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. 2024 Jul 31;14:17632. doi: 10.1038/s41598-024-67572-y

Effect of magnetic microbeads on sustained and targeted delivery of transforming growth factor-beta-1 for rotator cuff healing in a rat rotator cuff repair model

Jeongkun Lee 1, Jinwoo Park 2, Yeongjun Chang 2, Jong Pil Yoon 3, Seok Won Chung 1,✉
PMCID: PMC11292015  PMID: 39085278

Abstract

Structural failure is a well-established complication of rotator cuff repair procedures. To evaluate the effect of magnetic microbeads, designed for precise drug delivery via magnetic force, on sustained transforming growth factor-beta-1 (TGF-β1) release and rotator cuff healing in a rat rotator cuff repair model. TGF-β1 laden microbeads were prepared, and baseline in vitro experiments included the magnetization of the microbeads and TGF-β1 release tests. In an in vivo experiment using a rat rotator cuff repair model on both shoulders, 72 rats were randomly assigned to three groups (24 per group): group A, conventional repair; group B, repair with and simple TGF-β1 injection; and group C, repair with magnet insertion into the humeral head and TGF-β1 laden microbead injection. Delivery of TGF-β1 was evaluated at 1 and 7 days after the intervention using PCR, Western blot, and immunohistochemistry. At 6 weeks post-intervention, rotator cuff healing was assessed using biomechanical and histological analysis. The in vitro experiments confirmed the magnetization property of the microbeads and sustained delivery of TGF-β1 for up to 10 days. No difference in the TGF-β1 expression was found at day 1 in vivo. However, at day 7, group C exhibited a significantly elevated expression of TGF-β1 in both PCR and Western blot analyses compared to groups A and B (all P < 0.05). Immunohistochemical analysis revealed a higher expression of TGF-β1 at the repair site in group C on day 7. At 6 weeks, biomechanical analysis demonstrated a significantly higher ultimate failure load in group C than in groups A and B (P < 0.05) and greater stiffness than in group A (P = 0.045). In addition, histological analysis showed denser and more regular collagen fibers with complete continuity to the bone in group C than in groups A and B, a statistically significant difference according to the semi-quantitative scoring system (all P < 0.05). The use of the TGF-β1 laden magnetic microbeads demonstrated sustained delivery of TGF-β1 to the repair site, improving rotator cuff healing.

Keywords: Magnetic microbead, TGF beta 1, Rotator cuff tear repair, Sustained drug delivery

Subject terms: Biotechnology, Medical research

Introduction

Rotator cuff tear is a common condition that causes severe pain and disability.1–3. The prevalence of rotator cuff tear in the general population is reported to range from 20.7 to 22.2%, with the rate increasing with age4–6. Surgical repair is commonly performed to treat rotator cuff tears, aiming to achieve safe biologic tendon-bone healing7–9. Although surgical techniques and instruments have improved considerably, the failure rate of rotator cuff repair remains high and an unresolved problem10–12. Thus, the use of biological materials, such us stem cells, atelocollagen, platelet-rich plasma, and growth factors has been explored as novel ways to improve healing5,13,14. Transforming growth factor-beta-1 (TGF-β1) is a protein that performs various cell functions, including the control of cell growth and differentiation15.

The sustained and targeted delivery of different biological materials remains a challenge. Previous studies have evidenced that TGF-β1 is a key mediator in healing, promoting collagen production, angiogenesis, and tissue formation16; moreover, it has a positive effect on rotator cuff healing by increasing the synthesis of collagen and proteoglycan and stimulating cell proliferation17. Generally, local injections of various growth factors, platelet concentrates, and stem cells directly into the target site have been used to improve tendon healing18,19. However, it is challenging to accurately deliver the biological materials to the desired site through this conventional method, and the injected materials may be degraded over time20. Although biomaterials such as alginate, collagen gels, sponges, and various absorbable constructs have been used for sustained delivery21, these delivery methods have the disadvantages of being cumbersome, time-consuming, and have an increased risk of infection22,23.

