Sr/MOFs were synthesized using a previously reported method (17). SrCl₂ · 6H₂O (0.58 g; 2.2 mmol), H₂BDC (0.32 g; 1.93 mmol) and 56 ml dimethylformamide (DMF) were mixed together and stirred. This solution was then stored in a polytetrafluoroethylene-lined stainless-steel container at 100°C for 2 days. After cooling to room temperature, the synthesized MOFs were filtered, washed with DMF and vacuum-dried at 60°C.

An SF hydrogel was prepared as described previously (18). A silkworm cocoon was boiled in 2 l 0.02 M sodium carbonate solution for 1 h, and subsequently boiled in 1 l distilled water for 30 min. The cocoon was then rinsed with fresh distilled water and dried at room temperature in a culture dish for 2 days. Purified SF was dissolved in 9.3 M lithium bromide at 70°C for 1 h, which was then filtered and dialyzed with distilled water at room temperature for 48 h. After being concentrated with polyethylene glycol (20,000 g/mol), the dialysis tube was cleaned and SF was collected and stored at 4°C. SF hydrogel was prepared by mixing 1 ml SF solution, 100 µl HRP solution (Beijing Solarbio Science & Technology Co., Ltd.) and 65 µl hydrogen peroxide (H₂O₂) solution (Shanghai Aladdin Biochemical Technology Co., Ltd.).

Sr/MOF morphology was characterized via field emission scanning electron microscopy (SEM; Sigma 500; Zeiss GmbH) after being sputter-coated with a thin layer of gold (19). High angle annular dark field scanning transmission electron microscopy combined with energy dispersive spectroscopy was used for elemental mapping (20). The powder samples were dispersed in ethanol via ultrasonication (40 kHz for 5 min at 25°C) and drop-cast onto a carbon-coated copper grid prior to observation. The X-ray diffraction (XRD) pattern was recorded on an equipped X-ray diffractometer (D8 Discover; Bruker Corporation) (21). To determine the optimal MOF concentration, a series of MOF suspensions with varying concentrations (200, 300, 400, 500, 600, 700 and 800 µg/ml) were prepared. The concentration of Sr from these different groups was evaluated at predetermined time points (days 1, 4, 7, 14 and 21) using inductively coupled plasma mass spectrometry (ICP-MS) (iCAP RQ; Thermo Scientific; Thermo Fisher Scientific, Inc.) (22).

A total of 30.5 mM (8.2 g) Aln was dissolved in 50 ml methanol. Subsequently, 2 ml Aln solution (1.22 mM) was mixed with 100 mg prepared Sr/MOFs and placed on a magnetic stirrer for 48 h (37°C; 600 rpm) (23). The stirred mixture was then transferred to a centrifuge and centrifuged for 10 min at 12,000 × g at room temperature. This was followed by the removal of the supernatant to obtain the centrifuged product. After repeated washing with deionized water and methanol, the reactants were dried overnight in a vacuum freeze dryer to remove the solvent and subsequently stored at room temperature for further experiments. The success of Aln loading was confirmed via high-performance liquid chromatography (HPLC) (1260 LC system; Agilent Technologies, Inc.). The chromatographic separation was performed at a column temperature of 40°C using an ACQUITY UPLC HSS T3 column (1.8 µm, 2.1×100 mm; Waters Corporation). The mobile phase consisted of solvent A (10 mM ammonium acetate containing 0.1% formic acid) and solvent B (acetonitrile) at a flow rate of 0.300 ml/min, with a sample injection quantity of 10.0 µl. A total of 400 µg/ml Sr/MOF-Aln was dissolved in distilled water and HPLC analysis was performed at different time points (0, 4, 14, 15 and 18 min) to detect the sustained release of Aln.

Following induction of anesthesia with 4% isoflurane, which was maintained with 2% isoflurane, 6 female Sprague Dawley (SDa) rats (age, 3–4 weeks; weight, 40–60 g; Charles River Laboratories, Inc.) were euthanized by cervical dislocation. The animals were housed in a controlled environment with a temperature of 22±2°C, relative humidity of 50±10% and under a 12-h light/dark cycle. The rats were provided ad libitum access to standard rodent chow and water throughout the duration of the study. Primary rat BMSCs were subsequently isolated from the femoral and tibial bone marrow cavities of euthanized rats (24). BMSCs were cultured in a complete culture medium comprising DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.) and 10% fetal bovine serum (Wisent Biotechnology). BMSCs were cultured in an incubator at 37°C with 5% CO₂ and the culture medium was changed every 3 days. The BMSCs were obtained for the present study only.

