Abstract

   Osteochondral defects pose significant challenges in joint health,
   leading to pain, decreased mobility, and substantial healthcare costs
   due to surgical interventions. This study aimed to develop and evaluate
   Si-A/PUE@HA composite hydrogels as scaffolding materials for cartilage
   tissue engineering, addressing the limitations of current treatment
   options. Utilizing a multi-step synthesis method that incorporates both
   chemical and physical crosslinking techniques, we assessed the
   hydrogels’ cytocompatibility, chondrogenic differentiation potential,
   immunomodulatory properties, and in vivo cartilage regeneration
   capabilities. In vitro results demonstrated high cell viability and
   proliferation rates of bone marrow mesenchymal stem cells (BMSCs)
   cultured in Si-A/PUE@HA hydrogels, with significant expression of
   chondrogenic markers such as Acan, Sox9, and Col2a1. Notably, the
   hydrogels exhibited a favorable immunomodulatory effect, promoting an
   anti-inflammatory M2 macrophage phenotype, which is crucial for tissue
   regeneration. In vivo studies confirmed substantial new tissue
   formation and integration with surrounding cartilage, as evidenced by
   micro-CT analysis, alongside excellent biocompatibility with no
   significant adverse effects observed in major organs over a 12-week
   period. Single-cell RNA sequencing analysis revealed a favorable immune
   microenvironment and enhanced chondrogenesis linked to hydrogel
   treatment. In conclusion, Si-A/PUE@HA hydrogels represent a promising
   biomaterial with potential applications in cartilage repair and
   regenerative medicine, warranting further investigation in larger
   clinical trials to validate their efficacy and safety for future
   therapeutic use.

Graphical abstract

   [56]graphic file with name 12951_2025_3648_Figa_HTML.jpg

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12951-025-03648-9.

   Keywords: Cartilage regeneration, Tissue engineering, Chondrogenic
   differentiation, Stem cell therapy, Macrophage polarization,
   Single-Cell RNA sequencing

Introduction

   Osteochondral defects represent a significant clinical challenge due to
   their complex nature, involving damage to both articular cartilage and
   underlying subchondral bone [[57]1–[58]3]. These defects can arise from
   various etiologies, including trauma, osteoarthritis, and degenerative
   conditions [[59]4–[60]6]. The limited regenerative capacity of
   cartilage, compounded by its avascular nature, often leads to chronic
   pain, decreased mobility, and an overall decline in quality of life for
   affected patients [[61]7–[62]9]. The economic burden associated with
   these conditions is substantial, as they frequently necessitate
   surgical interventions and extensive rehabilitation efforts [[63]10].
   Thus, there is an urgent demand for innovative therapeutic strategies
   aimed at enhancing cartilage repair and regeneration. Current treatment
   modalities for osteochondral defects include microfracture, autologous
   chondrocyte implantation, and osteochondral grafting [[64]11–[65]13].
   Despite their widespread use, these approaches often yield variable
   success rates and may result in the formation of fibrocartilage instead
   of the desired hyaline cartilage [[66]14–[67]16]. This limitation
   underscores the need for further research to explore alternative
   methods that can effectively promote cartilage regeneration and restore
   joint function. Advances in biomaterials and tissue engineering have
   opened new avenues for the development of scaffolds that can mimic the
   native extracellular matrix, providing a conducive environment for
   cellular proliferation and differentiation.

   Recent studies have highlighted the potential of hydrogels as effective
   scaffolding materials in cartilage tissue engineering [[68]17]. Their
   unique properties, such as high water content, biocompatibility, and
   the ability to facilitate cell migration, make hydrogels an attractive
   choice for cartilage repair applications [[69]18–[70]20]. For example,
   hydrogels can be synthesized to possess specific mechanical and
   biochemical cues that promote chondrogenic differentiation of
   mesenchymal stem cells (MSCs) [[71]21, [72]22]. Moreover, the
   incorporation of bioactive agents within hydrogel matrices can enhance
   their regenerative potential by providing sustained release of growth
   factors that support cartilage formation [[73]23, [74]24].