Microbeads made from naturally occurring polymers have the potential to be drug delivery vehicles that can be administered by simple injection. In medical applications, microbeads are defined as spherical capsules or solid particles with a drug coated on the surface or encapsulated inside a core.24 Beads have a high surface to volume ratio, which enables encapsulation of large amounts of drug, allows for uniform distribution and sustained release of the drug25, and is one of the most widely studied carriers for various types of drug delivery26. A method to keep the microbeads in the desired area by adding an iron oxide component to the microbead configuration and driving the beads using a magnet has been studied27. If the driving microbead structure contains biostable iron oxide, magnetic force can be used to keep the beads in the target site28. This kind of magnetic microbeads as a scaffold may be good option for the sustained and targeted delivery of drugs, expecting a biological effect of a delivered drug on the target site, by a simple injection method. The biological effect of a TGF-β1 delivered to the rotator cuff repair site would be an increased rotator cuff healing by promoting collagen production, angiogenesis and tissue formation.

However, to the best of our knowledge, no study has evaluated the effects of magnetic microbeads on rotator cuff healing. Therefore, the objective of this study was to evaluate the effect of magnetic microbeads on the sustained and targeted delivery of TGF-β1 and rotator cuff healing using a rat rotator cuff repair model.

Methods

All animal experiments were approved by the Institutional Animal Care and Use Committee of the Senior Authors’ Institute (Konkuk University IACUC) (No. KU21235-1) and conducted according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health(This study is reported in accordance with ARRIVE guidelines).

Preparation of the TGF-β1 laden microbead

The magnetic microbead(Biot Korea Inc., Republic of Korea) is an carboxydextran-coating iron oxide (Ferucarbotran), sodium alginate (ALG) and sodium hyaluronate (HA) based microbead made by electrospinning method, and it has characteristics of being magnetic and ability to contain a drug in the alginate/hyaluronate structure. Mix HA solution-ALG solution-Ferucarbotran (weight ratio of 11.0:7.3:4.0) at room temperature for 1 h at 200 rpm using a Programmable Ball Mill. Disperse with a sonicator for 5 min at 40 °C. Drop into 1.5 kg of 0.99% CaCl2 solution at 110 rpm with Frequency (2200 Hz), Electrode (1000 V), Heating (70 °C), Nozzle hole size (150 μm), and drop distance (12.5 cm) (Fig. 1). The dropped beads are cured using a 200 rpm Programmable Ball Mill at room temperature for more than 16 h. Washing is repeated 3 or more times for 5 min using a 150 μm sieve and 5 L of ultrapure water. Freeze in an ultra-low temperature freezer at − 65 °C or lower for more than 2 h. Frozen beads are freeze-dried in a freeze dryer at temperatures below − 45 °C and below 100 mTorr for more than 72 h. After freeze-drying is complete, the beads are recovered and stored in a desiccator. Microscope images were taken to confirm the morphology and size of the microbead. The prepared microbeads were observed using a Microscope (KI3000, OPTINITY) at ×20 magnifications. To contain the TGF-β1 in this prepared magnetic microbead, we soaked the microbead in saline containing TGF-β1 dose of 400 ng/mL before use.

Figure 1.

Figure 1

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Schematic image of microbeads fabrication. Preparation of magnetic microbeads by using electrospinning technology. Disperse with a sonicator and drop into CaCl2 solution at 110 rpm with Frequency (2200 Hz), Electrode (1000 V), Heating (70 °C), Nozzle hole size (150 μm), and drop distance (12.5 cm).

Sustained release of TGF-β1 in magnetic microbeads

To confirm the capacity of the sustained release of TGF-β1 contained in the magnetic microbeads, we performed an in vitro experiment for sustained release in advance of the in vivo experiments. The magnetic microbeads (Biot Korea Inc., Republic of Korea) carrying 400 ng of TGF-β1 were incubated in 100 mL of deionized water at 4 °C for 10 days. The 400 ng/mL TGF-β1 dose has previously demonstrated to significantly enhance rotator cuff healing in animal models29,30. At various time points (0, 1, 3, and 6 h; and 1, 3, 6, 8, and 10 days) a 0.2-mL aliquot of incubation liquid was collected and stored at -20 °C for 10 days30. After 6 h and 10 days, all aliquots were analyzed using an enzyme-linked immunosorbent assay (ELISA) kit (Human LAP TGF-β1 Quantikine ELISA Kit; R&D Systems) to measure the resealed amount of TGF-β1.

