Loading Mesenchymal Stem Cell–Derived Exosomes Into a Traditionally Designed Rotator Cuff Patch: A Potential Strategy to Enhance the Repair of Chronic Rotator Cuff Tear Associated With Degenerative Changes

Abstract

Background:

Retraction and degenerative changes of chronic rotator cuff tears limit the healing capacity after routine surgical repair.

Purpose:

To fabricate a mesenchymal stem cell–derived exosome (MSC–Exos) loaded patch and evaluate the effect of this patch on the activity of rabbit tenocytes in vitro and on the repair of chronic rotator cuff tears associated with degenerative changes in vivo.

Study Design:

Controlled laboratory study.

Methods:

The MSC–Exos loaded patch was fabricated using a dynamic wet-spinning system. In the in vitro studies, the proliferation and migration activities of tenocytes were evaluated by culturing tenocytes with saline, a fiber-aligned patch, or an MSC–Exos loaded patch. In the in vivo studies, a rabbit model of chronic rotator cuff tear was established and directly repaired, repaired with fiber-aligned patch augmentation (RFPA group), and repaired with MSC–Exos loaded patch augmentation (REPA group). Histological and biomechanical analyses were performed at 4, 8, and 12 weeks after surgery.

Results:

An MSC–Exos loaded patch with inner aligned fibers, a loose microstructure, and reliable initial strength was fabricated using a dynamic wet-spinning system. The MSC–Exos loaded patch significantly promoted tenocyte proliferation and migration activities in vitro. In vivo, the REPA group exhibited significantly higher tendon maturing scores at 8 and 12 weeks after surgery compared with both the control and the RFPA groups. Fatty infiltration was significantly reduced in the REPA group at 4, 8, and 12 weeks compared with both the control and the RFPA groups. Biomechanical properties, including load to failure and stress, were also significantly improved at 12 weeks in the REPA group compared with both the control and the RFPA groups.

Conclusion:

Results in the present study suggested that an MSC–Exos loaded patch was able to enhance the repair of a chronic rotator cuff tear by providing mechanical support and minimizing degeneration.

Clinical Relevance:

This work supported the idea that loading bioactive MSC–Exos into a traditionally designed rotator cuff patch might exert a better effect on the repair of chronic rotator cuff tears than augmented patch repair alone.

Keywords

chronic rotator cuff tear mesenchymal stem cell–derived exosomes patch

Rotator cuff tear is a common cause of shoulder pain and dysfunction. Open or arthroscopic rotator repair is routinely performed to restore normal shoulder function. However, many such repairs fail, in particular, the failure rate for the repair of chronic large to massive tears is up to 90% at 3 to 5 years postoperatively.^10,27

An epidemiological study reported that there was a long delay of 6 months to more than 3 years between the initial rotator cuff injury and surgical management.²¹ During this period, the effect of surgical intervention and its healing potential were greatly compromised.^11,12,27 First, retraction of the musculotendinous tissues caused high tension at the repair site, resulting in large pull-out forces on the suture anchor during the acute rehabilitation stage that exceeded the strength of the repair.^26,27 Second, the quality of the rotator cuff tissue decreased because of potential irreversible degenerative changes, such as tendon degeneration and fatty infiltration.^7,9,28,35 The physiological properties of the torn rotator cuff never returned to preinjury levels, even after a perfect surgical repair.^10,11,27

Enhancement of rotator cuff repair can be achieved by augmentation with a patch that provides both acute mechanical support and a conductive scaffold for cell migration and tissue regeneration at the repair site.^1,4,24,27,31 The ideal patch has 3 requirements. First, the patch should be mechanically strong.^16,22 Second, the microstructure of the patch should be fiber-aligned and loose to allow the ingrowth of cells and induce tendon differentiation.^23,39 Third, the patch should be biocompatible so that it does not stimulate a foreign body reaction.^2,16,20 Waterborne polyurethane (WBPU) is a Food and Drug Administration–approved material that has been widely used in medical engineering fields owning to its good biocompatibility and strength.^15,18 Moreover, WBPU can be mixed with bioactive growth factors in water instead of in organic solvents with biological toxicity that could lead to the loss of the bioactivity of growth factors. An electrospinning technique that controls the fiber alignment and microstructure of fibrous scaffolds can be used to fabricate an ideal augmentation patch.^34,39

