Methods and compositions for repairing tendon-bone interfaces

Abstract

A method for repairing a damaged bone-tendon interface in a subject generally comprises contacting the damaged bone-tendon interface with an effective amount of a composition comprising a purified exosome product (PEP) and a pharma- ceutically acceptable carrier. In one or more embodiments, the damaged bone-tendon interface comprises a complete separation of the tendon from the bone, and the method further comprises surgically reattaching the tendon to the bone. In one or more embodiments, the damaged tendon-bone interface comprises a partial separation of the tendon from the bone, and the method comprises implanting the PEP composition at a site effective to contact the damaged tendon-bone interface.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 279,841, filed November 16, 2021, which is incorporated by reference in its entirety. Summary of the Invention

[0002] In one embodiment, the present disclosure describes a method for repairing a damaged bone-tendon interface in a subject. Typically, the method comprises contacting the damaged bone-tendon interface with an effective amount of a composition comprising a purified exosome product (PEP) and a pharma- ceutically acceptable carrier.

[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 include spherical or ellipsoidal exosomes having a diameter of 56 nm to 151 nm.

[0005] In one or more embodiments, the PEPs comprise spherical or ellipsoidal exosomes with a mean diameter of 97 nm. In one or more of these embodiments, the PEPs comprise spherical or ellipsoidal exosomes with a mean diameter of 97 nm ± 54 nm.

[0006] In one or more embodiments, the PEP comprises 1% to 20% CD63 - Exosomes and 80%-99% CD63 + Contains exosomes.

[0007] In one or more of the above-mentioned PEPs, at least 50% of the CD63 - Contains exosomes.

[0008] In one or more embodiments, the PEP is 1×1011 PEP exosomes ~ 1 × 10 13 Contains PEP exosomes.

[0009] In one or more embodiments, the PEP is 1×10 12 PEP exosomes ~ 1 × 10 13 Contains PEP exosomes.

[0010] In one or more embodiments, the composition further comprises a support matrix. In one or more of these embodiments, the support matrix comprises a collagen scaffold. The support matrix may further comprise a tissue sealant or a fibrin sealant.

[0011] In one or more embodiments, the effective amount is an amount effective to increase the osteoblast-tendon cell interface compared to the osteoblast-tendon cell interface at a bone-tendon interface treated without PEP.

[0012] In one or more embodiments, the effective amount is an amount effective to improve at least one histological measure of the tendon-bone interface compared to a bone-tendon interface treated without PEP.

[0013] In one or more embodiments, the histological measures include increased fiber continuity, increased parallel fiber orientation, increased collagen fiber density, decreased vascularity, or decreased cellularity when compared to bone-tendon interfaces treated without PEP.

[0014] In one or more embodiments, the effective amount is an amount effective to increase the expression of at least one gene that promotes repair of a damaged tendon-bone interface. In one or more of these embodiments, the gene encodes type I fibrillar collagen (Col1), type III fibrillar collagen (Col3), bHLH transcription factor scleraxis (SCX), tenomodulin (TNMD), decorin (DCN), or insulin-like growth factor I (IGF-I) in the tissue of the tendon-bone interface. In one or more embodiments, the effective amount is an amount effective to increase at least one biomechanical measure of the tendon-bone interface compared to a bone-tendon interface treated without PEP. Biomechanical measures may include, for example, maximum load or stiffness.

[0015] In one or more embodiments, the damaged bone-tendon interface comprises a complete separation of the tendon from the bone, and the method further comprises surgically reattaching the tendon to the bone.

[0016] In one or more embodiments, the damaged tendon-bone interface comprises a partial separation of tendon from bone, and the method comprises implanting the PEP composition at a site effective to contact the damaged tendon-bone interface.

[0017] 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]