Animal model and procedure

We used 72 Sprague Dawley rats (Orient Bio Inc., Seongnam Gyeonggi, Republic of Korea) aged 10 weeks with a mean body weight of 250 g: 48 for evaluating the TGF-β1 delivery and 24 for evaluating the effect of the TGF-β1 magnetic microbead on the rotator cuff repair model. Power analysis indicated that eight specimens per group were required to detect a significant difference in the ultimate load to failure at 6 weeks after repair (comparison between the two groups): mean difference, 7 N; standard deviation, 5 N; error = 0.05, b error = 0.2, 2-tailed comparison31. Accordingly, 8 rats per group for 6 weeks were required to evaluate the effect of TGF-β1 magnetic microbeads on rotator cuff repair, and the same number was used to evaluate TGF-β1 delivery on days 1 and 7.

The rats were acclimatized to a 12-h light–dark cycle at 22 °C with free access to food and drinking water. Surgical procedures were performed under anesthesia using an intraperitoneal injection of Zoletile 30 mg/kg (Virbac, Carros, France) and Rompun 10 mg/kg (Bayer Korea Ltd., Seoul, Korea). Subsequently, the shoulders of each rat were shaved and prepared in the usual sterile surgical technique. A 3-cm lateral skin incision was made on both shoulders, and the deltoid muscle was retracted to expose the supraspinatus tendon at its insertion into the greater tuberosity. The supraspinatus tendon was severed from the greater tuberosity by using a sharp scalpel. Using a motor drill, two bone tunnels were created at the articular margin of the footprint to the lateral humeral cortex. A suture (3–0 Ethilon; Ethicon Inc., Johnson & Johnson, Belgium) was passed through the bone tunnels and tied, reattaching the supraspinatus tendon to the footprint.

Seventy-two rats were randomly allocated to three groups (24 rats per group). Group A served as the control group and no additional procedures were performed after supraspinatus repair (repair only). In group B, we injected 400 ng of TGF-β1 in the repair site after supraspinatus repair. In group C, we injected TGF-β1 laden magnetic microbeads in the repair site after supraspinatus repair surgery. In Group C, a micro-magnet was implanted into the humeral head near the greater tuberosity to attract the magnetic microbeads to the repair site (Fig. 2). The experimental flowchart is illustrado in Fig. 3.

Figure 2.

Figure 2

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Surgical procedure for group C. After the supraspinatus tendon was cut at the insertion site (A), a magnet is inserted into the humeral head (B). After rotator cuff tear repair (C), TGF-β1 laden magnetic microbeads are applied locally at the repair site (D).

Figure 3.

Figure 3

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Experiment flowchart. At 1 and 7 days after the procedure, qRT PCR and western blot analyses were performed using the supraspinatus tendon of the left shoulder and immunohistochemical analysis was performed on the en-bloc bone-to-tendon repair site of the right shoulder (n = 8 for each group). At 6 weeks after the procedure, biomechanical analysis was performed on the en-bloc bone-to-tendon repair site of left shoulder and histological analysis was performed on the en-bloc bone-to-tendon repair site of the right shoulder (n = 8 for each group).

At 1 and 7 days after surgery (8 rats per group), the supraspinatus tendon of the left shoulder and the en bloc bone-to-tendon repair site of the right shoulder were harvested, PCR and Western blot analyses were performed using the supraspinatus tendon of the left shoulder, and immunohistochemical analysis was performed in the en bloc bone-to-tendon repair site of the right shoulder. Six weeks after surgery (8 rats per group), the en bloc bone-to-tendon repair sites of both shoulders were harvested, biomechanical analysis was performed on the left shoulder, and histological analysis was performed on the right shoulder.

Quantitative reverse transcription PCR: qRT PCR

To evaluate gene expression, Quantitative Reverse Transcription PCR (qRT-PCR) was performed on the TGF-β1 gene. Total RNA was extracted from the isolated deltoid muscles using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions, and cDNA was generated using the Maxime RT Premix Kit (iNtRON Biotechnology). qRT-PCR was performed on a Light Cycler 480 System (Roche Diagnostics) using 2X qPCR BIO SyGreen Mix Lo-ROX (PCR Biosystems). All data were normalized to glyceraldehyde3-phosphate dehydrogenase (GAPDH) expression levels. The primer sequence of TGF-β1 is (F) 5′-ATGCACACTGGTGCAGAGAG-3′ (R) 5′-′TGTAAGCACACAGGCAGGTC-3.