The efficacy of mesenchymal stem cells (MSCs) in tissue engineering has been further elaborated. Rather than directly differentiating into tissue-specific cells after implantation, MSCs secrete numerous growth factor exosomes (MSC–Exos), which are nanosized (50-200 nm) membrane vesicles that contain proteins, nucleic acids, and lipids and exert therapeutic effects by promoting cell proliferation, migration, and differentiation.^{28,29,33,36,37}

The application of an augmenting patch alone may enhance the strength of repair but cannot restore the degenerative changes of the torn tendon because the patch itself is nonbioactive. Furthermore, the injection of MSC–Exos alone prevents degenerative changes according to previous studies but has the problem of ingredient loss and lack of concentration at the repair site. Inspired by their complementary advantages, the fabrication of an exosome-loaded patch that can not only enhance the strength of the repair and provide a scaffold for cell ingrowth but can also improve the degenerative changes might be promising in chronic rotator cuff tear repairs.

This study was divided into 3 parts. In the first part, an MSC–Exos loaded patch with inner aligned fibers, a loose microstructure, and reliable mechanical strength was fabricated. In the second part, the effect of this patch on the proliferation and migration activities of rabbit tenocytes was evaluated in vitro. In the third part, the effect of this patch on chronic rotator cuff tears associated with degenerative changes was evaluated in vivo. A chronic rotator cuff tear animal model was established in accordance with a previous study.¹⁴ We hypothesized that this MSC–Exos loaded patch would be able to promote proliferation and migration activities of rabbit tenocytes and enhance the healing of chronic rotator cuff tears.

Methods

MSC–Exos Loaded Patch

Isolation and Identification of MSC–Exos

MSC–Exos were isolated from MSCs derived from a New Zealand White rabbit's (male, 4 weeks) bone marrow using the method described in previous studies (Appendix, available in the online version of this article).^28,38 The morphology of MSC–Exos was observed by transmission electron microscopy (H-7650; Hitachi High-Technologies Corporation). The particle size distribution was analyzed using nanoparticle tracking analysis (ZetaView PMX 120; Particle Metrix). The expression of exosomal markers—such as CD9, CD63, TSG-101, and ALIX—was detected by Western blotting.

Fabrication of the MSC–Exos Loaded Patch

WBPU synthesis was carried out mainly in accordance with a previous study⁴⁰ (Appendix, available online). The MSC–Exos loaded patch was fabricated using a dynamic wet-spinning system.³⁴ Briefly, sterile gelatin (Gel) (5% v/v) and WBPU (10% w/v) were dissolved in sterile distilled water and stirred overnight at 37°C to obtain a uniform solution. To prepare the MSC–Exos mixed solution, 10¹¹ pellets/mL of MSC–Exos (10% of the Gel mass) was added to the Gel–WBPU solution and further stirred for 1 hour before fabrication. After that, the MSC-Exos-Gel-WBPU or the Gel–WBPU solution was collected into a 10-mL sterile syringe and electrospun. During the process of dynamic wet spinning, nanomacro yarns were collected through a vortex of sterile water (0°C-4°C) and finally received onto a high-speed roller to obtain the fiber-aligned (without MSC–Exos loading) or MSC–Exos loaded patches.

Characterization of the MSC–Exos Loaded Patch

Scanning electron microscopy (JEOL; JSM-5600) was used to observe the inner fiber alignment of the patch. A transmission electron microscope (FEI, JEM-2100F, JEOL) was used to observe the surface morphology of the fiber.