[0018] [Figure 1-1]Figure 1. Co-culture model and cell names. (A) Schematic diagram of the co-culture model, including osteoblasts, tenocytes, and PEP gel cubes. (B) When the cells reached the boundary, the boundary was excised. (C) Schematic diagram of the osteoblast region, tenocyte region, and interface region in the co-culture model. (D) Photograph of the co-culture model before boundary removal. (E) Photograph of the co-culture model after boundary removal. [Figure 1-2] Figure 1. Co-culture model and cell names. (F) Alkaline phosphatase staining on days 7 and 14 after application of normal medium, medium containing PEP, or osteogenic induction conditions. (G) Relative mRNA expression levels of Col1, Col3, and SCX in primary tenocytes (with osteoblasts as control). Labels: ALP, alkaline phosphatase; PEP, purified exosome product; *, P<.1; **, P<.01; ***, P<.001. [Figure 2-1] Figure 2. Morphological characteristics of PEP. (A) PEP was prepared and stored in the form of a stabilized lyophilized powder in vials to allow room temperature storage. (B) Preparation of fibrin sealant (TISSEEL, Baxter International, Deerfield, IL) with or without PEP. A 100% (vol / vol) PEP solution was prepared by mixing one vial of sealed PEP powder with 1 mL of phosphate-buffered saline (PBS). The PEP solution (400 μL) was added to 600 μL of CaCl2 solution to adjust the solution to a 40% (vol / vol) concentration. The preparation was then completed using the manufacturer's instructions for TISSEEL preparation. The final concentration of PEP in TISSEEL was 20% (vol / vol). [Figure 2-2] Figure 2. Morphological characteristics of PEP. (C) PEP has a spherical vesicle structure with an intact lipid bilayer. (D) Particle size distribution analysis (Nanosite, Salisbury, UK) shows that the mean vesicle diameter of PEP ranges from 56.4 nm to 151 nm, with a mean diameter of 96.9 nm ± 2.8 nm, which is within the standard size range of exosomes. A 100% PEP solution was calculated to be 1.9 × 1011 particles / mL. [Diagram 3]Figure 3: Flowchart showing the experimental design of the in vivo model. The same rats were used for each evaluation marked with "*". Labels: PEP, purified exosome products; H&E, hematoxylin-eosin; RC, rotator cuff. [Figure 4-1] FIG. 4. Surgical procedure and biomechanical testing of tendon-bone interface repair in a rotator cuff model. (A) Localized PEP placement at the attachment of the supraspinatus tendon. (B) Modified Masson-Allen suture. (C) Macroscopic observation of the PEP gel cube before implantation in vivo. (D) Suturing after placement of the PEP gel cube. (E) Two double-ended 5-0 sutures (ETHIBOND, Ethicon, Raritan, NJ) were passed laterally through the tendon, creating small loops at both ends of the tendon. (F) The supraspinatus tendon was transected at its attachment on the greater tuberosity. (G) The sutures were passed through a 0.5 mm hole drilled laterally in the proximal humerus. (H) Macroscopic observation after careful suturing. [Figure 4-2] FIG. 4. Surgical procedure and biomechanical testing of tendon-bone interface repair in the rotator cuff model. (I) The biomechanical testing system shows the humerus embedded in a polymethylmethacrylate tube. The supraspinatus tendon is fixed to the attachment through a clamp at maximum breaking load. (J) The maximum breaking load at 6 weeks after surgery was significantly higher in the TISSEEL-PEP group compared to the repair alone group. Results in (J) and (K) are shown as mean (SD) (n=8 per group). Labels: PEP, purified exosome product; RC, rotator cuff; *, P<.1; ***, P<.001; ****, P<.0001. (K) Stiffness at 6 weeks after surgery was significantly higher in the TISSEEL-PEP group compared to the repair alone group. Results in (J) and (K) are shown as mean (SD) (n=8 per group). Labels: PEP, purified exosomal product; RC, rotator cuff; *, P<.1; ***, P<.001; ****, P<.0001. [Diagram 5]Figure 5. Cell proliferation and migration after exposure to PEP. Blue indicates osteoblast area at various time points (0, 2, 4, 6, and 8 days, n=6), and red indicates tenocyte area at various time points (0, 2, 4, 6, and 8 days, n=6). Left, PEP group. Right, control group (scale bar, 200 μm). Histograms show quantification of gap area and fusion area. [Figure 6-1] Figure 6 shows the results of quantitative RT-PCR validation in an in vitro study. (A) Real-time PCR results of Col1 mRNA expression 3 days after direct contact between osteoblasts and tenocytes in an in vitro study. (B) RT-PCR results of Col3 mRNA expression 3 days after direct contact between osteoblasts and tenocytes in an in vitro study. (C) RT-PCR results of DCN mRNA expression 3 days after direct contact between osteoblasts and tenocytes in an in vitro study. (D) RT-PCR results of TNC mRNA expression 3 days after direct contact between osteoblasts and tenocytes in an in vitro study. [Figure 6-2] Figure 6 shows the results of quantitative RT-PCR validation in vitro. (E) RT-PCR results of Spp1 mRNA expression 3 days after direct contact between osteoblasts and tenocytes in in vitro study. (F) RT-PCR results of EGR mRNA expression 3 days after direct contact between osteoblasts and tenocytes in in vitro study. (G) RT-PCR results of PPARG mRNA expression 3 days after direct contact between osteoblasts and tenocytes in in vitro study. RT-PCR, reverse transcription-polymerase chain reaction; *, P<.1; **, P<.01; ***, P<.001. [Figure 7-1]Figure 7 shows the results of histological analysis and quantitative RT-PCR validation in in vivo study. Histological images of rat rotator cuff tendons and their attachment to the humerus after 6 weeks. (A) Normal control group. (B) Repair alone group. (C) TISSEEL alone group. (D) TISSEEL-PEP group. Histological staining for each of (A)-(D): (1) H&E staining (10x magnification), (2) Masson-Trichosome staining (x10 magnification, black border, x20 magnification), (3) Picrosirius red staining (x10 magnification), and (4) Picrosirius red staining (under polarizing microscope; scale=100μM). [Figure 7-2] Figure 7: Histological analysis and quantitative RT-PCR validation results in in vivo study. (E) Histological findings were evaluated using a semi-quantitative scoring system. (F) Real-time PCR results of relative mRNA expression of Col1, Col3, SCX, Tnmd, TNC, DCN, Spp1, and IGF-I after 6 weeks in in vivo study. H&E, hematoxylin-eosin; PEP, purified exosome product; RC, rotator cuff; RT-PCR, reverse transcription-polymerase chain reaction; *, P<.1; **, P<.01; ***, P<.001; ****, P<.0001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present disclosure describes compositions and methods for promoting healing and / or repair of damage at tendon-bone interfaces. Although described herein in connection with an exemplary tendon-bone interface model involving repairing a rotator cuff tear, the methods described herein may be performed to repair and / or heal damaged tendon-bone interfaces with lesser degrees of damage. Furthermore, the methods described herein may be performed to repair and / or heal any damaged tendon-bone interface at any site in the body.

[0020] This disclosure describes the effects of purified exosome products (PEP) on osteoblasts and tenocytes in a novel co-culture model, using PEP to improve tendon-bone healing in a rat rotator cuff tear model, and the molecular mechanisms by which PEP induces tendon-bone healing. PEP upregulates tendon-bone interface healing by promoting tendon cell proliferation and migration. That is, treatment of the tendon-bone interface (e.g., by local implantation of PEP) increases the expression of genes and / or signaling pathways that promote tendon attachment healing.

[0021] PEP is a purified exosome product prepared using a cryodesiccation process that results in a product with a different structure than exosomes prepared using conventional methods. For example, PEP typically has a spherical or ellipsoidal structure rather than a crystalline structure, and an intact lipid bilayer that results from reaggregation of the lipids of the exosome lipid bilayer after exosomes are disrupted during conventional exosome preparation methods. As used herein, an "ellipsoidal" structure is shaped like a three-dimensional sphere with flattened poles. 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. An example of the size distribution of a PEP preparation is shown in Figure 2D. Here, the average particle size was 96.9 nm ± 52.2 nm. In some preparations, the PEP comprises spherical or ellipsoidal exosomal structures having a mean diameter of 110 nm±90 nm, with the majority of the exosomal structures having a mean diameter of 110 nm±50 nm, such as, for example, a mean diameter of 110 nm±30 nm.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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 + PEP with unaltered exosome ratio may be used.

[0026] 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.

