Enhanced rotator cuff tendon-bone interface regeneration with injectable manganese-based mesoporous silica nanoparticle-loaded dual crosslinked hydrogels

Rotator cuff tears (RCTs) represent one of the most prevalent tendon-bone injuries, frequently causing recurrent shoulder pain and restricted mobility. Statistical data indicate over 30 million annual global cases of tendon-bone interface (TBI) injuries (Maffulli et al., 2003), with more than 270,000 surgical procedures performed yearly in the United States alone for rotator cuff tendon repair (Saveh-Shemshaki and Laurencin, 2019). The TBI constitutes a structurally intricate and functionally essential transitional zone connecting tendon to bone, crucial for maintaining shoulder joint integrity. Despite advancements in surgical techniques for rotator cuff repair, RC prosthetic surgery failure rates remain elevated (20%–94%) (Galatz et al., 2004; Xu et al., 2021) due to inadequate bone-tendon healing and regeneration. This clinical challenge arises from the non-regeneration of the rotator cuff functionally graded tendon-bone attachment during postoperative healing, which fails to reconstruct the TBI's physiological layered architecture (tendon, uncalcified fibrocartilage, calcified fibrocartilage, and bone). Instead, fibrovascular scar tissue with inferior mechanical properties forms, substantially compromising load transfer efficiency and stress dissipation capacity (Zumstein et al., 2017), thereby increasing retear susceptibility. Recent years have witnessed growing interest in tissue engineering material applications for TBI treatment. Numerous material-based strategies have been developed to enhance TBI healing, demonstrating potential for functional reintegration of injured rotator cuffs (Zhao et al., 2014a; Wang et al., 2023; Zhu et al., 2021). Nevertheless, achieving functional regeneration of native tendon enthesis at the tendon-bone interface persists as a significant clinical challenge.

Optimal tendon-bone interface (TBI) healing is fundamentally determined by vascularization and osteogenesis (Zhao et al., 2022), with healing strength contingent upon bone tissue ingrowth, mineralization, and maturation (Rodeo et al., 1993). The regeneration extent of fibrocartilaginous zones and osseous regions critically influences clinical outcomes in injured patients (Xu et al., 2021; Shengnan et al., 2021). Rodeo et al. (1999) demonstrated that bone morphogenetic protein (BMP) treatment enhances peri-tendinous bone formation, improves bone-tendon integration, and increases biomechanical strength, thereby accelerating TBI healing. Calcium phosphate, recognized for its osteogenic properties, has been extensively employed to augment bone regeneration. Enhanced osteogenic differentiation in rat bone marrow mesenchymal stem cells (BMSCs) manifests through upregulated expression of Rux2, ALP, and OPN markers (Ren et al., 2023). Liao et al. (2021) reported histological improvements in bone tunnels using calcium phosphate-treated tendon grafts during goat anterior cruciate ligament (ACL) reconstruction, facilitating TBI healing. Innovative approaches utilizing growth factor-loaded nano-scaffolds have shown promise. Zhao et al. (2014b) developed basic fibroblast growth factor (bFGF)-incorporated electrospun poly (lactic-co-glycolic acid) (PLGA) membranes for rotator cuff repair, demonstrating enhanced cell adhesion/proliferation and accelerated tendon-bone reconstruction. Inflammatory responses and oxidative stress at the bone-tendon junction significantly impact repair outcomes. Inflammation critically influences tissue healing responses to transplants and medical devices. Connizzo and Grodzinsky (2018) revealed that pro-inflammatory factors in murine TBI models downregulate tenocyte markers (Scx, Tnmd, Col3α1) and induce cell death. Key inflammatory components in tendon repair include IL-6, IL-8, Tnf-α, MMP-1, and MMP-3 (Jensen et al., 2018), with MMP-1/3/13 exhibiting collagen/proteoglycan degradation capacity (Nissinen and Kähäri, 2014), potentially impairing TBI healing. IL-6, IL-1β, and Tnf-α stimulate receptor activator of nuclear factor κB ligand (RANKL) expression in synovial fibroblasts/osteoblasts, promoting osteoclastogenesis (Mori et al., 2011). IL-6 additionally inhibits synovial fibroblast proliferation while upregulating TIMP-1 in synovial/chondrocytic cells (Mori et al., 2011). Nikhil et al. (Oak et al., 2014) demonstrated that 5-LOX and COX-1/2 inhibition enhances post-repair fibrosis and adipogenesis in RCT models. Oxidative stress during healing modulates tendon stem cell apoptosis/autophagy (Li et al., 2020), with Ren et al. (2023) confirming that antioxidant intervention at injury sites promotes tendon regeneration. Upregulated SOD1/SOD2 expression enhanced TBI healing in rat RCT models, while the AMPK/Nrf2/GPX4 pathway critically regulates tenocyte apoptosis/senescence (Chen et al., 2024). Collectively, these findings underscore that coordinated osteochondral differentiation at TBI interfaces coupled with inflammatory/oxidative stress modulation represents a promising therapeutic strategy for bone-tendon integration.