   In this context, the development of composite hydrogels represents a
   promising strategy to optimize the mechanical properties and biological
   performance of scaffolds for cartilage regeneration. Therefore, we have
   developed a hydrogel named Si-A/PUE@HA. This composite hydrogel is
   designed through the combination of silane, arginine, puerarin, and
   hyaluronic acid (HA), utilizing both chemical and physical crosslinking
   methods to significantly enhance the material’s mechanical strength and
   bioactivity. Specifically, silane provides excellent structural
   stability and adjustable mechanical properties [[75]25]; arginine, a
   natural amino acid, promotes cell adhesion and proliferation while
   improving biocompatibility [[76]26]; puerarin, a natural isoflavone
   compound, offers strong antioxidant and anti-inflammatory effects,
   contributing to the regulation of the cellular microenvironment
   [[77]27, [78]28]; and hyaluronic acid, a natural polysaccharide,
   effectively promotes cell migration and cartilage tissue repair
   [[79]29]. The synergistic effect of these bioactive components allows
   the composite hydrogel to exhibit outstanding advantages in both
   mechanical performance and biological functionality. Such innovative
   materials are crucial as they have the potential to overcome the
   limitations of traditional scaffolds, which often lack sufficient
   support for cell adhesion, proliferation, and tissue repair [[80]30].

   The primary objective of this research is to evaluate the efficacy of
   Si-A/PUE@HA hydrogels in promoting chondrogenic differentiation of stem
   cells and enhancing cartilage repair in vivo. This multi-faceted
   approach will involve assessing cellular responses in vitro, as well as
   evaluating the functional outcomes of cartilage regeneration in
   appropriate animal models. By integrating advanced hydrogel technology
   with stem cell therapy, this study aims to contribute valuable insights
   into the development of effective treatment strategies for
   osteochondral defects.

   In summary, the increasing prevalence of osteochondral defects
   necessitates the exploration of novel therapeutic strategies that can
   facilitate cartilage regeneration. The use of advanced composite
   hydrogels offers a promising avenue for improving treatment outcomes,
   and the findings of this research may pave the way for future clinical
   applications in regenerative medicine. Continued investigation in this
   field is essential to address the existing challenges and enhance the
   quality of life for patients suffering from cartilage-related injuries.