Western blot analysis

To assess protein expression, the rats were euthanized in a CO2 vacuum cage, and the entire supraspinatus tendon of each rat was harvested and lysed in 20 mm Tris–HCl buffer (pH 7.4) containing a protease inhibitor mixture (0.1 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml pepstatin A, and 1 μg/ml chymostatin) and phosphatase inhibitors (5 mm Na3VO4 and 5 mm NaF) for 1 h. Subsequently, cell lysates were centrifuged at 13,200 × g for 5 min at 4 °C for extraction. The concentration of the extracted protein was measured using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Equal amounts of protein (30 μg) were loaded in each well of 9% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. The transferred membranes were blocked with 5% non-fat dry milk in Tris-buffered saline for 1 h at room temperature and incubated with primary antibody overnight at 4 °C. Each primary antibody was diluted 1:1000 with 1% bovine serum albumin. Antibodies used in this study were GAPDH, TGF-β1 (catalog no. 25778, 514302, respectively; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Subsequently, membranes were incubated with anti-rabbit or anti-mouse secondary antibodies for 1 h. Each secondary antibody was diluted 1:5000 with 5% nonfat dry milk in Tris-buffered saline. Finally, membranes with proteins were treated with enhanced chemiluminescence solution and exposed to X-ray film. The band intensities developed on the film were quantified using ImageJ software (NIH, Bethesda, MD, USA).

Immunohistochemistry

Immunohistochemistry was performed on the en bloc bone-tendon repair sites of the right shoulder to determine local expression of the target protein TGF-β1. For immunohistochemical analysis, 5-mm paraffin-embedded tissue sections were prepared, deparaffinized in xylene, and rehydrated in an ethanol series. Antigen retrieval was performed using citrate buffer (pH 6.0). The slides were incubated with primary antibodies for 1 h at room temperature, washed three times with phosphate-buffered saline, incubated with the corresponding secondary antibody conjugated to horseradish peroxidase for 30 min at room temperature, and washed with phosphate-buffered saline three times. Immunohistochemical staining was conducted using anti-TGF-β1 (Abcam, Cambridge, UK) on each slide. All slides were evaluated under an Eclipse Ni-U microscope (Nikon), and images were acquired using a DS-Ri1 camera (Nikon) and analyzed using NIS Elements (v 4.0; Nikon). Supraspinatus tendon repair site analysis was performed at ×20 and ×100 magnifications under a microscope.

Biomechanical analysis

Six weeks after the repair, the entire supraspinatus tendon of both shoulders with the humeral head of each rat was harvested, and the left shoulder was used for biomechanical analysis. The mechanical evaluation parameters were stiffness and load to failure (maximum load that could withstand external forces until failure, measured at a rate of 10 mm/s with a preload of 20 kg using a custom fixture clamping system and a universal test machine (Shimadzu AGS-X 500N, Japan). The supraspinatus tendon was wrapped with a cotton gauze sponge to minimize slippage and fixed to this system along its anatomical direction to allow tensile loading and the tendon-to-bone interface to form a right angle. The data were automatically collected using a personal computer-based data acquisition system.

Histological analysis

Six weeks after surgery, all right shoulder specimens from each group were fixed in 10% deionized buffered formalin (pH 7.4), decalcified, and paraffin blocks containing the repair site were prepared. Sections (4-µm thick) were cut in the coronal plane and stained with hematoxylin and eosin. Specimen slides were evaluated for fiber continuity, tissue cellularity, and collagen organization at the tendon-to-bone interface. The assessment included orientation and density of the collagen fibers, maturation of the tendon-to-bone interface structure, and cellularity. Each item was graded semi-quantitatively (0, 1, 2, or 3). For collagen fiber continuity and orientation (i.e., collagen fibers oriented in parallel), we divided their grades by percentages as follows: < 25%, 25–50%, > 50–75%, and > 75%, which corresponded to grades 0, 1, 2, and 3, respectively. Collagen fiber density was graded as very loose, loose, dense, and very dense, corresponding to grades 0, 1, 2, and 3, respectively30. The maturation of the tendon-to-bone interface structure was graded as poorly organized, mildly organized, moderately organized, and markedly organized, corresponding to grades 0, 1, 2, and 3, respectively. Cellularity was graded as none or minimally present, mildly present, moderately present, or markedly present, corresponding to grades 0, 1, 2, and 3, respectively (Table 1). To eliminate observer bias, all examinations were performed in a randomized fashion by a pathologist with 7 years of training (J.Y.K.) who was blinded to the group assignment. An Eclipse Ni-U microscope (Nikon) was used for histological analysis at ×100 magnification. The histological analysis was performed with referring to a previous study29.