[0027] Morphological characteristics of PEP In one or more embodiments, the preparation may be characterized by measuring the vesicle size of the PEP exosomes. The vesicle size may be measured, for example, by electron microscopy, such as transmission electron microscopy or scanning electron microscopy. Transmission electron microscopy images show that the PEP exosomes exhibit typical spherical vesicles with a complete lipid bilayer structure (Figure 2C). The PEP vesicle size ranged from 56.4 nm to 151 nm, with an average size of 96.9 nm ± 2.8 nm, indicating the standard size range for exosomes. A 100% PEP solution had a vesicle size of 1.9 x 10 per mL. 11 It was calculated to contain 10 PEP particles (Figure 2D).

[0028] In vitro experiments As described herein, primary osteoblasts and primary tenocytes can be used to generate an in vitro cell culture model of tendon-bone interface repair, which can be used to determine the effect of PEP or another pharmaceutical composition on tendon-bone interface repair.

[0029] Identification of primary rat osteoblasts and primary rat tenocytes Primary cells from any organism of interest, such as mouse, rat, horse, dog, pig, or primate, may be used. Calvarial bone cells isolated from neonatal rats were chosen as rat primary osteoblasts for in vitro studies. As the culture time was extended (days 7-14), the alkaline phosphatase (ALP) activity of osteoblasts gradually increased. In osteogenic induction medium, osteoblasts showed more bone formation, which was further enhanced with increasing culture time (Figure 1F). However, the PEP group did not show an increase in osteogenic potential. This result suggests that 20% TISSEEL-PEP did not enhance osteoblast activity when compared to the control under osteogenic conditions in an in vitro co-culture model.

[0030] PCR results showed that the relative mRNA expression of tendon-related genes was significantly upregulated compared to the control (osteoblast) group. Furthermore, SCX was directly linked to tendon development and differentiation, indicating that the identification of primary cell types corresponds closely to the tendon origin (Figure 1G).

[0031] Effect of PEP on the morphology and growth of the interfacial region The in vitro culture model illustrated in Figures 1A-1E allowed the evaluation of osteoblast and tenocyte migration and fusion with and without PEP treatment. The tissue growth patterns of osteoblast, tenocyte, and interface areas were manually pseudocolored at 0, 2, 4, 6, and 8 days (Figure 5). Area coverage was measured manually, and gap and fusion areas were calculated. The initial gap area in the intervention group (PEP group) was 5.17 ± 0.22 mm. 2 (n=6), whereas the initial gap area in the control group (without PEP) was 3.37±0.19mm 2 After exposure to PEP, cell proliferation was significantly increased in all areas. Cell migration was more pronounced at the interface in the PEP group compared with the control group. In the PEP group, cells were confluent on day 4, and the confluent area was 0.47 ± 0.14 mm on day 8. 2In the control group, however, the cells only started to become confluent on day 6, and the confluent area on day 8 was only 0.20 ± 0.17 mm 2 (n=6) (Figure 5).

[0032] Changes in mRNA levels In vitro studies showed that the mRNA levels of Col1, TNC, DCN, SCX, Spp1, and EGR were significantly increased in the PEP group on day 9 compared to the PEP group on day 3 in the interface region (P<.05). The mRNA levels of Col3, TNC, Spp1, EGR, and PPARG were significantly increased in the PEP group on day 6 compared to the PEP group on day 3 in the interface region (P<.05). In addition, in the PEP group, coculture with direct cell-cell contact increased the expression of Col3, TNC, Spp1, PPARG, and EGR compared to coculture without direct cell-cell contact (Figure 6A-G).

[0033] In vivo rat model Although described in connection with a preferred embodiment in which a damaged tendon-bone interface involves a rotator cuff tear, the methods described herein may be practiced to treat and / or repair a tendon-bone interface at any body site, whether the tendon-bone interface is at a natural site (e.g., an attachment or other natural tendon attachment) or an artificial tendon-bone interface (e.g., a surgically constructed tendon-bone interface). Additionally, any suitable animal model may be used to measure the efficacy of the compositions and methods described herein for tendon-bone interface repair.

[0034] Biomechanical testing Repair of the tendon-bone interface may be measured by changes to one or more mechanical properties of the tendon-bone connection, such as, but not limited to, maximum load, tensile load, and / or stiffness. In one or more embodiments, mechanical property testing may be used to compare the progress of tendon-bone interface 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 the healing tendon-bone interface compared to compositions and methods that do not include PEP.

[0035] The maximum load of the repaired rotator cuff 6 weeks after surgery showed no statistically significant difference between the TISSEEL-PEP group and the normal healthy control group. The maximum tensile load was 22.36 N ± 1.51 N (n = 8) in the normal control group and 21.83 N ± 1.78 N (n = 8) in the TISSEEL-PEP group. The maximum load of the repair alone group (16.63 N ± 0.67 N, n = 8) was the smallest when compared with the other three groups, and was statistically significant when compared with both the TISSEEL-PEP group and the normal healthy control group (P < .01; Figure 4J). The load of the TISSEEL group (18.62 N ± 0.77 N, n = 8; P = .03) was intermediate between the repair alone group and the TISSEEL-PEP group and was significantly different from the normal healthy control group (Figure 4J). The stiffness evaluation in these four groups is shown in Figure 4K. The TISSEEL-PEP group demonstrated a stiffness value of 10.41 N / mm ± 4.71 N / mm, similar to the normal healthy control group (14.06 N / mm ± 3.31 N / mm; P = .11). Stiffness values ​​were lowest in the repair alone group, with the TISSEEL group achieving stiffness values ​​between those in the repair alone and TISSEEL-PEP groups.

[0036] Tissue analysis Repair of the injured tendon-bone interface may be measured by histological analysis of the injured area. Histological characteristics that may be measured include, but are not limited to, inflammation, scar formation, collagen fiber arrangement, vascularity, and mineralization. In one or more embodiments, tissue analysis may be used to compare the progress of tendon-bone interface 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 histological measures of the healing tendon-bone interface compared to compositions and methods that do not include PEP.