Martials and methods

Preparation and characterization of Si-A/PUE@HA composite hydrogels

   The Si-A/PUE@HA composite hydrogels were synthesized through a
   precisely controlled multi-step process that integrated both chemical
   and physical crosslinking to create a structurally robust and bioactive
   material. For comparison, a negative control hydrogel (NC@HA), composed
   solely of hyaluronic acid (HA) without silane, arginine, or puerarin,
   was prepared using the same physical processing steps but without the
   addition of bioactive components. Initially, 1.0 g of hyaluronic acid
   (HA, Sigma-Aldrich, USA) was fully dissolved in 100 mL of deionized
   water under continuous magnetic stirring at room temperature (~ 25 °C)
   to form a clear and homogeneous solution. Subsequently, 2.0 mL of
   polyethoxysilane (silanol, Aladdin, China) was gradually added dropwise
   into the HA solution under vigorous stirring for 2 hours, facilitating
   pre-crosslinking through silanol condensation reactions. To impart
   bioactivity, 0.5 g of L-arginine (Sigma-Aldrich, USA) and 0.3 g of
   puerarin (Yuanye Bio-Technology, China) were incorporated into the
   pre-crosslinked solution and stirred for an additional 1 hour to ensure
   uniform distribution throughout the hydrogel matrix. Chemical
   crosslinking was then induced by adding 0.2 mL of hexamethylene
   diisocyanate (HDI, Sigma-Aldrich, USA), followed by thorough stirring
   for 1 hour. The resulting mixture was cast into polytetrafluoroethylene
   (PTFE) molds and allowed to cure at room temperature for 24 hours. Upon
   curing, the hydrogels were demolded and extensively washed with
   phosphate-buffered saline (PBS, pH 7.4, Gibco, USA) to eliminate
   unreacted monomers. To further reinforce mechanical strength, the
   hydrogels were subjected to three freeze–thaw cycles, consisting of
   freezing at − 20 °C for 12 hours and thawing at room temperature for 6
   hours. The microstructure and surface morphology of the Si-A/PUE@HA
   composite hydrogels were analyzed using scanning electron microscopy
   (SEM, Hitachi SU8010, Japan). Prior to imaging, the hydrogel samples
   were freeze-dried using a lyophilizer (Labconco FreeZone 2.5, USA),
   fractured in liquid nitrogen, and sputter-coated with a thin layer of
   gold using a Quorum Q150R Plus sputter coater (UK) to enhance
   conductivity. SEM imaging was conducted at an accelerating voltage of
   3.0 kV. Elemental distribution within the hydrogels was examined using
   energy-dispersive X-ray spectroscopy (EDS) integrated with the SEM
   system (Hitachi SU8010, Japan). This analysis was used to confirm the
   presence and uniform dispersion of key elements in the hydrogel matrix.
   The surface chemical composition of the hydrogels was investigated
   using X-ray photoelectron spectroscopy (XPS, Thermo Scientific ESCALAB
   250Xi, USA). Spectra were recorded to identify characteristic binding
   energy peaks corresponding to carbon (C1s), oxygen (O1s), nitrogen
   (N1s), and silicon (Si2p), verifying the successful incorporation of
   functional components. The mechanical and viscoelastic properties of
   the hydrogels were assessed using a rotational rheometer (TA
   Instruments Discovery HR-2, USA) equipped with a parallel plate
   geometry (diameter: 20 mm). Frequency sweep measurements were conducted
   at room temperature to evaluate the storage modulus (G’) and loss
   modulus (G”) across a strain range of 0.1–1000%. The release profile of
   puerarin from the hydrogel was measured by immersing 1 g of the
   hydrogel in 10 mL of phosphate-buffered saline (PBS, pH 7.4) at 37°C.
   At designated time intervals, aliquots were withdrawn and replaced with
   fresh PBS. The concentration of puerarin released was quantified using
   a UV-Vis spectrophotometer (Shimadzu UV-2600, Japan) at a detection
   wavelength of 250 nm. The adhesive properties of the hydrogels were
   evaluated by applying the hydrogel to different substrates, including
   human skin, polymer (latex gloves), glass, and metal. The adhesion
   performance was qualitatively assessed and recorded using a Canon EOS
   90D digital camera (Japan).

Preparation of hydrogel extract for injection

   The Si-A/PUE@HA hydrogel was sterilized under ultraviolet (UV) light.
   After sterilization, both Si-A/PUE@HA and NC@HA hydrogels were added to
   DMEM at a concentration of 0.1 g/mL and incubated at 37 °C for 24 h.
   The supernatant was then collected and filtered through a 0.22 μm
   membrane filter. Standard DMEM medium without any hydrogel extract was
   used for the Control group in all in vitro experiments. The filtered
   solution was stored at 4 °C–− 20 °C for long-term preservation.

Cell isolation and culture

   RAW 264.7 macrophage cells and rat bone marrow mesenchymal stem cells
   (BMSCs) were used for in vitro experiments. RAW 264.7 cells were
   obtained from the Cell Bank of the Chinese Academy of Sciences and
   cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Gibco,
   USA) and 1% (v/v) penicillin-streptomycin (P/S, Gibco, USA). Rat BMSCs
   were purchased from Shanghai Fuyu Biotechnology Co., Ltd. and cultured
   according to the manufacturer’s instructions. BMSCs were maintained in
   α-minimal essential medium (α-MEM, Gibco) supplemented with 10% FBS and
   1% penicillin-streptomycin. Both cell types were incubated at 37 °C
   with medium changes every two days. Only early-passage cells (passages
   3–6) were used in the experiments.