Table 1.

Histological grading for the tendon-to-bone interface.

0 1 2 3
Cellularity No/Minimal Mild Moderate Marked
Continuity  < 25% 25–50% 50–75%  > 75%
Orientation  < 25% 25–50% 50–75%  > 75%
Density Very loose Loose Dense Very dense
Maturation Poor Mild Moderate Marked
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Statistical analysis

Data were evaluated using SPSS software (version 20.0; IBM Corp., Armonk, NY, USA). A Kruskal–Wallis test was performed to determine whether there were differences between groups, followed by a post-hoc Mann–Whitney U test with Bonferroni correction for multiple comparisons. Data were expressed as the mean ± standard error, and P < 0.05 was considered statistically significant.

Results

Magnetization test of magnetic microbeads and analyze uniform shape and spherical morphology

Electromagnetic tests were performed to confirm the magnetization of the magnetic microbeads. Magnetization was confirmed through the detection of magnetic microbeads that moved according to the direction of the external magnetic force (Fig. 4). Microscopic observations were conducted to analyze the morphology and size of the magnetic microbeads. Uniform shape and spherical morphology were observed (Fig. 4D).

Figure 4.

Figure 4

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Photographs showing the responses of magnetic microbeads to the external magnetic attraction. Initial state (A), magnetic force direction left (B), and magnetic force direction right (C). Optical microscope image of the magnetic microbeads (D). Magnification: ×20 original. Scale bar, 500 µm.

Confirmation of TGF-β1 sustained release in vitro

The level of the TGF-β1 release from the microbeads was measured using ELISA and a 450-nm ultraviolet spectrophotometer at numerous time points. Sustained release of TGF-β1 up to 6 h and 10 days was observed (Fig. 5). These results may suggest that TGF-β1 would have the possibility of being continuously released to the repair site when the magnetic microbead-based delivery system is applied in-vivo as well.

Figure 5.

Figure 5

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Sustained release of TGF-β1 in the magnetic microbead.

Biomolecular analysis: qRT-PCR analysis showed higher expression of TGF-β1 in group C

TGF-β1 gene mRNA levels were evaluated using qRT-PCR analysis at days 1 and 7. Although there were no differences between groups at day 1, at day 7, we found a significantly higher expression of TGF-β1 in group C compared with that in groups B (P = 0.011) and A (P = 0.003) (Fig. 6).

Figure 6.

Figure 6

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Ratios of the relative mRNA expression level for days 1 (A) and 7 (B). Results are the means of three independent experiments (bars represent SEM). *P < 0.05, **P < 0.01.

Biomolecular analysis: Western blot analysis showed higher expression of TGF-β1 in group C

TGF-β1 protein levels were evaluated using Western blot analysis at days 1 and 7. Similar results were obtained by qRT-PCR. Even though there was no significant difference between groups at day 1, we observed a significantly higher expression of TGF-β1 in group C compared to that in groups B (P = 0.027) and A (P = 0.005) at day 7 (Fig. 7).

Figure 7.

Figure 7

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The TGF-β1 protein levels by Western blot analysis for days 1 (A) and 7 (B). Each value is illustrated as a ratio of the relative protein level. Results are the means of three independent experiments (bars represent SEM). *P < 0.05, **P < 0.01.

Biomolecular analysis: immunohistochemical analysis showed higher staining intensity of TGF-β1 at day 7

We performed immunohistochemistry for TGF-β1. The expression of TGF-β1 at the repair site was not evident in all groups at day 1. However, a higher staining intensity at the repair site was observed in group C than in groups B and A on day 7 (Figs. 8 and 9).

Figure 8.