It is well-known that the primary factors determining perfect healing of the tendon-bone interface are vascular and bone formation (Zhao et al., 2022). The healing strength of the tendon-bone interface is determined by the ingrowth, mineralization, and maturation of bone tissue (Rodeo et al., 1993). The degree of regeneration in the fibrocartilaginous zone and bone tissue area plays a crucial role in satisfying clinical outcomes for injured patients (Xu et al., 2021; Shengnan et al., 2021). Scott A and colleagues (Rodeo et al., 1999) found that treatment with Bone morphogenetic protein (BMP) could lead to more extensive bone formation around the tendon, tighter integration between the new bone and the tendon, and higher biomechanical strength, significantly promoting tendon-bone healing. Additionally, calcium phosphate, known for its bone formation-promoting advantages, has been widely used to enhance bone regeneration. Promoting bone regeneration of rat BMSCs can be manifested as increased expression of osteogenic markers Rux2, ALP, and OPN (Ren et al., 2023). Researchers have also experimented with growth factor-loaded nano scaffolds to promote tendon-bone healing. Zhao et al. (2014b) developed an electrospun polylactic acid-glycolic acid (PLGE) fiber membrane loaded with basic fibroblast growth factor (bFGF) for rotator cuff repair, showing that the product facilitated cell attachment and proliferation, accelerating tendon-bone reconstruction. Inflammation and oxidative stress at the bone-tendon junction also play a crucial role in repair outcomes. Inflammation is a key factor in determining tissue healing outcomes and responses to transplants and other medical devices. For example, the study by Brianne K et al. found that in a mouse tendon-bone injury model, pro-inflammatory factors released by muscles and bones can lead to a decrease in the expression of tendon cell markers Scx, Tnmd, and Col3a1, and even cause cell death (Connizzo and Grodzinsky, 2018). Regarding tendon injury and repair, many inflammation-associated mediators and cell types have been identified, such as IL-6, IL-8, TNF-α, matrix metalloproteinase (MMP)-1, MMP-3, etc. (Jensen et al., 2018); MMP-1, MMP-3, and MMP-13 can degrade proteins such as types 1, 2, and 3 collagen and proteoglycans (Nissinen and Kähäri, 2014), which may not be good news for TBI healing. IL-6, IL-1, and tumor necrosis factor have also been shown to stimulate RANKL expression on synovial fibroblasts and osteoblasts, leading to osteoclastogenesis (Mori et al., 2011). IL-6 can inhibit the proliferation of synovial fibroblasts and stimulate the expression of MMP 1 tissue inhibitor (TIMP-1) in synovial cells and chondrocytes (Mori et al., 2011). Nikhil R and others reported that inhibiting inflammatory factors 5-LOX, COX-1, and COX-2 can increase fibrosis and fat formation after rotator cuff injury repair (Oak et al., 2014); furthermore, during the injury repair process, oxidative stress and reactive oxygen species can affect the apoptosis and autophagy of tendon stem cells (Li et al., 2020). Research by Xunshan R and others (Ren et al., 2023) has proven that counteracting oxidative stress damage at the site of tendon injury can effectively promote tendon regeneration. Enhanced expression of antioxidant enzymes such as SOD1 and SOD2 promoted tendon-bone healing after rotator cuff repair in rats. In addition, the oxidative stress signaling pathway AMPK/Nrf2/GPX4 also plays a crucial role in tendon cell apoptosis and senescence (Chen et al., 2024). In summary, promoting bone and cartilage differentiation at the TBI interface, and regulating the inflammatory and oxidative stress in the microenvironment, will be effective therapeutic means to promote tendon-bone healing.