Firstly, 96-well plates were pre-coated with 50 µl/well of the respective hydrogels (SF, SF + Sr/MOF and SF + Sr/MOF-Aln) and incubated at 37°C for 24 h. Subsequently, the wells were washed with a PBS solution. Primary BMSCs (passage 3) were then harvested, and a total of 1×10³ BMSCs were added to each well corresponding to the different treatment groups and cultured at 37°C in a 5% CO₂ incubator. The culture medium was changed on days 1, 3 and 5. Subsequently, the original culture medium was discarded and the cells were incubated with culture medium containing 10% Cell Counting Kit-8 (CCK-8) solution (Abbkine Scientific Co., Ltd.) for 1 h at 1, 4 and 7 days of culture. Following this incubation, 100 µl culture medium was taken from each well and added to a new 96-well plate, after which, optical density values were detected using a microplate reader (Model 680; Bio-Rad Laboratories, Inc.) at 450 nm absorbance (25).

BMSCs were seeded into 96-well plates, which were pre-coated with SF hydrogel at 37°C for 24 h, at a density of 1×10³ cells/well. After 48 h of incubation at 37°C, the cell survival rate was measured using the Calcein AM/PI dual staining kit (cat. no. C2015S; Beyotime Biotechnology) according to the manufacturer's instructions. The culture medium was replaced with a working solution and incubated at 37°C with 5% CO₂. Subsequently, an inverted fluorescence microscope (MF53-N; Guangzhou Micro-shot Technology Co., Ltd.) was used to observe the cells and capture images.

BMSCs were seeded into 96-well plates, which were pre-coated with SF hydrogel at 37°C for 24 h, at a density of 1×10³ cells/well. Subsequently, 100 µM H₂O₂ was added to the culture medium and cells were stimulated at 37°C for 24 h, whereas cells in the control group were cultured in normal medium without H₂O₂ treatment. Cells were incubated with a 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe (Beyotime Biotechnology) at 37°C in 5% CO₂ for 20 min and ROS levels were measured according to the manufacturer's instructions. Fluorescence images were obtained for each group using an inverted fluorescence microscope (MF53-N; Guangzhou Micro-shot Technology Co., Ltd.).

To determine whether MOF-based hydrogels promoted osteogenesis, alkaline phosphatase (ALP) activity (at weeks 1 and 2) and extracellular-matrix mineralization (at weeks 2 and 3) were evaluated using ALP (cat. no. P0321S; Beyotime Biotechnology) and Alizarin Red staining kits (cat. no. C0148S; Beyotime Biotechnology), respectively, according to the manufacturer's instructions. For quantification, a total of 100 µl of the respective reaction solution from each sample was transferred to a new 96-well plate. Subsequently, a microplate reader (Model 680; Bio-Rad Laboratories, Inc.) was utilized to measure the absorbance at 410 nm for ALP activity and 560 nm for mineralization.

BMSCs were seeded into 96-well plates pre-coated with SF hydrogel at 37°C for 7 days. Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. cDNA was synthesized from 500 ng total RNA using the PrimeScript™ RT Reagent Kit (Takara Bio, Inc.) according to the manufacturer's protocol. Subsequently, qPCR was performed using SYBR Green Master Mix (Takara Bio, Inc.) on a StepOnePlus™ Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) to detect the expression levels of runt-related transcription factor 2 (Runx2), osteocalcin (OCN), collagen type I α1 chain (COL1A1) and collagen type II α1 chain (COL2A1). The primers used are presented in Table I. The amplification protocol consisted of an initial denaturation step at 95°C for 30 sec, followed by 50 cycles of denaturation at 95°C for 5 sec and amplification at 60°C for 30 sec. GAPDH was used as the internal reference gene. All reactions were conducted in triplicate and relative gene expression levels were calculated using the 2^−ΔΔCq method and normalized to the control group (26).

A rat RCT model was established to evaluate the ability of MOF-based hydrogels to promote RCT repair in vivo according to a previously described method (27). In the present study, 48 female SDa rats (age, 4–5 months; weight, 300–350 g; Charles River Laboratories, Inc.) were randomly divided into the following four groups: i) Control group (n=12); ii) SF group (n=12); iii) SF + Sr/MOF group (n=12); and iv) SF + Sr/MOF-Aln group (n=12). This sample size was consistent with previously reported studies on rat models of RCT and TBI healing (28,29). The allocation of rats to each group was concealed using opaque envelopes to ensure that the researchers conducting the experiment were blinded to group assignments, preventing any bias in the outcome assessment.