The porosity of the patch was calculated using the following formula (n = 3 for each group): Porosity (%) = (V₀ - V) / V * 100%, where V₀ is the apparent volume of the patch, and V is the true volume of the patch. The pore diameter of the patch was measured using image analysis software (Image-Pro Plus; Media Cybernetics).

The mechanical properties of the patch, including load to failure, stiffness, and stress, were evaluated using a custom-designed uniaxial testing machine (Instron 5569; Instron Co).

In Vitro Studies

Cell Proliferation

The Cell Counting Kit–8 (CCK8) assay (Dojindo Molecular Technologies Inc) was performed to evaluate the effect of the MSC–Exos loaded patch on the tenocytes’ proliferation activity isolated from rabbit rotator cuff tendons (Appendix, available online). Briefly, cells were plated into 96-well plates at an initial density of 1000 cells/well and cultured at 37°C with a fiber-aligned patch, with an MSC–Exos loaded patch, or without a patch as control. Also, a 10-μL CCK8 solution was added to each well and incubated for a further 1 hour at 37°C on days 1, 3, and 5. The absorbance of the wells was measured at 450 nm using a spectrophotometric microplate reader (Bio-Rad 680; Bio-Rad Laboratories Inc). The optical density of the tested well minus the absorbance of the blank wells represented the proliferation intensity of cells.

Cell Migration In Vitro Studies

The effect of the MSC–Exos loaded patch on tenocytes migration was analyzed using a transwell assay. Briefly, 1 × 10⁶ tenocytes were plated into the upper chamber of a 96-well transwell plate (Corning; Corning Inc). The fiber-aligned patch or the MSC–Exos loaded patch was then added to the lower chamber. The lower chamber without a patch served as a control. After incubation at 37°C for 24 hours, cells from the upper surface of the filter membranes were discarded. Cells that migrated to the lower surface of the filter membrane were fixed in 4% formaldehyde for 5 minutes and then stained with 0.5% crystal violet for 5 minutes. The migratory activity was evaluated by observing the stained cells under an optical microscope.

In Vivo Studies

Animal Model and Power Analysis

The animal experimental protocol was approved by the Animal Ethical and Welfare Department.

To calculate the sample size required for the animal experiments, a power analysis was performed, with α = .05, 1 –β = 0.8, an assumed dropout rate = 25%, a mean difference = 10 N, and SD = 6.59 N, in accordance with a previous study.³² A sample size of 8 shoulders was required to detect a significant difference in the biomechanical analysis in terms of the ultimate load to failure. Additionally, 4 shoulders were allocated for histological analysis in each group. Thus, 108 male New Zealand White rabbits, with a mean age of 16 weeks and a mean weight of 2.5 kg were included in the present study. Rabbits were randomly allocated into 3 groups: (1) the repair group (R group); (2) the repair + fiber-aligned patch augmentation group (RFPA group); and (3) the repair + MSC–Exos loaded patch augmentation group (REPA group). All experimental procedures were performed on the left shoulder. Biomechanical and histological analyses were performed at 4, 8, and 12 weeks.

Establishment of Chronic Rotator Cuff Tear Model and Surgical Repair

With the animal under general anesthesia and with sterile conditions, a 1-cm longitudinal skin incision was made between the greater tubercle and the acromioclavicular joint. The supraspinatus tendon was exposed by incising and retracting the deltoid muscle. The rotator cuff tear model was established by completely cutting the supraspinatus tendon from the greater tubercle. The torn tendon was left unrepaired for 12 weeks to establish a chronic tear model that simulated the degenerative changes observed in humans. The muscle, subcutaneous tissue, and skin were then closed as separate layers.