This study employed hyaluronic acid (HA) and polyethylene glycol (PEG) as bioactive substrates to fabricate an adhesive hydrogel system via click chemistry, combining biological functionality with enhanced mechanical properties and handling characteristics. The hydrogel system enables rapid in situ adhesion at injury sites post-injection, providing a three-dimensional scaffold that facilitates cellular attachment and proliferation, thereby accelerating tissue repair. To optimize mechanical performance and nano-structural modification, manganese-doped mesoporous silica nanoparticles (Mn-MSN) were incorporated into the HA/PEG hydrogel matrix, yielding manganese-based mesoporous silica nanoparticle-incorporated dual-crosslinked hydrogels (Mn-MSN@gel). Characterization revealed that Mn-MSN@gel possesses a porous three-dimensional architecture with homogeneous distribution of Si, O, and Mn elements. The composite material demonstrated favorable mechanical strength, injectability, self-healing capability, biodegradability, and biocompatibility. Mn-MSN@gel enables controlled Mn⁴⁺ release, exhibiting anti-inflammatory effects through IL-6 and TNF-α suppression coupled with IL-10 elevation, while mitigating oxidative stress via Nrf2, GPX4, and SOD2 activation. The hydrogel promoted osteogenic differentiation in rat bone marrow mesenchymal stem cells (BMSCs), evidenced by upregulated Rux2, ALP, and Sox9 expression, and enhanced tenogenic differentiation in tendon-derived stem cells (TDSCs) through increased Scx, Tnmd, Col3α1, and MMP3 markers. In rat rotator cuff repair models, Mn-MSN@gel significantly improved biomechanical properties of regenerated tissue and facilitated tendon-bone interface (TBI) reconstruction. These findings collectively demonstrate Mn-MSN@gel's potential as a foundational biomaterial for TBI repair in rotator cuff injuries.

Due to the specificity of combined local drug administration, drug localization and sustained release present significant challenges, underscoring the critical importance of developing biomaterials and compounds for adjuvant therapy. Injectable bio-repair materials with robust tissue adhesion, designed to facilitate gradient transitions between tendons and bone tissues and ultimately achieve mature healing, have garnered increasing attention in scientific research. Hydrogels, hydrophilic polymeric scaffold materials featuring a three-dimensional interconnected porous structure (Ahmed, 2015), exhibit water-swelling behavior without dissolution. Their extracellular matrix-mimetic properties, coupled with prolonged drug retention times and high drug-loading efficiency, render them promising candidates as biomedical drug carriers (El-Sherbiny and Yacoub, 2013). Hyaluronic acid (HA), a naturally occurring non-sulfated linear polysaccharide, has been widely utilized in clinical applications. For osteoarthritis treatment, intra-articular HA injections provide joint lubrication and pain relief (Altman et al., 2015). In trauma repair and tissue engineering, HA promotes cell migration and proliferation, accelerating wound healing (Murakami, et al., 2019). Additionally, HA serves as an effective drug delivery system carrier, enabling sustained drug release via its porous structure and biodegradability (Hemshekhar et al., 2016). However, HA exhibits extremely poor mechanical properties due to its high solubility and rapid in vivo degradation. To address these limitations, a dual-crosslinked hyaluronic acid/polyethylene glycol hydrogel (Gel) scaffold was synthesized using HA and polyethylene glycol as raw materials, mal-PEG-mal as a crosslinker, and Diels-Alder click chemistry combined with Schiff base reactions. This approach endowed the Gel with adhesive properties and reduced degradation rates. Further, to enhance mechanical strength and microstructure, mesoporous silica nanoparticles (MSN) and manganese-doped MSN (Mn-MSN) were incorporated into the hydrogel, yielding a manganese-based mesoporous silica nanoparticle-loaded dual-crosslinked hydrogel (Mn-MSN@Gel). Enhancing HA properties while leveraging its inherent advantages is a common strategy. For instance, Liu et al. (Liu et al., 2012) developed a tough, highly stretchable hydrogel by integrating graphene oxide (GO) into the hydrogel network, which resisted fracture even under strains exceeding 3000%. Mesoporous silica (MSN) has attracted extensive research interest owing to its tunable morphology, high specific surface area, large pore volume, thermal stability, and facile surface functionalization (Slowing et al., 2010). Notably, MSN synthesis is cost-effective, scalable, and environmentally abundant. Its porous architecture provides cavities for hosting bioactive molecules or drugs, positioning MSN as a promising platform for small-molecule delivery (Chen et al., 2020). MSN size can be precisely controlled from nanometers to micrometers by adjusting synthesis conditions, enabling broad biomedical applications (Wu et al., 2013). Furthermore, the morphological characteristics of nano-scaffolds significantly influence the adhesion, proliferation, and differentiation of active cells (Mahmoodinia Maymand et al., 2018).