[0037] In the normal control group, highly aligned collagen fibers passed through the non-calcified fibrocartilage region and intruded into the calcified fibrocartilage. After 6 weeks, specimens from the TISSEEL-PEP group showed that the collagen fibers were more organized and denser at the tendon-bone interface, with fewer inflammatory cells and a vascularity similar to that of the native interface. Furthermore, the appearance of a natural tendon attachment was also observed in the TISSEEL-PEP group compared with the repair alone and TISSEEL groups (all P<.05) (Figure 7).

[0038] In the repair alone and TISSEEL groups, clumps of inflammatory cells, mainly consisting of polymorphonuclear leukocytes, were present. In addition, a looser, scar-like, irregular meshwork of collagen fibers was observed in the repair alone group. The TISSEEL group showed dense inflammatory cells, a relatively organized collagen fiber arrangement, and scar tissue with newly formed fibrovascular tissue at the tendon-bone interface compared with the repair alone group (P<.05) (Figure 7).

[0039] Under polarized microscopy, the collagen network in the repair alone and TISSEEL groups appeared shorter and thinner than that in the normal control and TISSEEL-PEP groups. In comparison with the normal control group, the collagen fiber structure in the TISSEEL-PEP treatment group showed restoration of fiber continuity, parallel orientation, and robust density, comparable to the native control. Restoration of native structure in the TISSEEL-PEP group can be seen in the microscopic images (Figure 7).

[0040] Based on these results, PEP+TISSEEL promotes the remodeling of collagen fibers and new cartilage-like tissue at the tendon-bone interface after 6 weeks.

[0041] Changes in mRNA levels Repair of the injured tendon-bone interface may be measured by changes in gene expression in the injured area. Changes to gene expression may be measured by quantifying the levels of messenger RNA (mRNA). Increased expression of multiple genes may indicate improved repair. Genes that may be quantified include Col1, Col3, SCX, Tnmd, TNC, DCN, and IGF. In one or more embodiments, gene expression levels may be used to compare the progress of tendon-bone interface 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 increase the expression of genes associated with repair of the healing tendon-bone interface compared to compositions and methods that do not include PEP.

[0042] In the in vivo study, the TISSEEL-PEP group showed a significant increase in the expression of Col1, Col3, SCX, Tnmd, TNC, DCN, and IGF compared with all other groups (P<.05) (Figure 7F). The expression of osteogenesis-related genes (Spp1, Runx2) and chondrogenesis genes (COMP and Col2) was not detected.

[0043] This study evaluated the response to PEP at the tendon-bone interface. Treatment with PEP in a rotator cuff repair model at the tendon-bone interface resulted in significant improvements in biomechanical characteristics. Histological observation revealed well-organized collagen fibers along with increased expression of tendon- and tendon-bone-associated markers. The diversity of components in exosomes provides exosomal multipotency, and as a result, PEP-exosomes can accelerate the healing process at the tendon-bone interface by acting on multiple targets and multiple pathways.

[0044] The strength of the TISSEEL-PEP group is close to that of normal healthy rotator cuff. These histological results indicate a more organized and tightly packed collagenous tissue at the tendon-bone interface of the TISSEEL-PEP group. That is, the TISSEEL-PEP treatment group showed strength and histological similarity to normal healthy rotator cuff compared to the other treatment groups (repair alone and TISSEEL alone). Furthermore, these gene expression results were confirmed through biomechanical and histological test results. In addition to enhancing IGF expression, PEP was found to promote increased expression of tendon-related genes (Col1, Col3, SCX, Tnmd, and DCN), resulting in collagen rearrangement and extracellular matrix matrix components during healing of the tendon-bone interface of the rotator cuff. Thus, PEP is involved in the release of components that help remodel the tendon-bone interface structure. Furthermore, PEP and direct cell-cell contact correlated and strengthened each other in vitro, which may explain why greater recovery of rotator cuff attachment area occurred in vivo.

[0045] Thus, the present disclosure describes compositions and methods for improving the repair of damaged tendon-bone interfaces. 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., collagen scaffold, hydrogel, etc.).

[0046] That is, the method comprises administering an effective amount of the composition to tendon tissue in need of repair, wherein an "effective amount" in this embodiment is an amount effective to increase the osteoblast-tendon cell interface, improve at least one histological measure of the tendon-bone interface, increase expression of at least one gene that promotes repair of a damaged tendon-bone interface, or increase at least one biomechanical measure of the tendon-bone interface, compared to the osteoblast-tendon cell interface of a bone-tendon interface treated without PEP.

[0047] Exemplary histological measures include, but are not limited to, increased fiber continuity, increased parallel orientation of fibers, increased collagen fiber density, decreased vascularity, or decreased cellularity when compared to bone-tendon interfaces treated without PEP.

[0048] In one or more embodiments, the progress of tendon-bone repair may be measured by gene expression. For example, expression measurements of tendon-related genes, bone formation-related genes, and / or chondrogenesis genes may be used to indicate the progress of repair to the injured tendon-bone interface. Exemplary genes that promote the repair of the injured tendon-bone interface include, but are not limited to, type I fibrillar collagen (Col1), type III fibrillar collagen (Col3), bHLH transcription factor scleraxis (SCX), tenomodulin (TNMD), decorin (DCN), or insulin-like growth factor I (IGF-I) in the tissue of the tendon-bone interface.

[0049] In one or more embodiments, biomechanical measures may be used to characterize the repair of a damaged tendon-bone interface. Exemplary biomechanical measures include, but are not limited to, maximum load or stiffness. Typically, as a damaged tendon-bone interface heals, the interface becomes stronger and maximum load and stiffness increase.

[0050] In one or more embodiments, the compositions and methods described herein may increase the rate of repair of tendon-bone injuries.

[0051] 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).

[0052] 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- ceutical active substances is well known in the art. Any conventional media or agent is contemplated for use in a therapeutic composition unless it is incompatible with PEP. Additional active ingredients may also be incorporated into the composition. As used herein, "pharma- ceutical 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 that contains the substance. As mentioned above, in a surgical setting, exemplary suitable carriers include surgical adhesives, tissue adhesives, or support matrices (e.g., collagen scaffolds). As used herein, "collagen scaffold" refers to a three-dimensional network comprising collagen, such as a hydrogel.