After 12 weeks, the torn tendon was surgically repaired. The torn supraspinatus tendon was approached as mentioned above, and the detached tendon appeared proximally retracted with some adhesions to the surrounding tissues. The tendon was freed from the fibrotic adhesions and mobilized. Decortication was performed on the greater tubercle to create a fresh bleeding surface, and a 1.0 K-wire was used to make 2 parallel bone tunnels approximately 5 mm apart oriented from the original footprint of the supraspinatus tendon to the lateral aspect of the greater tubercle. In the R group, the torn tendon was directly reattached to the footprint by 2 parallel transosseous sutures and 2 crossed transosseous sutures (2-0/T; Jinhuan), as described in a previous study³² (Figure 1A). In the RFPA and REPA groups, the repair was augmented with a fiber-aligned patch or an MSC-Exos-loaded patch on top of the repaired tendon, and the proximal part of the patch was fixed on the muscular part of the supraspinatus using 2 interrupted sutures (Figure 1B). The muscle, subcutaneous tissue, and skin were closed in separate layers, and the rabbits were returned to their cages without immobility until the day of harvesting.

At each determined time point, rabbits were euthanized with an overdose of intravenous pentobarbital. Histological and biomechanical analyses were performed after a macroscopic evaluation of the harvested sample.

Histological Analysis

Samples were fixed in 4% buffered paraformaldehyde for 24 hours, fully decalcified in 0.25 mol/L ethylenediaminetetraacetic acid in phosphate-buffered saline for 8 weeks, dehydrated with graded alcohol, cleared using Xylene, and embedded in paraffin. Consecutive 3 μm–thick sections, including the musculotendinous part of the supraspinatus and the proximal humerus, were cut parallel to the long axis of the supraspinatus tendon in the coronal plane and stained with hematoxylin and eosin, safranin O, picrosirius red, and oil red O for the analysis of the general morphology, fibrocartilage regeneration, collagen deposition and maturation, and fatty infiltration, respectively. Picrosirius red-stained sections were observed and evaluated under polarized microscopy (Imager.M1; ZEISS), as described in previous studies.^8,19

The tendon maturing scoring system proposed by Ide et al¹⁷ was used to semiquantitatively analyze the healing of the repaired rotator cuff (Table 1). Each variable was respectively evaluated and scored. The final sum of the scores was obtained, with a score of 28 indicating perfect healing, and a score of 7 indicating maximum abnormality. The semiquantitative analysis was performed by 2 independent pathologists who were blinded to the present study.

Table 1

The Tendon Maturing Scoring System^a

	1	2	3	4
Cellularity	Marked	Moderate	Mild	Minimal
The proportion of cells resembling tenocytes, %	<25	25-50	50-75	>75
The proportion of parallel cells, %	<25	25-50	50-75	>75
Vascularity	Marked	Moderate	Mild	Minimal
The proportion of large-diameter fibers, a characteristic of mature tendon fibers, %	<25	25-50	50-75	>75
The proportion of parallel fibers, %	<25	25-50	50-75	>75
Remodeling of the tendon-to-bone insertion	C(+) I(-)	C(+) I(+) F(-)	C(+) I(+) F(+) T(-)	C(+) I(+) F(+)T(+)

C, continuity; I, ingrowth; F, fibrocartilage; T, tidemark.

A quantitative analysis of fatty infiltration was performed using Image J software (National Institutes of Health). Digital images were converted into 8-bit digitization, and the threshold was adjusted to assess the area that was stained with oil red O.

Biomechanical Analysis

A biomechanical analysis was commenced immediately after harvesting samples of the scapula, the complete musculotendinous part of the supraspinatus, and the humerus. The cross-sectional area of the tendon at the repair site was measured using digital calipers. All sutures were cut and removed from the specimen before testing. A custom-designed uniaxial testing machine (Instron 5569) was used to perform the biomechanical analysis. The humerus was mounted in a cylindrical holder using 6 interference screws, and the scapula was gripped in a clamping device connected to the sensor system. The musculotendinous part of the supraspinatus was at an angle of approximately 135° to the humerus to mimic the anatomic direction of tensile force. After the application of a preload of 5 N for 10 minutes, the sample received 10 cycles of loading ranging from 5 N to 20 N for preconditioning to minimize the viscoelastic effects. Immediately after preconditioning, the load to failure test was performed using uniaxial tension at 10 mm/min. The ultimate load to failure was defined as the first significant decrease in the load-displacement curve, and the stress was calculated by dividing the ultimate load to failure by the initial cross-sectional area. The mode of failure was also recorded.

