar luxation at necropsy. Eighteen (40%) rabbits required cast revision (12 for misalignment and 6 for toe swelling). Four rabbits from group 2 were found to have postoperative hematuria on postoperative day 1 but recovered spontaneously. These four rabbits were part of a group of nine rabbits in group 2 operated on the same day. The mean weight loss was 0.21 ± 0.14 grams with no significant difference between treatment groups (p = 0.49). Three weeks after surgery, six rabbits from each group were sacrificed. From each group, three rabbits were used for histological examination and three rabbits for mechanical property testing. After the rabbits were sacrificed, the Achilles tendons were detached from the hindlimbs 3 cm proximal to the musculotendinous junction and cut distal to the calcaneal tubercle to obtain a distal 1 cm × 1 cm bone block. FDS was removed from the specimens as it was believed to interfere with the mechanical property test results. The remaining 9 rabbits in each group were sacrificed 6 weeks after surgery. In each group, 3 rabbits were used for histological testing and the remaining 6 rabbits were used for mechanical property testing. All rabbits that survived to the assigned time point (3 or 6 weeks) were included in the data analysis.

[0097] Mechanical Property Testing Testing was performed on a servohydraulic testing machine (MTS Systems, Eden Prairie, MN). The MTS fixture configuration consisted of a toothed clamp on the distal side and a grooved plate for the proximal bone block (Figure 9). The distal clamp was augmented with dry ice to increase friction between the clamp and the specimen. Prior to each test, the cross-sectional area of ​​each specimen was measured, as well as the initial length, and strain and stiffness were determined. Ultimate tensile strength, failure mode, failure location, stress, strain, and Young's modulus were recorded for all post-operative specimens and several un-operated specimens.

[0098] The breaking load and ultimate tensile strength were found to be similar across all groups (p≧0.15), but the tensile strength at 6 weeks was significantly higher than at 3 weeks in the collagen and collagen+PEP groups (p<0.05) (Table 2, Figure 2A, Figure 2B). The cross-sectional area measured before MTS testing was found to be smaller by 6 weeks in Group 3 specimens compared to Groups 1 or 2 (p=0.04) (Table 1, Figure 2C). Young's modulus increased with time in all groups (p≦0.03) (Table 1, Figure 2D). There was a significant group interaction for Young's modulus and cross-sectional area (p=0.03), resulting in higher stiffness per cross-sectional area in the PEP-treated group compared to the control group. There was no significant group interaction for ultimate tensile strength and cross-sectional area (p=0.84). The most common failure mode was at the repair site (65%, n=17) (Table 2). Other failure modes included calcaneal avulsion (n=6) and slippage at the distal toothed clamp (n=3). [Table 1] [Table 2-1] [Table 2-2]

[0099] Histological analysis Tendons from sacrificed rabbit specimens were placed in 10% formalin solution and then embedded in paraffin. Tissue samples were cut longitudinally at 8–10 μm with a rotary microtome (Cryocut 1800; Leica Microsystems, Buffalo Grove, IL) and mounted on glass slides. After deparaffinization, specimens were stained with hematoxylin and eosin (H&E) to assess cellularity and Masson's trichrome to assess collagen content and organization. All slides were analyzed by light microscopy (Olympus DP25; Olympus Americas, Melville, NY) and digital images were obtained (cellSens version 1.9, Olympus Americas, Melville, NY). These stains allowed characterization of tendon structure, collagen density, and cellularity. Tendon adhesions were graded both macroscopically and microscopically using a validated adhesion grading scale.

[0100] Six specimens from each group were subjected to histological analysis by both hematoxylin-eosin and Masson's trichrome staining. PEP-treated tendons contained dense collagen fibers with a parallel organization (Figure 4), which more closely resembled normal tendons compared to the disorganized structure commonly seen in groups 1 and 2. Over time, the PEP-treated group showed mature (flattened) nuclei (Figure 4), approaching the acellular nature of normal tendons.

[0101] The tendons treated with PEP were found to have lower adhesion grades (p≦0.006) both macroscopically and microscopically compared to groups 1 and 2 (Table 3, Figure 5, Figure 6). Microscopic adhesion grade assessment was performed by three physicians and showed a mean interrater coefficient of variation of -0.16. [Table 3]

[0102] immunohistochemistry Specimens were prepared, paraffin-embedded, sectioned, and deparaffinized. Antigen retrieval was not performed because it was known to significantly alter tissue integrity. Multiple combinations of primary and secondary antibodies were applied. Primary antibodies used included anti-type I collagen (mouse monoclonal, 1:400; AB90395, Abcam, Cambridge, MA), anti-type III collagen (goat polyclonal, 1:400; Southern Biotech, Birmingham, AL), Ki-67 (mouse monoclonal, 1:100; Novus Biologicals, Centennial, CO), and P-selectin (sheep polyclonal, 1:100; R&D Systems, Minneapolis, MN). Secondary antibodies used were Cy3 (goat anti-mouse polyclonal, 1:100; A10521, Invitrogen, Carlsbad, CA), Alexa Fluor 680 (donkey anti-sheep polyclonal, 1:100; A21102, Invitrogen), Alexa Fluor 555 (donkey anti-goat polyclonal, 1:400; ab150130, Invitrogen), and Alexa Fluor 647 (goat anti-mouse polyclonal, 1:400; ab150115, Invitrogen) (Table 1). Slides were mounted with DAPI-added adhesive (ProLong Gold, Invitrogen). Slides were analyzed under a contrast fluorescence microscope (Axio Observer Z1, Carl Zeiss Microscopy, Thornwood, NY) at 25x magnification. These slides were characterized for collagen type I and III staining intensity, cell proliferation, and the presence or absence of PEP.