[0053] 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 type I collagen, type III collagen, 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 components may be provided in any suitable form, such as purified recombinant protein.

[0054] 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.

[0055] That is, the pharmaceutical composition may be provided in any suitable form, including but not limited to a 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, for example, a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion, and the like. In embodiments in which the formulation is a gel, the gel may have any suitable density. For example, in certain embodiments in a surgical setting, the pharmaceutical composition may be formulated as a gel with sufficient density to maintain the formulation in the desired location. The formulation may further include one or more additives, such as, for example, an adjuvant, a skin penetration enhancer, a colorant, an aroma, a flavoring, a moisturizer, a thickener, and the like.

[0056] Suitable excipients include, for example, human or bovine collagen, hyaluronic acid-based compounds, human fibrinogen, or human thrombin.

[0057] In one or more embodiments, the compositions described herein may be lyophilized. The lyophilized compositions containing PEP may be combined with additional excipients, which may be further lyophilized. The components of the lyophilized compositions 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 a biocompatible gel upon contact with bodily fluids (e.g., blood or interstitial fluid).

[0058] In one or more embodiments, the compositions described herein are administered via injection into / on the tendon-bone interface during arthroscopic or open surgical repair. Compositions as described herein may be administered alone or in addition to traditional surgical repair methods such as suturing or stapling. Compositions as described herein may also be used to enhance the biocompatibility and therapeutic efficacy of tendon sutures, anchors, patches, or other devices used to repair tendon injuries.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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 9 PEP 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 11PEP 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.

[0063] 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.

[0064] 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 13In some 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 involve administering a single dose of PEP exosomes.

[0065] 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 11 exosomes (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.

[0066] 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%.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] In one or more particular embodiments in which multiple doses of the PEP composition are administered to a subject, the PEP composition may be administered as needed to heal and / or repair the tendon-bone interface 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.

[0071] In one or more embodiments, the method may include multiple administrations of PEP at an interval (in the case of two administrations) or at intervals (in the case of three or more administrations) characterized by a range defined by any minimum intervals as above and any maximum intervals greater than the minimum intervals, inclusive of the endpoints. 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.

[0072] In one or more embodiments, the method may include administering a cocktail of PEPs prepared from different cell types, each with a unique profile (e.g., protein composition and / or gene expression) that repairs the tendon-bone interface. In this manner, the PEP composition may provide a broader range of tendon-bone interface repair than if the PEP composition was prepared from a single cell type.

[0073] 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.).

[0074] In the above description, certain embodiments may be described separately for clarity. When used herein as "one embodiment," "an embodiment," "a particular embodiment," or "some embodiments," 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 the present specification does not necessarily refer to the same embodiment of the present disclosure. Furthermore, certain features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Furthermore, certain 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] Preferred Embodiments Embodiment 1 is a method of repairing a damaged bone-tendon interface in a subject, comprising: The damaged bone-tendon interface is Purified exosome product (PEP), and A pharma- ceutically acceptable carrier; contacting the subject with an effective amount of a composition comprising The method includes:

[0079] Embodiment 2 is the method of embodiment 1, wherein the PEP comprises spherical or ellipsoidal exosomes having a diameter of 300 nm or less.

[0080] Embodiment 3 is the method of embodiment 1, wherein the PEP comprises spherical or ellipsoidal exosomes having a diameter of 56 nm to 151 nm.

[0081] Embodiment 4 is the method of any one of embodiments 1 to 3, wherein the PEP comprises spherical or ellipsoidal exosomes having an average diameter of 97 nm.

[0082] Embodiment 5 is the method of embodiment 4, wherein the PEPs comprise spherical or ellipsoidal exosomes having an average diameter of 97 nm±54 nm.

[0083] In a sixth embodiment, the PEP is 1%–20% CD63 - Exosomes, and 80%-99% CD63 + Exosomes, The method according to any one of embodiments 1 to 5, comprising:

[0084] In embodiment 7, the PEP is at least 50% CD63 - The method according to any one of embodiments 1 to 5, comprising exosomes.

[0085] In embodiment 8, the PEP is 1×10 11 PEP exosomes ~ 1 × 10 13 The method according to any one of embodiments 1 to 7, comprising administering to the subject a therapeutically effective amount of PEP exosomes.

[0086] In embodiment 9, the PEP is 1×10 12 PEP exosomes ~ 1 × 10 13 The method according to any one of embodiments 1 to 8, comprising administering to the subject a therapeutically effective amount of PEP exosomes.

[0087] Embodiment 10 is the method of any one of embodiments 1 to 9, wherein the composition further comprises a support matrix.

[0088] Embodiment 11 is the method of embodiment 10, wherein the support matrix comprises a collagen scaffold.

[0089] Embodiment 12 is the method of embodiment 10, wherein the support matrix comprises a tissue sealant or a fibrin sealant.

[0090] Embodiment 13 is a method according to any one of embodiments 1 to 12, wherein the effective amount is an amount effective to increase the osteoblast-tendon cell interface compared to the osteoblast-tendon cell interface at a bone-tendon interface treated without PEP.

[0091] Embodiment 14 is the method of any one of embodiments 1 to 13, wherein the effective amount is an amount effective to improve at least one histological measure of the tendon-bone interface compared to a bone-tendon interface treated without PEP.

[0092] Embodiment 15 is the method of embodiment 14, wherein the histological measures include increased fiber continuity, increased parallel orientation of fibers, increased collagen fiber density, decreased vascularity, or decreased cellularity when compared to a bone-tendon interface treated without PEP.

[0093] Embodiment 16 is the method according to any one of embodiments 1 to 15, wherein the effective amount is an amount effective to increase the expression of at least one gene that promotes repair of a damaged tendon-bone interface.

[0094] Embodiment 17 is the method of embodiment 16, wherein the gene encodes type I fibrillar collagen (Col1), type III fibrillar collagen (Col3), the bHLH transcription factor scleraxis (SCX), tenomodulin (TNMD), decorin (DCN), or insulin-like growth factor I (IGF-I) in the tissue of the tendon-bone interface.

[0095] Example 18 is the method of any one of Examples 1-17, wherein the effective amount is an amount effective to increase at least one biomechanical measure of the tendon-bone interface compared to a bone-tendon interface treated without PEP.