Statistical Analysis

All data are expressed as mean and standard deviation. SPSS software (Version 15; SPSS Inc) was used to conduct the statistical analysis. A one-way analysis of variance with post hoc testing was performed using the Bonferroni method. Significance was set at P < .05.

Results

Characterization of MSC–Exos

Transmission electron microscopy and nanoparticle tracking analysis results revealed spherical vesicles with a particle size distribution of 50 to 150 nm (Figure 2, A and B). Western blotting analysis showed that these vesicles had a strong surface expression of CD9, CD63, TSG-101, and ALIX (Figure 2C).

Characterization of the MSC–Exos Loaded Patch

Using a dynamic wet-spinning system, the patch was successfully fabricated. Both the fiber-aligned patch and the MSC–Exos loaded patch exhibited a smooth, fiber-aligned appearance (Figure 3). Furthermore, MSC–Exos were attached to the surface of a single fiber in the MSC–Exos loaded patch, suggesting that MSC–Exos were successfully loaded into the patch (Figure 4).

Both the fiber-aligned patch and the MSC–Exos loaded patch exhibited loose porosity (fiber-aligned patch: 76.58% ± 3.95%; MSC–Exos loaded patch: 78.18% ± 4.17%; ns) (Figure 5A) and pore size (fiber-aligned patch: 26.30 ± 9.59 µm; MSC–Exos loaded patch: 27.88 ± 14.13 µm; ns) (Figure 5B).

There were no significant differences in mechanical properties between the fiber-aligned patch and the MSC–Exos loaded patch with regard to load to failure (fiber-aligned patch: 30.30 ± 2.41N; MSC–Exos loaded patch: 29.70 ± 2.96 N), stiffness (fiber-aligned patch: 330.33 ± 22.12 N/m; MSC–Exos loaded patch: 328 ± 17.52 N/m), and stress (fiber-aligned patch: 10.15 ± 1.38 MPa; MSC–Exos loaded patch: 10.16 ± 1.69 MPa) (Figure 5C, 5D, 5E).

Cellular Responses to the MSC–Exos Loaded Patch

The CCK8 assays showed that the fiber-aligned patch significantly increased the proliferation intensity of tenocytes compared with the control group. The MSC–Exos loaded patch further significantly increased the proliferation intensity of tenocytes compared with both the control and the fiber-aligned patch groups (Figure 6A).

The transwell assays showed that the fiber-aligned patch significantly increased the migration activity of tenocytes compared with the control group. The MSC–Exos loaded patch further significantly increased the migration activity of tenocytes compared with both the control and the fiber-aligned patch groups (Figure 6, B and C).

These results indicated that loading MSC–Exos into a fiber-aligned patch exerted a stronger positive effect on the proliferation and migration activities of tenocytes.

Effects of the MSC–Exos Loaded Patch on the Repair of Chronic Rotator Cuff Tears

Macroscopic Evaluation

There was no infection, foreign body reaction, or retear observed at the surgical site in any group at any harvest time point. The repaired tendon showed great integrity and continuity to the greater tubercle in all groups. The biodegradable augmentation patch was completely surrounded by fibrous tissues, and its general appearance could not be discerned (Figure 7).

Histological Observation

At 4 weeks, all groups showed great hypercellularity and hypervascularity. There were more fibroblasts and fibers in the RFPA and REPA groups than those in the R group, and their arrangement was more orderly organized in the RFPA and REPA groups than that in the R group. The continuity between the tendon and the bone was complete in all groups, with the repaired tendon better integrated into the bone in the RFPA and REPA groups than that in the R group. A few positively stained collagen fibers were observed in the RFPA and REPA groups, but not in the R group. Compared with the R group, the degree of fatty infiltration was reduced in the REPA group but not in the RFPA group.