Cell proliferation and viability assays

   The biocompatibility of the Si-A/PUE@HA hydrogel was evaluated using a
   live/dead cell staining kit (Invitrogen, USA). In brief, BMSCs were
   cultured in the Si-A/PUE@HA hydrogel for 1, 3, and 7 days. Cells were
   washed three times with PBS and stained with PBS containing 2 mM
   calcein-AM (green fluorescence) and 4 mM propidium iodide (PI, red
   fluorescence) for 15 min at 37 °C. After another gentle PBS wash, the
   cells were visualized under a fluorescence microscope (Leica, USA).
   Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8,
   Beyotime, China) on days 1, 3, and 7. Cells treated under different
   conditions were incubated in medium containing 10% CCK-8 at 37 °C for
   2 h, and absorbance was measured at 450 nm using a microplate reader
   (BioTek, USA). Normal growth medium served as the control group. For
   the BMSCs proliferation assay, a bromodeoxyuridine (BrdU) incorporation
   kit (Cell Signaling Technology, USA) was used following the
   manufacturer’s instructions. BMSCs were seeded at 1 × 10^6 cells per
   well in a 6-well plate and exposed to various treatments for 24 h. BrdU
   was added to each well for the last 12 h of treatment. Cells were then
   fixed, washed twice with PBS, and incubated with mouse anti-BrdU
   antibody for 1 h at room temperature. Nuclei were stained with
   4′,6-diamidino-2-phenylindole (DAPI) for 5 min. Immunofluorescence
   images were captured using a fluorescence microscope (Leica, Germany),
   and proliferation was quantified by calculating the percentage of
   BrdU-positive cells relative to DAPI-stained cells, or by counting
   BrdU-positive cells per square millimeter.

Reactive oxygen species (ROS) assay

   To evaluate the hydrogel’s effect on reactive oxygen species (ROS)
   scavenging, BMSCs were seeded at 1 × 10^6 cells per well in a 6-well
   plate and incubated for 24 h to ensure proper attachment. After
   replacing the medium with extracts from Gel and Si-A/PUE@HA hydrogels,
   cells were incubated for an additional 6 h. Intracellular ROS levels
   were visualized using 2′,7′-dichlorofluorescin diacetate (DCFH-DA,
   Beyotime, China). Cells were incubated with 10 µM DCFH-DA in the dark
   for 20 min, and fluorescence images were captured using a fluorescence
   microscope (Leica, Germany) from five randomly selected areas per
   group. Quantification of fluorescence intensity was performed using
   ImageJ software.

Wound healing assay

   BMSCs were seeded into 6-well culture plates at an appropriate density
   and incubated under standard conditions (37 °C, 5% CO₂) until a
   confluent monolayer was formed. A uniform linear scratch was created
   across the cell monolayer using a sterile 200 µL pipette tip to
   simulate a wound. Detached cells were carefully removed by washing the
   wells twice with sterile phosphate-buffered saline (PBS, pH 7.4). Fresh
   culture medium specific to each experimental group was then added to
   the wells. Immediately after scratching, images of the wound area were
   captured at 0 h using an inverted phase-contrast microscope. The cells
   were subsequently incubated, and additional images of the scratch area
   were taken at 12 h and 24 h to monitor cell migration and wound
   closure. The extent of wound closure was quantitatively analyzed using
   ImageJ software (NIH, USA) by calculating the percentage of the wound
   area covered over time.

Chondrogenic differentiation of BMSCs

   To investigate the effects of various extracts on chondrogenic
   differentiation, BMSCs were harvested and pelleted after
   trypsinization, following a previously described protocol. The pellets
   were cultured in 15 mL centrifuge tubes, centrifuged (200 g, 5 min),
   and after 24 h, the medium was replaced with chondrogenic
   differentiation medium (CM) prepared with Si-A/PUE@HA extracts. The CM
   contained 10 ng/mL TGFβ3 (Sigma, USA), 1 mM sodium pyruvate (Sigma,
   USA), 0.1 µM dexamethasone (Sigma, USA), 1% ITS Premix (Sigma, USA),
   and 1 µM ascorbate-2-phosphate (Sigma, USA). CM without extract served
   as the control group. After three weeks, the chondrogenic pellets were
   cryo-sectioned (7 μm thickness) and subjected to histological analysis,
   including hematoxylin and eosin (HE), alcian blue (AB), safranin O
   staining, and immunohistochemistry (IHC). The Bern score was used to
   assess in vitro chondrogenesis. Real-time quantitative polymerase chain
   reaction (RT-qPCR) was conducted to assess the expression of
   chondrogenesis-related genes, including collagen type I α1 (Col1a1),
   collagen type II α1 (Col2a1), SRY-related HMG-box gene 9 (Sox9), and
   aggrecan (Acan). Total RNA was extracted using the EZ-press RNA
   Purification Kit (EZBioscience), followed by cDNA synthesis using a 4×
   Reverse Transcription Master Mix (EZBioscience). RT-qPCR was performed
   using a 2× SYBR Green qPCR Master Mix (EZBioscience) on a Light Cycler
   system (Bio-Rad, USA). Relative mRNA expression levels were calculated
   using the 2-ΔΔCt method and normalized to the housekeeping gene GAPDH.
   Primer sequences are listed in Table S1.