Figure 8

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An example of group C immunohistochemical analysis, (A) day 1, (B) day 7. The red squares indicate the supraspinatus tendon repair site. The blue arrows indicate the location where the magnet was inserted. Detection using anti-TGF-β1. Magnification: ×20 original. Scale bar, 50 µm.

Figure 9.

Figure 9

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Immunohistochemical analysis of the supraspinatus tendon repair site at day 1 (A, B, and C) and day 7 (D, E, and F) between groups (Left, group A; Middle, group B; and Right, group C). Detected using anti-TGF-β1. Magnification: ×100 original. Scale bar, 100 µm.

Evaluation of the rotator cuff healing: biomechanical analysis showed higher score in group C

In the biomechanical evaluation at 6 weeks after surgery, the stiffness in group C was significantly higher than in group A (P = 0.045). In addition, the ultimate failure load of group C was significantly higher than that of group B (P = 0.016) as well as the group A (P = 0.005) (Fig. 10).

Figure 10.

Figure 10

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Results of the biomechanical analysis. Results are the means of three independent experiments (bars represent standard error of the mean). *P < 0.05, **P < 0.01.

Evaluation of the rotator cuff healing: histological analysis showed higher score in group C

Histological analysis showed denser and regularly oriented collagen fibers with complete continuity to the bone in group C compared to groups A and B. In the semi-quantitative scoring system comparing cellularity, collagen fiber continuity, orientation, density, and repair site maturation, the differences were statistically significant (Fig. 11 and Table 2).

Figure 11.

Figure 11

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Histology at the repair site in hematoxylin and eosin stain (A, group A; B, group B; C, group C). Magnification: ×100 original. Histology at the repair site in Masson trichrome stain (D, group A; E, group B; F, group C). Magnification: ×100 original. (G) The graph shows the difference of the histology scores between groups. *P < 0.05, **P < 0.01, ***P < 0.001.

Table 2.

Results of semi-quantitative histological grading for rotator cuff tendon-to-bone healing.

A B C P value
Cellularity 0.7 ± 0.4 1.6 ± 0.4 2.5 ± 0.7a  < 0.001
Continuity 0.5 ± 0.3 1.8 ± 0.5 2.9 ± 0.5a,b  < 0.001
Orientation 0.7 ± 0.3 1.9 ± 0.7 2.2 ± 0.6a 0.002
Density 0.5 ± 0.2 1.5 ± 0.5 2.8 ± 0.8a 0.002
Maturation 0.5 ± 0.3 1.9 ± 0.8 2.1 ± 0.7a 0.003
Total score 3.3 ± 1.7 8.7 ± 1.7 12.5 ± 1.3a,b  < 0.001
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Values are presented as mean ± SD. Comparisons between groups were performed using the Kruskal–Wallis test.

aSignificant difference between A (Repair) and C (TGF + bead) groups (P < 0.05).

bSignificant difference between B (TGF) and C (TGF + bead) groups (P < 0.05).

Discussion

In this study, we demonstrated the therapeutic effectiveness of magnetic microbeads in the sustained and targeted release of TGF-β1 at a desired repair site with enhanced healing. Among various growth factors, TGF-β1 has been of interest due to its previously proven effectiveness in enhancing rotator cuff healing29,30. However, the effectiveness of TGF-β1 may be limited when administered through a single injection due to its short half-life, in vivo instability, and challenges in accurately reaching the target site32. Consequently, various scaffolds, such as collagen gels and sponges, have been used for the sustained delivery of growth factors to the target site33,34. However, the reliability of growth factors delivery to the tendon through the scaffold may still be uncertain, necessitating additional efforts to affix the scaffold to the repaired tendon30. The delivery of growth factors through scaffolds has the inherent disadvantage of being feasible only during the surgery, which can be cumbersome, time-consuming, and pose an increased risk of infection22,23. In this study, we used magnetic microbeads loaded with TGF-β1, delivering the growth factor to the repair site through a simple injection. This method offers the advantage of sustained and targeted delivery of the growth factor.