At 8 weeks, cellularity and vascularity were markedly reduced in all groups. Compared with the R group, the RFPA and REPA groups still had more abundant fibrous tissues and a better arrangement. The repaired tendon in the R group still did not show signs of ingrowth into the bone. A newly formed fibrocartilage was seen in a few samples in the REPA group, but not in the RFPA group. Positively stained collagen was observed in all groups at this time point, with the most collagen seen in the REPA group. Fatty infiltration was still not reduced in the R or RFPA groups.

At 12 weeks, there was mild cellularity and vascularity in all groups; the tissue was generally better arranged in all groups; and fibroblasts and the extracellular matrix were organized along the longitudinal axis of the tensile force. The repaired tendon in the R group still showed no signs of ingrowth into the bone. Fibrocartilage formation was still only observed in the REPA group. There was no evidence of the formation of a tidemark gradually changing from the tendon to the bone in any of the 3 groups. The RFPA and REPA groups had more abundant positively stained collagen fibers than the R group. Fatty infiltration was only reduced in the REPA group (Figures 8, 9, 10, and 11).

Semiquantitative Histological Score

The histological score increased in a time-dependent manner in all groups. At 4 weeks, the histological score was significantly higher in the RFPA and REPA groups than in the R group; however, it did not significantly differ between the REPA and RFPA groups. At 8 and 12 weeks, both the REPA and the RFPA groups exhibited significantly higher histological scores than the R group. The REPA group also had a significantly higher histological score than the RFPA group at 8 and 12 weeks (Figure 12A).

Fatty Infiltration

At 4, 8, and 12 weeks, the degree of fatty infiltration was significantly lower in the REPA group than in the RFPA and control groups. The amount of fatty infiltration did not significantly differ between the RFPA and control groups at any time point (Figure 12B).

Biomechanical Analysis

At each time point, the cross-sectional area of the tendon at the repair site did not significantly differ between the 3 groups (Figure 13A).

The ultimate load to failure increased in a time-dependent manner in all groups. At 4 weeks, the ultimate load to failure in the RFPA and REPA groups tended to be higher than that in the R group, but this difference was not statistically significant. At 8 and 12 weeks, both the RFPA and REPA groups exhibited a significantly better ultimate load to failure than the R group; furthermore, the REPA group exhibited a significantly better ultimate load to failure compared with the RFPA group at 12 weeks (Figure 13B).

At 12 weeks, the stress in the REPA group was significantly higher than that in the R and RFPA groups (Figure 13C).

All specimens failed at the tendon-to-bone interface.

Discussion

In the present study, the MSC–Exos loaded patch was successfully fabricated using a dynamic wet-spinning system. Compared with previous studies, we believed that the patch in the present study had reliable mechanical properties that were sufficient to support the augmented repair of rotator cuff tears^5,39 (Table 2). The in vitro study showed that the MSC–Exos loaded patch had a positive effect on the proliferation and migration activities of tenocytes. In the in vivo study, we found that compared with the control group, the application of the fiber-aligned patch resulted in significantly improved healing after rotator cuff tear repair but did not improve the fatty infiltration. Furthermore, the MSC–Exos loaded patch augmentation not only improved tendon healing but also reduced fatty infiltration after surgery.

Table 2

List of Mechanical Properties of Some Different Kinds of Patches^a

Name	Application Style	Load to Failure, N	Stiffness, N/m	Stress, MPa
Allogenic patches
Human dermis⁵	Augmenting or bridging	27.85 ± 6.77	−	27.45 ± 6.55
Xenogenic patches
Porcine small intestine submucosa⁵	Augmenting or bridging	13.04 ± 0.9	−	26.7 ± 3.8
Porcine dermis⁵	Augmenting or bridging	50.26 ± 12.21	−	32.65 ± 7.43
Synthesized patches
Sponge collagen-silk scaffold³⁹	Bridging	18.09 ± 2.49	−	−
Aligned collagen-silk scaffold³⁹	Bridging	21.65 ± 2.7	−	−
Polyurethanes patch⁵	Augmenting or bridging	17.66 ± 7.55	−	11.86 ± 4.64

Data are presented as mean ± SD. Dashes indicate values not reported in this table.