The TBI exhibits slow healing due to challenges in cartilage regeneration and limited vascularization, particularly within fibrocartilaginous regions. Consequently, TBI healing predominantly occurs via type I collagen scar formation (Zumstein et al., 2017; Chen H. et al., 2021; Patel et al., 2018). The inferior biomechanical properties of fibrovascular scars result in immature interfacial healing, significantly elevating risks of reinjury and rupture. Current TBI repair strategies are further limited by deficient gradient structure and disorganized collagen fiber alignment (C et al., 2019). Biomaterial applications frequently focus on regulating osteoinductive capacity through controlled material properties, including chemical composition, surface microporosity, and geometric architecture (Zhang et al., 2014). For instance, pore diameters of 150–500 μm, porosity exceeding 50%, and high pore connectivity have been demonstrated to directly enhance bone mineralization and vascularization (Bai et al., 2010; Mastrogiacomo et al., 2006). Notably, ceramic biomaterials featuring micro/nanostructured surfaces with interconnected macropores significantly improve cell adhesion, proliferation, and in vitro osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), while augmenting in vivo bone regeneration (Lin et al., 2013; Xia et al., 2013). Consistent with these findings, the Mn-MSN@Gel developed in this study demonstrated a suitable three-dimensional mesoporous structure, excellent biocompatibility, and enhanced BMSC adhesion, proliferation, and osteogenic differentiation. The silicon component in Mn-MSN@Gel has been previously reported to exert osteogenic effects (Xia et al., 2016).

Silicon (Si) exhibits immunomodulatory properties during early inflammatory phases post-implantation, suppressing macrophage-mediated pro-inflammatory responses (Huang et al., 2018) and activating monocytes (Song et al., 2020). Our experimental results indicate that Mn-MSN@gel significantly enhances the expression of IL-6 and IL-10 in BMSCs, while reducing Tnf-α expression, thereby exerting an anti-inflammatory effect. As you pointed out, IL-6 possesses dual functions in inflammation—both pro-inflammatory and anti-inflammatory. The co-induction of IL-6 and IL-10 in the BMSC model essentially reflects the dynamic balancing mechanism of the immune system. Milwid et al. reported that IL-6 secreted by BMSCs binds to sIL-6R on surrounding cells, activating the STAT3 pathway, which in turn induces IL-10 secretion and establishes a negative feedback loop to suppress excessive inflammatory responses (Milwid et al., 2012). Furthermore, in a murine arthritis model, elevated serum IL-6 levels indicate the initiation of local inflammation. BMSCs exert a paracrine effect by secreting IL-10 and PGE2, thereby promoting a Th2-type immune shift and systemically steering the immune response toward anti-inflammation (Bouffi et al., 2010). Interestingly, excessive IL-6 secretion has been found to inhibit β-catenin activity, impairing the osteogenic capacity of osteoporotic BMSCs (Li et al., 2016). Neutralizing antibodies against IL-6 have been shown to rescue vertebral osteoporotic phenotypes in mice (Li et al., 2016). Additionally, high levels of IL-6 expression are known to trigger chondrocyte apoptosis, cartilage matrix degradation, cartilage collapse, and synovial infiltration (Noronha et al., 2019). While low levels of IL-6 may induce anti-inflammatory responses, overexpression can exacerbate inflammatory damage. This suggests that IL-6 may play opposing roles in tendon-bone healing, warranting further mechanistic studies to better elucidate its contributions. Additionally, Si stimulates collagen synthesis and organic matrix production through silicate signaling pathway modulation, thereby improving bone mechanical properties and biocompatibility (Reffitt et al., 2003). Si also facilitates tendon-bone healing by enhancing tenocyte proliferation and differentiation, as well as augmenting tendon tissue regeneration (Wan et al., 2024; Gereli, 2014). Corroborating these reports, our experimental results confirmed that Mn-MSN@Gel effectively promoted