Initially, rats were anesthetized with isoflurane (induction, 4%; maintenance, 2%). Subsequently, the deltoid muscle was exposed under sterile conditions and incised to access the supraspinatus muscle. The supraspinatus tendon was then carefully dissected from its insertion site on the humeral bone using a surgical blade. A tunnel was drilled into the greater tubercle of the humerus and, for experimental groups, the relevant hydrogel was inserted into the tunnel. Finally, the supraspinatus tendon was secured back to the greater tubercle using a non-absorbable suture. Rats in the control group underwent simple suturing of the supraspinatus tendon to the humerus without the addition of any hydrogel. Rats in the SF group were subject to suturing with the incorporation of SF hydrogel at the interface. Similarly, the SF + Sr/MOF group received suturing with the addition of the SF + Sr/MOF hydrogel at the interface, whereas the SF + Sr/MOF-Aln group were sutured with insertion of the SF + Sr/MOF-Aln hydrogel at the interface. Rats were administered aspirin (100 mg/kg; once daily postoperatively for 3 days) via oral gavage to manage pain (30).

The rats were housed in a controlled environment that was maintained at a temperature of 22±2°C, relative humidity of 50±10% and subject to a 12-h light/dark cycle. All rats were provided ad libitum access to standard rodent chow and water throughout the duration of the study. Rats exhibiting signs of notable weight loss (>20% baseline body weight), an inability to move or infection were humanely euthanized immediately. After 8 weeks, the rats were sacrificed by cervical dislocation under anesthesia, which was induced with 4% isoflurane and maintained with 2% isoflurane. Of these, 6 rats per group were allocated for micro-MRI, micro-CT and biomechanical testing, whereas samples from the remaining 6 rats per group were used exclusively for histological analysis.

Imaging of the supraspinatus tendon-bone complex was performed using a 9.4 T BioSpec 94/30 MRI scanner and positron emission tomography insert (Bruker Corporation). Image acquisition and reconstruction were processed using ParaVision software (version 6.0.1; Bruker Corporation). MRI images of each sample were independently evaluated and graded by two radiologists who were both blinded to the group allocation of the animals. MRI images of the supraspinatus tendon-bone complex were assessed using a semi-quantitative grading system, which included the following three parameters: i) Signal intensity; ii) tendon thickness; and iii) tendon retraction. The detailed grading criteria for sample signal intensity, tendon thickness and tendon retraction are provided in Table SI (31). To ensure the reliability of MRI scoring, inter- and intra-observer consistency were evaluated by calculating the intraclass correlation coefficient based on the assessments of two independent, blinded observers.

Imaging of shoulder joint samples was performed using a micro-CT system (Quantum GX2; PerkinElmer, Inc.) with a scanning resolution of 18 µm at a voltage of 50 kV and a current of 100 µA. Image reconstruction and analysis were performed using the Quantum GX2 software (version 2.0; PerkinElmer, Inc.). The parameters analyzed included bone volume/tissue volume (BV/TV) and bone mineral density (BMD), which were quantified according to a previously described method (32).

Samples of the supraspinatus tendon-humerus complex were collected from SDa rats for biomechanical testing using a universal testing machine (UTM6104; Shenzhen SUNS Technology Co. Ltd.) (27). The maximum load (N) and load deformation curve at the time of failure were recorded. The stiffness (N/mm) of each specimen was determined by the slope of the linear region of the load deformation curve until the point of maximum load failure (33). Biomechanical parameters were intentionally reported without normalization to the cross-sectional area of the samples, as successful TBI healing is typically accompanied by an increase in tissue volume; therefore, non-normalized values more accurately reflect the functional repair outcome (33).