It is widely established that most torn rotator cuffs are surgically treated under nonacute conditions, as the injury probably occurred months to years before the patient became symptomatic and considered surgical intervention.^21,27 During this delayed stage, the quality of the torn tendon is greatly compromised, leading to decreases in both the quality and the quantity of the contractile units.^{9-11,27,28,35} Recent studies have reported that even after a perfect repair, fatty infiltration may continue, especially in patients with chronic massive tears, and the best result is to not have an increase over the preoperative amount of fatty infiltration.⁹ Various modified fixation techniques developed in an attempt to achieve the strongest initial mechanical properties of the repair include the single row, independent double row, transosseous equivalent linked double row, double-row equivalent, and even triple-row techniques.^3,30 However, these modified surgical techniques have a limited effect on the improvement of the biochemical properties of torn tendons.

A fiber-aligned augmentation patch shares the stress at the repair site and provides a scaffold for cell ingrowth, with clinical applications already demonstrating promising results.^1,22,27,39 Loading with bioactive factors imparted bioactivity to the fiber-aligned patch. However, in the conventional electrospinning system, bioactive factors are directly dissolved in the organic solvent with other raw electrospinning materials, which may lead to the inactivation of bioactive factors.^34,39 Therefore, in the present study, WPBU—a water-soluble raw material—was used to avoid the inactivation of MSC–Exos caused by direct contact with toxic organic solvents during the co-electrospinning process. The in vitro study results showed that the proliferation and migration activities of tenocytes were significantly stronger in the MSC–Exos loaded patch group than in the fiber-aligned patch group, indicating that the bioactivity of MSC–Exos was not adversely affected during the fabrication process.

This is further supported by the results of the present in vivo study—which showed that augmented repair with a fiber-aligned patch improved both the histological and the biomechanical properties of the rotator cuff after surgery compared with single repair—while the MSC–Exos loaded patch not only improved fibrocartilage and collagen regeneration at 8 and 12 weeks but also significantly reduced fatty degeneration at 4 weeks after surgery.

Both animal and clinical studies have proved that augmented repair with a single patch enhances the outcome of routine surgery.^1,4,22,24,31 Patch augmentation repair has been proven to reduce pain, improve shoulder function scores, and reduce postoperative retear rates.^1,4,22,31 However, some poorer outcomes of patch augmentation repair have also been seen in clinical practice, especially regarding patches derived from xenografts.^{1,2,13,16,20,24} A multicenter study comparing open rotator cuff repair with augmented repair with a small intestine submucosal patch found that both groups had similar functional scores at 1 year postoperatively, while the augmented group had higher failure rates than the nonaugmented group.¹³ Several recent studies have confirmed the efficacy of synthetic grafts or allografts using the augmentation or interposition technique, but not of xenografts.^{1,2,13,16,20,24} The potential mechanism for this is that xenografts contain too much xenogeneic deoxyribonucleic acid residue to provoke a vigorous immunogenic response, leading to scar tissue formation at the repair site.^16,20