tendon-derived stem cell (TDSC) differentiation, accompanied by upregulated expression of Col3a1, Scx, and Tnmd. Manganese (Mn), an essential trace element in biological systems, participates in enzymatic catalysis and plays critical roles in redox reactions and antioxidant mechanisms (Burda et al., 2005). Tendon and bone healing are potentially associated with oxidative damage and oxidative stress (Lui et al., 2022; Callaway and Jiang, 2015). Secondly, Mn is involved in various physiological processes, including the regulation of cellular activities (He et al., 2023). For instance, Mn functions directly as a cofactor for several key enzymes involved in metabolic activities such as gluconeogenesis and chondrogenesis (Horning et al., 2015). Moreover, Mn-based materials have demonstrated significant promotive effects on tissue healing (M et al., 2021). Finally, Mn ions possess notable anti-inflammatory properties. For example, Luo et al. found that the degradation and release of Mn ions from metal/metal oxide nanoparticles can alleviate the progression of osteoarthritis through anti-inflammatory effects, cartilage repair promotion, and inhibition of cartilage ossification (Luo et al., 2021). In summary, considering Mn's antioxidant capacity, its facilitation of tendon-bone healing, and its anti-inflammatory effects, we have selected Mn as the active metallic component in our materials for tendon-bone repair. In this study, the Mn-MSN@Gel demonstrated significant efficacy in promoting cartilage formation in a rat rotator cuff injury model. It alleviated oxidative stress in BMSCs and TDSCs under inflammatory conditions by upregulating antioxidant enzymes (Sod2, Gpx4) and the core antioxidant transcription factor Nrf2, thereby exerting dual antioxidant and cartilage-repair functions. In tendon stem cells (TSCs), downregulation of MMP3 is closely associated with anti-degradation effects, and its regulatory mechanisms involve multi-level signaling cascades and transcriptional regulation. For example, activation of the TGF-β1/Smad2,3 pathway can upregulate anabolic markers in chondrocytes such as Col2a1 and Sox9, while simultaneously downregulating catabolic markers like MMP3 and MMP13 (Rodeo et al., 1999). In addition, inhibition of the Wnt/GSK-3β/NF-κB signaling axis can directly suppress MMP3 promoter activity, thereby ameliorating the progression of osteoarthritis (Ren et al., 2023). Interestingly, inflammatory cytokines also show potential links with MMP3 expression. BMSCs can secrete IL-1ra to block interactions between IL-1α/IL-1β and their receptors, effectively inhibiting the release of MMP3 and chemokines (Ren et al., 2023). In the context of tendinopathy, IL-1β promotes MMP3 expression by activating the MAPK/NF-κB pathway, whereas TGF-β1 exerts anti-degradation effects by suppressing this pathway (Liao et al., 2021). Collectively, the Mn-MSN@Gel that can slowly release Si and Mn elements is a promising multi-mechanism injectable biomaterial for structural TBI healing. Future integration of bioactive factors could further advance its potential as a therapeutic strategy for TBI injuries.

The study demonstrated that Mn-MSN@Gel possesses a three-dimensional porous structure with uniform distribution of silicon, oxygen, and manganese elements, enabling sustained slow release of Si⁴⁺ and Mn⁴⁺ ions. This hydrogel is facile to fabricate and exhibits favorable mechanical strength, injectability, self-healing capability, degradability, and cytocompatibility. It upregulates osteogenic differentiation genes in rat-BMSCs and tenogenic differentiation genes in rat-TDSCs. Additionally, Mn-MSN@Gel modulates oxidative stress- and inflammatory cytokine-related genes, demonstrating anti-inflammatory effects and mitigating oxidative stress damage (Figure 8). Mn-MSN@Gel injection markedly enhanced biomechanical recovery and stimulated robust tissue regeneration at the tendon-bone interface (TBI) in a rat rotator cuff repair model. These results underscore Mn-MSN@Gel as an innovative biomaterial platform with significant translational promise for achieving structural and functional restoration of TBI in rotator cuff injuries.