On the day of sacrifice in week 8, supraspinatus TBI samples from each group were fixed in 4% neutral buffered formalin at room temperature for 48 h. Following dehydration, the samples underwent decalcification for ~60 days. Subsequently, specimens were vertically sectioned and embedded in paraffin wax to obtain tissue blocks with a thickness of ~5 µm using a Leica SP1600 microtome (Leica Microsystems, Inc.). Tissue sections were stained with toluidine blue at room temperature for 5 min to evaluate morphological changes in newly formed fibrocartilage (33). Additional sections were stained with hematoxylin and eosin (H&E) at room temperature for 5 min to assess the maturation of the repaired rotator cuff tissues. Images were captured using a light microscope (Leica TCS SP2; Leica Microsystems, Inc.). Finally, tendon maturation scores were determined using a modified scoring system adapted from a previous study (28). Specifically, this system evaluates five distinct histological parameters: Cellularity, vascularity, continuity, fibrocartilage and tidemark. Each parameter is graded on a scale of 0 to 4, yielding a cumulative total score with a maximum possible value of 20 for each sample.

All data in the present study were analyzed using SPSS 17.0 statistical software (SPSS, Inc.). Continuous data are presented as the mean ± standard deviation, and comparisons between groups were conducted via one-way ANOVA followed by Tukey's post hoc test. Categorical data, such as the histological maturation scores and semi-quantitative MRI scores, are presented as the median and interquartile range, and were evaluated using the non-parametric Kruskal-Wallis test followed by Dunn's post hoc test for multiple comparisons. All experiments were independently repeated three times to ensure the reliability of the results. P<0.05 was considered to indicate a statistically significant difference.

In order to obtain information regarding the microstructure of Sr/MOFs, both Sr/MOF and Sr/MOF-Aln were analyzed by SEM, as shown in Fig. 1A. Sr/MOFs were found to be rod-shaped, indicating that they exhibited notable crystalline morphology. Elemental mapping illustrated the uniform distribution of each element detected within the Sr/MOFs. When loaded with Aln, the phosphorus signal of Sr/MOFs markedly increased, indicating that the loading of Aln onto MOFs was successful (Fig. 1A). The XRD pattern of the synthesized Sr/MOFs also reflected a notable crystal structure, indicating that functional MOFs had been successfully synthesized (Fig. 1B). Upon loading with Aln, no shifts in diffraction peaks were observed. However, observed changes in the Bragg reflectance intensity ratio may have been caused by variations in interatomic distance and bond angle or the preferential orientation of Aln particles. The ICP-MS results demonstrated a general concentration-dependent increase in Sr ion release as the Sr/MOF concentration rose from 200 to 700 µg/ml. For visual conciseness, only the statistical comparisons between the higher concentration groups (600 vs. 700 µg/ml and 700 vs. 800 µg/ml) are explicitly indicated in Fig. 1C. Specifically, significant differences were observed between the 600 and 700 µg/ml groups at days 1, 4,7 and 21. Notably, there was no statistical difference in Sr ion release between the 700 and 800 µg/ml groups at days 1, 4, 7, 14 and 21, indicating that the ion release reached a distinct plateau at concentrations above 700 µg/ml (Fig. 1C). Therefore, a MOF concentration of 700 µg/ml was selected for use in SF hydrogels in later experiments. HPLC results revealed that MOFs loaded with Aln exhibited the same pattern of peaks as Aln, indicating that the MOFs were successfully loaded with Aln (Fig. S1). Further analysis indicated that the drug loading capacity of Sr/MOFs with Aln reached 39% (data not shown). Subsequently, Sr²⁺ ion release was detected via HPLC. The cumulative release of Sr²⁺ ions increased progressively over the 21-day period, with larger increases in cumulative release observed on days 14 and 21 compared with earlier time points (Fig. 1D). The cumulative release of Aln from Sr/MOF-Aln also increased over the 21-day period; however, this release became more gradual at later time points compared with earlier time points (Fig. 1E).

The osteogenic properties of the SF + Sr/MOF group were investigated by measuring the ALP activity of BMSCs. The present study found that ALP staining increased in intensity between 1 and 2 weeks, demonstrating that the degrees of mineralization and osteogenic differentiation increased with time (Fig. 2A). In addition, compared with in the SF group, the ALP activity of BMSCs in the SF + Sr/MOF group significantly increased after 1 and 2 weeks of co-culture, and ALP activity in the SF + Sr/MOF-Aln group was significantly higher than the SF + Sr/MOF group after 2 weeks (Fig. 2B). In addition, the mineralization activity was evaluated to detect osteogenic differentiation status in weeks 2 and 3, with the staining intensity of cells gradually increasing over time (Fig. 2C). Furthermore, quantitative analysis confirmed a significantly higher mineralization degree in the SF + Sr/MOF group at both time points, as evidenced by the increased OD values (Fig. 2D). Additionally, the SF + Sr/MOF-Aln group exhibited denser mineral nodules and significantly higher mineralization levels compared with in the SF + Sr/MOF group at both time points. Furthermore, the expression levels of Runx2, OCN and COL1A1 gradually increased across the three experimental groups (Fig. 2E). Specifically, the SF + Sr/MOF group exhibited significantly increased gene expression levels compared with the SF group, whereas the SF + Sr/MOF-Aln group demonstrated the highest expression levels. By contrast, COL2A1 expression remained consistent across all treatment groups, demonstrating no statistically significant differences.