Single augmentation repair reportedly provides strong support and reduces retear rates but cannot prevent degeneration changes such as fatty infiltration, which is consistent with the present findings.^{1,4,9,22,24,31} As an innovative area of interest in the tissue-engineering field, exosomes have attracted attention in recent years. Studies have reported that exosomes show promising efficacy in wound healing, cartilage regeneration, and tendon remodeling.^{28,29,33,36,37} Sevivas et al²⁸ first studied the effect of a single injection of human MSC–Exos on muscle degenerative changes and shoulder function in a rat model of massive rotator cuff tear. Their data suggested that a single MSC–Exos injection effectively decreased the degree of muscular atrophy and fatty infiltration. The authors then fabricated an electrospun keratin membrane that was preconditioned with human MSC–Exos before implanting.²⁹ Such an exosome-preconditioned scaffold improved both the histological and the mechanical properties of the tendon-bone healing interface.²⁹ The degeneration-preventing effects of exosomes on the torn rotator cuff were also reported by Wang et al.³⁵ These previous results are consistent with the present findings that loading MSC–Exos into a fiber-aligned patch not only promoted the deposition of tenogenic tissues but also reduced the development of fatty infiltration. The basic mechanisms underlying the contribution of the MSC–Exos to improved healing are probably attributable to MSC–Exos regulated immunity effects.^6,28,29 Chen et al⁶ found that medium conditioned with human bone marrow-derived stem cells induced greater infiltration of anti-inflammatory M2 macrophages rather than proinflammatory M1 macrophages at the repair site to support the tendon-to-bone healing of the rotator cuff. Anti-inflammatory intervention is considered crucial to mitigate degenerative changes and create a mild environment for the subsequent tissue repair stage.²⁵ Furthermore, the anti-inflammatory effect of MSC–Exos represses the infiltration of lipid-laden macrophages, which may further differentiate into adipocytes that result in a fatty deposition in muscles.³⁵ This might explain the reduced fatty degeneration in the REPA group. Besides, various growth factors and micro-RNA contained in MSC–Exos also play a major role in physiological effects.^28,29,33,35

The present study had the following limitations. First, although results of the present in vitro studies indicated that the bioactivity of MSC–Exos was not adversely affected by the co-electrospinning process because it showed positive effects on the proliferation and migration activities of tenocytes, it was still difficult to directly prove that this fabrication process did not result in the inactivation of MSC–Exos. Exosomes are known to contain various bioactive components, including cytokines, growth factors, and nucleic acids, which are highly susceptible to stimulation of the physical and chemical environment and easily become inactivated during various processing procedures. However, even if some small part of their bioactive components was inactivated, the MSC–Exos could still exert positive effects on cell growth. Thus, there is a need for the development of a more sensitive method to accurately evaluate the bioactivity of MSC–Exos after processing. Second, although the chronic rotator cuff injury model was established using a 12-week timescale, the practical clinical situation varies, as most surgeries are performed on nonacute disruptions that occurred months to years before surgery. Cautions should be taken to see whether the animal model used is a human analog or not, and there is a need for further studies to standardize the appropriate timescale to establish an animal model of chronic rotator cuff tear. Third, although a 4-, 8-, and 12-week timescale was used to evaluate the effects of the MSC–Exos loaded patch on the repair of chronic rotator cuff tears in the present study, attention should also be paid to earlier stages postoperatively, as both augmenting patches and bioactive factors were able to exert their therapeutic effects in earlier stages. Fourth, the present study was only at the animal experimental stage. Caution is needed before the results are extended to clinical practice.

Conclusion

The present results suggest that loading MSC–Exos into a fiber-aligned rotator cuff patch enhanced the repair of chronic rotator cuff tears by improving both the histological and the biomechanical properties by providing mechanical support and bioactive stimulation.

Supplemental Material

sj-pdf-1-ajs-10.1177_03635465221096490 – Supplemental material for Loading Mesenchymal Stem Cell–Derived Exosomes Into a Traditionally Designed Rotator Cuff Patch: A Potential Strategy to Enhance the Repair of Chronic Rotator Cuff Tear Associated With Degenerative Changes

Supplemental material, sj-pdf-1-ajs-10.1177_03635465221096490 for Loading Mesenchymal Stem Cell–Derived Exosomes Into a Traditionally Designed Rotator Cuff Patch: A Potential Strategy to Enhance the Repair of Chronic Rotator Cuff Tear Associated With Degenerative Changes by Xuancheng Zhang, Zhengzhe Han, Kang Han, He Zhang, Jinghuan Huang, Xiaoqiao Huangfu and Jinzhong Zhao in The American Journal of Sports Medicine

Footnotes

Submitted October 7, 2021; accepted March 16, 2022.

The authors declared that they have no conflicts of interest in the authorship and publication of this contribution. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto.

ORCID iD

Kang Han

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