[0096] Embodiment 19 is the method of embodiment 18, wherein the biomechanical measure comprises maximum load or stiffness.

[0097] A 20th embodiment is a method according to any one of the first to 19th embodiments, the damaged bone-tendon interface comprises a complete separation of the tendon from the bone; The method further comprises surgically reattaching the tendon to the bone. This is the method.

[0098] Embodiment 21 is the method according to any one of embodiments 1 to 20, the damaged bone-tendon interface comprises a partial separation of the tendon from the bone; The method includes implanting the PEP composition at a site effective to contact the PEP composition with the damaged tendon-bone interface. This is the method. EXAMPLES

[0099] Example 1 In this example, PEP particles were measured and counted using transmission electron microscopy.

[0100] Transmission electron microscopy Observations by transmission electron microscopy (TEM) were performed with a transmission electron microscope (JEM-1400Plus 120 kV Transmission Electron Microscope, JEOL, Tokyo, Japan). Before TEM examination, one vial of sealed PEP was mixed with 1 mL of PBS (Gibco, Thermo Fisher Scientific, Waltham, MA) to prepare a 100% (vol / vol) PEP solution. 50 μL of the above PEP solution was transferred to a microcentrifuge tube, and 1 mL of 2.5% glutaraldehyde in 0.1 M sodium cacodylate solution (pH 7.0) was added, followed by mixing for 1 h at 4 °C. The fixed samples were washed three times with sodium cacodylate buffer (pH 7.4) for 10 min each. The samples were then postfixed with 2% osmium tetroxide for 1 h at 4 °C, washed with buffer, dried, and stained with 2% uranyl acetate according to standard protocols. The PEPs (50 μL sample / grid) were examined under an 80 kV transmission electron microscope, and electron micrographs were taken.

[0101] Nanoparticle tracking analysis The size distribution and concentration of PEP were determined using a nanoparticle tracking characterization system (NS300, Nanosite, Malvern, UK). The PEP solution (100%, vol / vol) was diluted 1,000-fold with 1 mL of PBS diluent and then loaded into the sample chamber. The PEP concentration, mean value, and mode PEP size were analyzed using NTA3.2 analysis software (Nanosite, Malvern, UK).

[0102] Example 2 In this example, the effect of PEP on tendon-bone interface repair was investigated using a cell culture model.

[0103] Preparation of PEP and PEP-containing and non-PEP-containing fibrin sealants (TISSEEL) PEP was obtained from API at the Mayo Clinic Center for Regenerative Medicine. The product was prepared and stored in the form of a stabilized lyophilized powder in vials to allow room temperature storage until processing (Figure 2A).

[0104] Fibrin sealant is a biodegradable pulp-like tissue that can be used as a drug delivery carrier and is highly effective in achieving localized and sustained release of exosomes. In this example, the TISSEEL kit, fibrin sealant product, was used with or without the addition of PEP. Prior to preparation of the TISSEEL kit (Baxter International, Deerfield, IL), one vial of sealed PEP powder was mixed with 1 mL of PBS (Gibco, Thermo Fisher Scientific, Waltham, MA) to prepare a 100% (vol / vol) PEP solution. 400 μL of PEP solution was added to 600 μL of CaCl2 solution (one of the contents of the TISSEEL kit) to adjust the solution to a concentration of 40% (vol / vol). The kit preparation was completed according to the manufacturer's instructions (Figure 2B). The final concentration of PEP in the TISSEEL was 20% (vol / vol). The gel was manually cut into small cubes (3 × 3 × 3 mm). For the co-culture model, one cube was placed in a small hole in one well of a 6-well plate. The medium and PEP gel were used to simulate the in vivo PEP microenvironment. For osteogenic induction, medium and PEP as above were used as positive controls. For in vivo studies, the cubes were placed directly into the RC repair site, between the supraspinatus tendon and the greater tuberosity.

[0105] Primary cells, culture conditions, and identification Primary osteoblasts were isolated from the parietal bones of neonatal rats that were euthanized (a process that does not affect osteoblasts) according to guidelines approved by the Institutional Animal Care and Use Committee (IACUC) using a method previously described (Liu et al., 2019, Cell and Tissue Banking 20:173-182). To collect osteoblasts, parietal bone segments were immersed in a mixture of 0.1% (wt / vol) collagenase I, 0.05% trypsin, and 0.004% ethylenediaminetetraacetic acid for 60 min. Cells were then collected from the third to fifth soaks and cultured at 37°C and 5% CO2 in Minimum Essential Medium Alpha (Invitrogen, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA) and 1% penicillin-streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA). To test the osteogenic potential of primary osteoblasts + PEP, three groups were established: the above medium serving as the negative control group; the above medium supplemented with PEP serving as the positive control group; and the StemPro Osteogenesis Differetiation Kit (Thermo Fisher Scientific, Waltham, MA) serving as the osteogenic induction group.

[0106] Osteoblasts were identified by alkaline phosphatase (ALP) staining. After 7 or 14 days of culture, primary osteoblasts were washed twice with cold phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 30 min, rinsed with deionized water, and stained with an ALP staining kit (Abcam, Cambridge, UK) for 30 min under protection from direct light according to the manufacturer's instructions. Images were then captured with a Nikon camera (Nikon, Minato-ku, Japan).

[0107] Primary tenocytes were isolated from euthanized 8-week-old female Sprague-Dawley rats using a previously described method (Zhang et al., 2010, BMC Musculoskeletal Disorders 11:10) based on IACUC-approved guidelines. Rat flexor tendons were harvested and the paratenon sheath layer was separated by gentle scraping. Tendons were washed three times with sterile PBS, cut into small fragments, and cultured with conditioned medium as described above until confluent growth. Cell medium was refreshed every 3 days. Cells from passages 3–6 were used in all studies. Tenocytes were identified by detection of tendon-specific genes: collagen type 1 (Col1), collagen type 3 (Col3), and Scleraxis (SCX) expression. In quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), the respective mRNA expression values ​​were normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression; rat primary osteoblasts were used as a control group.