Macrophage polarization assay

   Macrophage polarization induced by Si-A/PUE@HA hydrogel extracts was
   assessed via immunofluorescence staining, specifically evaluating the
   expression of M1 markers (iNOS, CD86) and M2 markers (CD163). To
   simulate post-implantation inflammation, RAW 264.7 cells were seeded in
   6-well plates and treated with 100 ng/mL lipopolysaccharide (LPS,
   Sigma, USA) for 24 h. Cells were then exposed to normal growth medium
   or Si-A/PUE@HA hydrogel extracts for 3 days. After treatment, cells
   were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton
   X-100, and blocked with 5% bovine serum albumin (BSA). Primary
   antibodies against iNOS (Abcam, UK), CD86 (Abcam, UK), F4/80 (Abcam,
   UK), and CD163 (Abcam, UK) were applied at a 1:500 dilution and
   incubated overnight at 4 °C. Secondary antibodies conjugated with Alexa
   Fluor 488 or Alexa Fluor 594 (Abcam, UK) were applied for 1 h, followed
   by DAPI staining for 5 min. Fluorescence images were captured using a
   microscope. Flow cytometry was used to further assess CD86 (PE-labeled,
   eBioscience, USA) and CD163 (FITC-labeled, eBioscience, USA) expression
   levels. Cells were scraped, centrifuged, blocked with 5% BSA, stained
   with the appropriate antibodies, and analyzed using a flow cytometer
   (BD, USA). CytExpert 2.4 software was used for data analysis.
   Additionally, the expression of polarization-related genes (TNF-α,
   IL-6, IL-1β, IL-4, IL-10, and IL-1ra) was evaluated by RT-qPCR.

Surgical procedure for osteochondral defect modeling

   Forty 6-week-old male Sprague Dawley (SD) rats were randomly assigned
   into groups according to the experimental design. The procedure was as
   follows: after anesthetizing the rats, shaving the fur, and
   disinfecting the surgical area, a medial incision was made on the knee
   joint. The patellar ligament and patella were everted to expose the
   femoral condyle. A micro electric drill was then used to create a
   defect (2 mm in diameter, 1.5 mm in depth) at the center of the
   trochlear groove. In the model group, the defect site was rinsed with
   PBS. In the two experimental groups, the defects were filled with 20 µL
   of NC@HA or Si-A/PUE@HA hydrogel using a microinjector. In the control
   group, the knee joint was exposed but no drilling was performed. The
   patella was repositioned, the incision was sutured, and the area was
   disinfected again. Postoperatively, all rats were housed in a specific
   pathogen-free (SPF) animal facility and allowed to move freely. Rats
   were sacrificed at 4 and 8 weeks after surgery for further analysis.