According to CCK-8 assay results, compared with in the SF group, the SF + Sr/MOF and SF + Sr/MOF-Aln groups demonstrated no significant differences in proliferation rate after incubation for 1, 4 and 7 days (Fig. 2F). Additionally, according to the results of calcein AM/PI staining, no notable differences in cell survival rate were observed between groups after 48 h of treatment (Fig. 2G).

The present study subsequently investigated whether SF + Sr/MOF or SF + Sr/MOF-Aln could reduce the increase in ROS caused by H₂O₂. Analysis of DCFH-DA staining revealed that the proportion of cells that stained positive for ROS following H₂O₂ treatment were increased compared with in the untreated control group. In addition, compared with the H₂O₂ + SF group, the relative number of BMSCs that stained positively for ROS was markedly reduced in the H₂O₂ + SF + Sr/MOF and SF + Sr/MOF-Aln groups (Fig. 2H).

Biomechanical testing of the collected SDa rat supraspinatus tendon-humerus complexes was performed via a universal testing machine (Fig. 3A). After 8 weeks of rotator cuff tendon repair, the maximum load values in the SF + Sr/MOF and SF + Sr/MOF-Aln groups were significantly increased compared with in the control group (Fig. 3B). The stiffness of rotator cuffs across the four groups also showed similar results; the stiffness values gradually increased from the control group to the SF + Sr/MOF-Aln group, and a significant difference was observed between the control group and the SF + Sr/MOF group (Fig. 3C). In addition, a significant increase in rotator cuff stiffness was observed between the control group and the SF + Sr/MOF-Aln group.

A total of 8 weeks after rotator cuff surgery, rats were examined using a micro-MRI system to observe the healing of the TBI (Fig. 4A). Subsequently, CT imaging of rat shoulder joints was used to examine bone repair at the TBI (Fig. 4C). Analysis of the regeneration status of tendons was evaluated using an MRI scoring system, in which higher MRI scores corresponded with poorer TBI healing. The control group scored the highest, followed by the SF and SF + Sr/MOF groups, whereas the SF + Sr/MOF-Aln group had the lowest MRI score (Fig. 4B). Notably, compared with in the control group, the MRI scores of the SF + Sr/MOF and SF + Sr/MOF-Aln groups were significantly decreased. However, the difference between the SF group and the control group was not statistically significant, and no significant differences were observed among the three treatment groups (SF, SF + Sr/MOF and SF + Sr/MOF-Aln) (Fig. 4B). Micro-CT images were used to generate 3D reconstructions of the humerus bones of rats in order to evaluate the growth of new bone tissue in each treatment group. There was no statistically significant difference in BV/TV and BMD between the control group and the SF group (Fig. 4D and E). However, the BV/TV and BMD values of the SF + Sr/MOF group were significantly increased compared with in the control group and the BMD of the SF + Sr/MOF-Aln group was significantly increased compared with the SF + Sr/MOF group, which indicated that the loading of Sr/MOFs with Aln was therapeutically effective.

In order to observe the microstructure after surgery, the local tissues were stained and analyzed. H&E staining indicated that collagen fibers in the SF + Sr/MOF-Aln group appeared to be more organized compared with the other treatment groups (Fig. 5A). According to tendon maturation score, the SF + Sr/MOF group showed a significant increase in tendon maturity 8 weeks after RCT surgery compared with in the control group (Fig. 5B). Toluidine blue staining was used to evaluate newly formed fibrocartilage, as shown in Fig. 5C. While cartilage matrix deposition was limited in the control group, the SF + Sr/MOF and SF + Sr/MOF-Aln groups exhibited a robust and continuous fibrocartilage layer. After 8 weeks, the fibrocartilage area of the SF + Sr/MOF and SF + Sr/MOF-Aln groups was significantly increased compared with the control group (Fig. 5D). These results indicated that, compared with in the other treatment groups, the SF + Sr/MOF-Aln group may have demonstrated an improved ability to repair RCTs.