[0108] Co-culture model An in vitro co-culture model based on a previously described model (Bogdanowicz et al., 2014, Methods in Molecular Biology 1202:29-36) was used. The co-culture model used 6-well plates and cryopreservation tubes. In addition to the bone region, interface region, and tendon cell region, this new model added an inlet for drug carriers. Cell culture grade agarose (Sigma-Aldrich, St. Louis, MO) was poured into a 6-well plate and cut with a cryopreservation tube to create three holes, leaving a 3 mm wide partition. The model was then transferred to a new 6-well plate and fixed to the bottom of the plate with unsolidified agarose (Figure 1A-E). Rat primary osteoblasts were seeded in the slightly larger hole on the left, and tendon cells were seeded in the slightly larger hole on the right. The small hole in the middle contained 20% PEP and a TISSEEL cube (Figure 1A). After 30 min of cell attachment, medium was added until the model was almost submerged. The co-culture was incubated for 2 days, the partition between the two cell populations was cut, and PEP vehicle was added to the small holes. No drugs were added to the control group. The medium was overflowed from the model and replaced every 3 days. Cell migration into the interface area was recorded twice daily using an IncuCyte HD system (IncuCyte ZOOM, Essen Biosciences, Ann Arbor, MI). Cell boundaries were manually tracked using Photoshop CS6 (Adobe, San Jose, CA). For PCR testing, the model was removed and washed with ice-cold PBS at the time point 3 days after the two cell populations came into direct contact. Cells were then detached from the plate by a cell scraper within each area and stored in tubes with TRIzol (TRI Reagent, Sigma-Aldrich, St. Louis, MO) and stored at -80°C for PCR testing. The results are shown in FIG. 5 and described in more detail herein.

[0109] Example 3 In this example, a rat model of rotator cuff injury was used to compare the rate of repair when animals were treated with sutures alone, sutures and TISSEL, or sutures, TISSEL, and PEP.

[0110] Animal study design and rat RCT model Thirty-six Sprague-Dawley rats (adult females, 4–5 months old, weighing 258–552 g) were used. They were randomly divided into three groups, with the right shoulder serving as the surgical side (n=12 in all groups): repair alone; repair + TISSEEL (Baxter International, Deerfield, IL) (TISSEEL group); and repair + TISSEEL + PEP (TISSEEL-PEP group). Rats were anesthetized via an induction chamber with 2%–3% isoflurane in 100% oxygen at 2 L / min delivered via mask until loss of toe pinch reflex. Preemptive analgesia was administered by intramuscular injection of meloxicam (1 mg / kg). Each rat was placed on a warm plate to maintain body temperature and reduce the risk of hypothermia. Anesthesia was maintained by a continuous flow of a mixture of 1.5%–2% isoflurane in 1 L of 100% oxygen via a nose cone.

[0111] The surgical site was cleaned with 2% chlorhexidine gluconate, and the skin was incised laterally 1 cm lateral to the deltoid using a sterile #15 scalpel blade. The supraspinatus tendon was identified and separated from the subscapularis tendon anteriorly and the infraspinatus tendon posteriorly. The supraspinatus tendon was then transected at its attachment on the greater tuberosity. To freshen the attachment, the tendon fibers at the attachment were scraped with a scalpel. One end of a double-ended needled 5-0 suture (ETHIBOND, Ethicon, Raritan, NJ) was then passed laterally through the tendon, and a small loop was made on either side of the tendon using a modified Masson-Allen suture (Figure 4B, Figure 4E, and Figure 4F). A 0.5 mm hole was drilled transversely in the anterior-posterior direction in the proximal part of the humerus, and the other end of the suture was passed through the 0.5 mm hole (Figure 4G). After placing TISSEELs (Baxter International, Deerfield, IL) with or without added PEP at the repair site, sutures were tied to the tendon at its attachment point on the greater tuberosity (Figure 4A, 4C, and 4D). TISSEELs with or without PEP were prepared as described in Example 2. The isolated deltoid muscle was repaired with 4-0 polyglactin 910 sutures (VICRYL, Ethicon, Raritan, NJ), and the skin was repaired with 3-0 polyglactin 910 sutures (VICRYL, Ethicon, Raritan, NJ) (Figure 4H). A water-ibuprofen mixture (15 mg / kg) was administered daily for 1 week after surgery in all groups. These doses were recommended by the laboratory veterinarian and approved by the IACUC protocol.

[0112] Six weeks after surgery, the rats were euthanized by CO2 asphyxiation. Eight rats from each group were used for biomechanical testing, and four rats from each group were used for both histological analysis and qPCR measurement of mRNA expression. The left shoulder served as a normal control group (Figure 3).

[0113] Biomechanical testing of rotator cuff tear (RCT) repairs Six weeks after the procedure, rats were euthanized by carbon dioxide inhalation to assess tissue healing. The supraspinatus tendon and peritendinous tissue of the humerus were then completely removed using a surgical loupe. The humerus was embedded in polymethylmethacrylate in a specially designed fixture, and the proximal end of the tendon was fixed in a spring-loaded clamp specially made for the test. After that, the specimens were preconditioned with a preload of 0.2 N and five cycles of 0.1 mm displacement at a rate of 0.1 mm / s, and then tested to failure under uniaxial tension at a rate of 0.1 mm / s (Figure 4I). The maximum failure load and stiffness were then calculated from the force-displacement curves generated by a custom MATLAB program (The MathWorks, Natick, MA).

[0114] Histological analysis After euthanasia at 6 weeks, the supraspinatus tendon and the repair site at the bone attachment of the supraspinatus tendon were carefully dissected in each group. The specimens were fixed overnight in 10% formaldehyde and then placed in 14% ethylenediaminetetraacetic acid. The specimens were placed in 30%, 50%, and 70% ethanol for at least 30 min each and then submitted to the core laboratory for paraffin embedding. All specimens were embedded in tissue embedding matrix (TISSUE-TEK, Sakura Finetech, Tokyo, Japan) and cut into coronal sections (10 μm thick) on a cryostat (Leica Biosystems, Wetzlar, Germany). Histological changes were analyzed by hematoxylin-eosin staining, Masson-Trichosome staining, and picrosirius red staining. Picrosirius red stained tissue sections were observed under polarized light microscopy (BH2, Olympus, Shinjuku, Japan). Representative microscopic images are shown in FIG.