Micro-CT analysis

   At 6 and 12 weeks post-surgery, the Sprague Dawley (SD) rats were
   euthanized, and their femurs were harvested. Samples were fixed in 4%
   paraformaldehyde solution (Servicebio, China) at 4 °C overnight to
   preserve their structural integrity. Morphological evaluation was
   subsequently performed using a micro-computed tomography (Micro-CT)
   scanner (SkyScan 1276, Bruker, Germany). The scanning parameters were
   set as follows: a voxel size of 10 μm, voltage of 70 kV, current of 200
   µA, a 1 mm aluminum filter, and an integration time of 350 ms to ensure
   high-resolution imaging. The raw Micro-CT data were reconstructed using
   NRecon software (version 1.7.4.2, Bruker, Germany). For
   three-dimensional visualization, CTvox software (version 3.3, Bruker,
   Germany) was utilized to convert the contoured two-dimensional images
   into detailed 3D models. Specific regions of interest (ROIs) were
   extracted from the 3D reconstructions using DataViewer software
   (version 1.5.1.9, Bruker, Germany). Quantitative analysis of bone
   regeneration was conducted using CTAn software (version 1.20.3, Bruker,
   Germany). Key parameters, including the bone volume fraction (BV/TV),
   were calculated to assess subchondral bone reconstruction.
   Additionally, the area of bilateral bone defects in the coronal
   sections was measured using ImageJ software (NIH, USA) for further
   analysis.

In vivo cartilage regeneration evaluation

   At 6 and 12 weeks post-surgery, the repaired articular cartilage was
   harvested for morphological evaluation. The samples were fixed in 4%
   paraformaldehyde solution for 5 days to preserve tissue integrity.
   Following fixation, the samples underwent continuous trimming during a
   2-month decalcification process. Upon completion of decalcification,
   the specimens were dehydrated through a graded ethanol series, embedded
   in paraffin, and sectioned into slices of 5 μm thickness using a
   microtome. For histological analysis, the sections were stained
   following standard protocols with hematoxylin and eosin (H&E) for
   general morphology, Safranin O/Fast Green for glycosaminoglycan
   content, and Toluidine Blue (TB) for cartilage matrix evaluation.
   Immunohistochemical staining for type II collagen (COL2) was performed
   to assess cartilage-specific matrix formation. Briefly,
   paraffin-embedded sections were dewaxed and rehydrated. Endogenous
   peroxidase activity was quenched using hydrogen peroxide (H₂O₂).
   Permeabilization was achieved with Triton X-100, followed by
   phosphate-buffered saline (PBS) washing. The sections were then blocked
   with a suitable blocking buffer and incubated overnight at 4 °C with a
   primary anti-collagen type II antibody (1:200, Abcam, USA).
   Subsequently, the sections were incubated with a secondary antibody,
   and signal detection was conducted using a chromogenic substrate. The
   stained sections were visualized and imaged under a light microscope.
   All histological images were independently assessed by a pathologist
   with extensive experience in cartilage histopathology, who was blinded
   to the experimental groups. Cartilage repair quality was quantitatively
   evaluated using the Modified O’Driscoll Scoring System and the
   International Cartilage Repair Society (ICRS) Visual Histological
   Assessment Scale (Table [81]S2−3).

Single-Cell RNA sequencing with 10x genomics chromium

   Articular cartilage samples were collected from 10 experimental rats,
   ground, and prepared for single-cell RNA sequencing (scRNA-seq) to
   evaluate cellular responses. Viable cells were isolated and treated
   with red blood cell lysis buffer, followed by magnetic bead separation
   to remove erythrocytes and dead cells. Cell viability was assessed
   using trypan blue staining (Thermo Fisher Scientific, Waltham, MA, USA)
   and a hemocytometer (Thermo Fisher Scientific). After counting, cell
   suspensions were adjusted based on a target of 7000 cells per capture.
   For samples with concentrations below the recommended 700–1200
   cells/µL, the cells were centrifuged and resuspended in a smaller
   volume before loading into the 10x Genomics Single Cell A Chip. Reverse
   transcription and library preparation were performed using the 10x
   Genomics Single Cell v2 kit, following the manufacturer’s protocol. The
   libraries were multiplexed and sequenced using an Illumina NextSeq-500
   platform with a high-output (400 m) kit in a single lane. Quality
   control of the scRNA-seq data was performed using the Cell Ranger
   pipeline (10x Genomics), which aligned the reads to the mm10 reference
   genome and generated a digital gene expression matrix. This matrix was
   filtered and normalized using the Seurat R package (version 2.3.4).
   Cells with fewer than 500 unique genes, greater than 50,000 UMI counts,
   or over 10% mitochondrial reads were excluded from further analysis.
   Genes that were not detected in any cells were also removed from
   subsequent analysis.