The microbeads used in this study contained alginate and hyaluronic acid, which are known to effectively carry and release various growth factors35, and additionally contained iron oxide so that the microbeads can be delivered and fixed to the desired site using magnetic force (magnetic microbeads). Iron oxide use in microbeads is an FDA-approved biosafe material that is used as a component of contrast agents36,37. The magnetic property of the microbeads was proven in a previous study, evidencing targeted movement of the magnetic microbeads to knee cartilage lesions38. In the in vitro magnetization test for our study, we confirmed that the microbeads were well magnetized through the microbeads movement toward the outside magnet. The magnetic microbeads should not easily move from the target repair site and have sustained release of TGF-β1, improving rotator cuff healing. Our in vivo results evidenced enhanced expression of TGF-β1 at the repair site in the group C on day 7, suggesting sustained and targeted delivery of TGF-β1 by the magnetic microbeads. On day 1, we could not find differences in the expression of TGF-β1 between groups, which suggests that 1 day was not enough time for the growth factor to be sufficiently absorbed to the repair site. In immunohistochemical analysis, we confirmed that TGF-β1 was present at both the repair interface and tendon substance, where growth factors may directly stimulate cell differentiation and matrix synthesis39,40. The vascularization or cell infiltration within the tendon may be accelerated by TGF-β1 released from the magnetic microbeads, improving the healing process41. We think the prolonged time of the magnetic microbeads at the repair site due to the magnetic force minimizes the loss of TGF-β1, allowing maximum TGF-β1 transfer to the repair site and enabling efficient and improved tissue healing42.

Magnetic microbead-laden growth factors offer several advantages. They can carry various types of growth factors and therapeutic drugs in the alginate and hyaluronic acid components and can be applied to various areas, including the shoulder joint we studied. We believe that it will be possible to use magnetic microbeads for the treatment of rotator cuff disease, cartilage diseases, cancers, and other diseases that require effective targeted drug delivery43. Magnetic microbeads can be easily delivered to the desired area through injection. Scaffolds for delivering growth factors or drugs can only be used during surgery; however, magnetic microbeads can be used in outpatient clinics without surgery44,45. However, a magnet is needed for localization; in our experiment, it was inserted into the humeral head near the greater tuberosity of the animals. Because it is still not possible to insert a magnet into human bones, there has been ongoing research into the development of orthoses employing external magnets to concentrate the magnetic field at the target site46,47. Thus, to implement magnetic microbeads in humans, the development of magnet-mounted braces or guidance devices is required38.

Our study is the first to evaluate the effect of the TGF-β1-laden magnetic microbeads on sustained and targeted delivery to the repair site and rotator cuff healing in a rat rotator cuff repair model. This study has some limitations. First, as this was an animal study, differences in the anatomical features, reactions to injury, and immobilization between humans and rats limit the generalization of the results. Second, shoulder trauma and surgical procedures are commonly encountered in older patients. This study was conducted in young rats; therefore, comparing the results with those of older rats may be necessary. Third, other additional biomechanical tests such as gait analysis or staircase test were not performed, which may strengthen the conclusion of this study. Fourth, 6 weeks of evaluation for rotator cuff healing may not have been sufficient, and further long-term studies are needed to confirm our results. Fifth, those conducting the biomechanical testing were not blinded, unlike those conducting the histological analyses; this may have decreased the reliability of the biomechanical results. Sixth, the in vivo release kinetic properties are unknown and are likely to differ from those in the in vitro environment. Finally, other growth factors and extracellular matrix proteins not analyzed in this study may be involved in the rotator cuff healing process, and the molecular pathways by which TGF-β1 affects rotator cuff tendon-to-bone healing were not identified. TGF beta target genes such as Snail or Twist would be beneficial to strengthen the results; therefore, this should be further researched.

Conclusion

We demonstrated the therapeutic effectiveness of TGF-β1 laden magnetic microbeads in the sustained and targeted release of TGF-β1 at a desired repair site with enhanced rotator cuff healing in a rat rotator cuff repair model. Considering the effectiveness and convenience of its use, the findings of this study may provide another possible solution for the treatment of rotator cuff tears.

Supplementary Information

Supplementary Information. (931KB, zip)

Acknowledgements

The authors would like to thank all those who helped conduct this study.

Author contributions

All authors contributed to the study’s conception and design. J.L., J.P., Y.C., J.P.Y., and S.W.C. performed material preparation, data collection, and analysis. J.L. wrote the first draft of the manuscript. All authors commented on previous versions and read and approved the final manuscript.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (No. RS-2023-00249219 and NRF-2022R1A6A3A01087378).

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-67572-y.

References

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Data Availability Statement

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

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