[0115] The continuity, parallel orientation, density, vascularity, and cellularity of collagen fibers at the tendon-bone interface were evaluated. Histological findings were evaluated using a semiquantitative scoring system (0-3 scale for each item). For collagen fiber continuity and collagen fibers oriented parallel to each other, the scores were defined by the percentage: 0 = 0%-25%; 1 = 25%-50%; 2 = 50%-75%; and 3 = 75%-100%. For collagen fiber density, the scores were defined by the percentage: 0 = very sparse, 1 = sparse, 2 = dense, and 3 = very dense. For vascularity and cellularity, the scores were defined by the percentage: 0 = absent or minimally present, 1 = mildly present, 2 = moderately present, and 3 = severely or markedly present. Each side was examined under a microscope (Olympus, Shinjuku, Japan) and analyzed using ImageJ software (Schneider et al., 2012, Nature Methods 9(7), 671-675). Four specimens were evaluated per group by two independent observers. Histological quantification is shown in Figure 7E.

[0116] RNA isolation and quantitative PCR In the in vitro study, after the measurement time point was reached, the cells in the corresponding regions (osteoblast region, tendon cell region, and interface region) were washed with PBS and detached separately by scraping. In the in vivo study, the tendon-bone tissues were dissected and flash frozen in liquid nitrogen, then pulverized with an abrasive tool. After homogenization, total RNA was extracted and purified using an RNA isolation kit (TRIZOL Plus, Invitrogen, Thermo Fisher Scientific, Waltham, MA). Then, the total RNA was quantified using a spectrophotometer (NANODROP1000, Thermo Fisher Scientific, Waltham, MA), and cDNA synthesis (RT-PCR) was performed using a cDNA synthesis kit (ISCRIPT, Bio-Rad Laboratories, Hercules, CA). Total RNA (1 μg) was reverse transcribed into complementary DNA using a kit (THERMOSCRIPT, Invitrogen, Thermo Fisher Scientific, Waltham, MA). Real-time PCR was performed in triplicate. Briefly, total RNA was extracted from cells using TRIzol Reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized from equal amounts of RNA (1 μg) using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). All reactions were performed using SYBR Green PCR Master Mix (Quantabio, Beverly, MA) on a C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Hercules, CA). Data obtained from target genes were normalized to GAPDH and then 2x the basal control. -ΔCtThe expression of gene markers was calculated using the formula: For detection of gene marker expression, primers for tenocyte-related gene markers (Col1, Col3, SCX, tenomodulin (Tnmd), EGR1 (early growth response protein 1), decorin (DCN)), osteoblast-related gene markers (secreted phosphoprotein 1 (Spp1), tenascin-C (TNC), RUNX family transcription factor 2 (Runx2), insulin-like growth factor I (IGF-I)), lipid metabolism-related gene markers (peroxisome proliferator-activated receptor gamma (PPARG)), and chondrogenesis-related gene markers (Col2, cartilage oligomeric matrix protein (COMP)) were performed. The primers used in the qPCR experiments were as previously reported (Shi et al., 2021, J Orthop Res. 39(8):1825-1837. doi:10.1002 / jor.24859).

[0117] statistical analysis Data are expressed as mean values ​​(SD). Each test was performed at least three times independently of each other. Kruskal-Wallis one-way ANOVA test and Dunn's test were used to determine the statistical significance of two-group and multiple-group comparisons, respectively. Statistical comparisons between two groups were analyzed by unpaired Student's t test or Mann-Whitney test. All statistical tests were performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA). These data are shown in Figure 7F. Results marked with an asterisk were considered statistically significant ( * P < .05, ** P < .01, *** P < .001, **** P<.0001).

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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. 1. A composition for repairing a damaged bone-tendon interface in a subject, comprising: Purified exosome product (PEP), and a pharmaceutically acceptable carrier; and contacting the injured bone-tendon interface with an effective amount of the composition.

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 a diameter of 56 nm to 151 nm.

4. 2. The composition of claim 1, wherein the PEP comprises spherical or ellipsoidal exosomes having an average diameter of 97 nm.

5. 5. The composition of claim 4, wherein the PEP comprises spherical or ellipsoidal exosomes having an average diameter of 97 nm±54 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 composition further comprises a support matrix.

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

12. The composition of claim 10 , wherein the support matrix comprises a tissue sealant or a fibrin sealant.

13. 10. The composition of claim 1, wherein the effective amount is an amount effective to increase the osteoblast-tendon cell interface compared to the osteoblast-tendon cell interface at a bone-tendon interface treated without PEP.

14. 10. The composition of claim 1, wherein the effective amount is an amount effective to improve at least one histological measure of the tendon-bone interface compared to a bone-tendon interface treated without PEP.

15. 15. The composition of claim 14, wherein the histological measures include increased fiber continuity, increased parallel fiber orientation, increased collagen fiber density, decreased vascularity, or decreased cellularity when compared to a bone-tendon interface treated without PEP.

16. 10. The composition of claim 1, wherein the effective amount is an amount effective to increase the expression of at least one gene that promotes repair of a damaged tendon-bone interface.

17. The composition of claim 16, wherein the gene encodes type I fibrillar collagen (Col1), type III fibrillar collagen (Col3), the bHLH transcription factor scleraxis (SCX), tenomodulin (TNMD), decorin (DCN), or insulin-like growth factor I (IGF-I) in the tissue of the tendon-bone interface.

18. 10. The composition of claim 1, wherein the effective amount is an amount effective to increase at least one biomechanical measure of a tendon-bone interface compared to a bone-tendon interface treated without PEP.

19. The composition of claim 18 , wherein the biomechanical measure comprises maximum load or stiffness.

20. the damaged bone-tendon interface comprises a complete separation of the tendon from the bone; The composition of any one of claims 1 to 19, wherein the tendon is surgically reattached to the bone.

21. the damaged bone-tendon interface comprises a partial separation of the tendon from the bone; The PEP composition is implanted at a site effective to contact the injured tendon-bone interface. The composition according to any one of claims 1 to 19.

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