[0103] In the case of immunohistochemical analysis combining multiple antibodies, six specimens from each group were analyzed. Analysis under a fluorescent microscope showed that the PEP-treated tendons stained more similarly to normal tendons in terms of the ratio of collagen types I and III compared to groups 1 and 2 (Figure 7). Although there was significant cell proliferation in all groups as indicated by the Ki-67 marker, the antibody marker for PEP (P-selectin) was not visualized at any time point in group 3, indicating that exosomes had been resorbed by the 3-week time point (Figure 8).

[0104] Sample size adjustment Sample size adjustments were made based on the breaking loads of biomechanical testing results and variability estimates from similar studies. If we expected similar variability in the present study, a sample size of n=3 per group at 3 weeks and n=6 per group at 6 weeks would provide 80% power to detect differences of at least 100N and at least 171N, respectively, between any two of the three study groups. At 6 weeks, more rabbits were selected for biomechanical testing than for histological analysis because increased variability was expected and increasing sample size would decrease variability.

[0105] statistical analysis Statistical analyses were primarily focused on comparing the three study groups at each of the two time points separately. Data consisting of continuous variables were analyzed using one-way analysis of variance (ANOVA). If the overall F-test was significant, further analyses were performed using appropriate multiple comparison procedures to maintain the probability of an overall type I error. Categorical data were analyzed using chi-square tests. Interaction analyses between groups were performed when examining Young's modulus and cross-sectional area, and ultimate tensile strength and cross-sectional area. All statistical tests were two-sided, and a p-value of less than 0.05 was considered statistically significant.

[0106] The complete disclosures of all patents, patent applications, and published applications cited herein, as well as electronically available materials (including, for example, nucleotide sequence submissions in GenBank and RefSeq, and amino acid sequence submissions in, for example, SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) are incorporated by reference in their entirety. In the event of a discrepancy between the disclosure of this application and the disclosure of a document incorporated herein by reference, the disclosure of this application shall control. The foregoing detailed description and examples are given for clarity of understanding only. No unnecessary limitations should be understood therefrom. The invention is not limited to the exact details shown and described, since variations obvious to one skilled in the art are included within the invention defined by the claims.

[0107] Unless otherwise indicated, all numerical values ​​expressing amounts of ingredients, molecular weights, and the like used in the specification and claims are to be understood as being modified in all instances by the word "approximately" or "about." Accordingly, unless specifically indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations and may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0108] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain ranges necessarily resulting from the standard deviation found in their respective testing measurements.

[0109] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless specifically stated.

Claims

1. Purified exosome product (PEP), and a pharmaceutically acceptable carrier comprising a support matrix; A composition comprising:

2. 2. The composition of claim 1, wherein the PEP comprises spherical or ellipsoidal exosomes having a diameter of 300 nm or less.

3. 2. The composition of claim 1, wherein the PEP comprises spherical or ellipsoidal exosomes having an average diameter of 110 nm±90 nm.

4. 4. The composition of claim 3, wherein the PEP comprises spherical or ellipsoidal exosomes having an average diameter of 110 nm±50 nm.

5. 5. The composition of claim 4, wherein the PEP comprises spherical or ellipsoidal exosomes having an average diameter of 110 nm±30 nm.

6. The PEP is 1% to 20% CD63 － Exosomes, and 80% to 99% CD63 ＋ Exosomes, The composition of claim 1 comprising:

7. The PEP is at least 50% CD63 － The composition of claim 1 , comprising exosomes.

8. The PEP is 1×10 １１ PEP exosomes ~ 1 x 10 １３ The composition of claim 1, comprising PEP exosomes.

9. The PEP is 1×10 １２ PEP exosomes ~ 1 x 10 １３ The composition of claim 8, comprising PEP exosomes.

10. The composition of claim 1 , wherein the support matrix comprises a collagen scaffold.

11. The composition of claim 10 , wherein the collagen scaffold comprises type I fibrillar collagen.

12. A composition according to any one of claims 1 to 11 for treating damaged tendon tissue, which is applied to the damaged tendon tissue.

13. The composition of claim 12, wherein the composition is applied in an amount effective to reduce adhesion incidence compared to injured tendon tissue treated without the composition.

14. 13. The composition of claim 12, wherein the composition is applied in an amount effective to increase the ratio of type I collagen to type III collagen compared to damaged tendon tissue treated without the composition.

15. The composition of claim 12, wherein the composition is applied in an amount effective to result in a more organized collagen architecture compared to damaged tendon tissue treated without the composition.

16. The composition of claim 12 , wherein the damaged tendon tissue comprises a disrupted tendon.

17. 17. The composition of claim 16, wherein the disruption of the tendon comprises a rupture of the tendon.

18. 18. The composition of claim 17, wherein the tendon rupture comprises an Achilles tendon rupture.

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