Single-cell RNA sequencing data processing

   The gene expression matrix was normalized and scaled using the
   NormalizeData and ScaleData functions in Seurat. Principal component
   analysis (PCA) was conducted using the RunPCA function for
   dimensionality reduction and clustering. DecontX was employed to
   predict contamination levels and remove abnormally expressed marker
   genes from mixed datasets. Batch effects at the sample level were
   corrected using Harmony. Cell subpopulations were visualized using
   UMAP, and characteristic gene expression was evaluated using Seurat
   functions such as Dotplot and FeaturePlot. Differential expression
   analysis was performed with Seurat’s FindAllMarkers function, assuming
   a negative binomial distribution for likelihood ratio testing. Only
   genes expressed in more than 25% of cells in a cluster with a log2 fold
   change greater than 0.25 were considered for differential expression.
   Cell type identification was carried out using the “Single R” software,
   along with insights from published studies and expert annotations.

Pathway enrichment analysis

   Functional enrichment analysis, including Gene Ontology (GO) and Kyoto
   Encyclopedia of Genes and Genomes (KEGG) pathway analysis, was
   conducted to determine whether differentially expressed genes (DEGs)
   were significantly enriched in specific GO terms or metabolic pathways.
   GO is an internationally standardized classification system for gene
   function. DEGs were mapped to GO terms representing biological
   functions, and gene counts within each term were calculated. A
   hypergeometric test was then performed to identify GO terms that were
   significantly enriched in the gene list compared to a reference gene
   list. GO terms and KEGG pathways with a false discovery rate (FDR) of
   P < 0.05 were considered significantly enriched.

Toxicity evaluation of Si-A/PUE@HA

   At 12 weeks post-surgery, the rats were euthanized, and major organs,
   including the heart, liver, spleen, lungs, and kidneys, were collected.
   These organs were subjected to hematoxylin and eosin (H&E) staining and
   Masson staining, as well as F4/80 and α-SMA immunofluorescence
   staining, to comprehensively evaluate the biosafety of the Si-A/PUE@HA
   composite hydrogel.

Statistical analysis

   All data were expressed as the mean ± standard deviation (SD).
   Statistical analyses were performed using one-way analysis of variance
   (ANOVA) followed by Tukey’s post hoc test. SPSS 22.0 software was used
   for statistical analysis. A P-value of less than 0.05 was considered
   statistically significant Fig. [82]1.

Fig. 1.

   [83]Fig. 1
   [84]Open in a new tab

   Schematic illustration of the synthesis and therapeutic application of
   the Si-A/PUE@HA hydrogel. Hyaluronic acid and siliconol are combined
   with arginine through physical crosslinking and puerarin through
   self-assembly to form an injectable hydrogel. The hydrogel is injected
   into the osteochondral defect site, where it promotes macrophage
   polarization from M1 to M2 and facilitates the differentiation of bone
   marrow mesenchymal stem cells (BMSCs) into chondrocytes, thereby
   accelerating cartilage regeneration

Result

Structural and physicochemical characterization of the Si-A/PUE@HA hydrogel

   The Si-A/PUE@HA hydrogel was developed through a self-assembly and
   physical crosslinking strategy involving hyaluronic acid, silicool,
   arginine, and puerarin (Fig. [85]2A), resulting in a multifunctional
   material with enhanced mechanical strength, bioactivity, and adhesive
   properties. The hydrogel exhibited a distinct sol–gel transition,
   transitioning from a flowable sol to a stable gel (Fig. [86]2B),
   enabling in situ application and conformal coating on irregular
   surfaces. Its excellent injectability was further demonstrated by
   smooth extrusion and the formation of self-supporting structures
   (Fig. [87]2C), highlighting its potential for minimally invasive
   delivery in clinical settings. Scanning electron microscopy (SEM)
   revealed a highly porous and interconnected network structure
   (Fig. [88]2D, Supplementary Fig. [89]1), which is critical for
   facilitating nutrient diffusion, cell infiltration, and sustained drug
   release. Energy-dispersive X-ray spectroscopy (EDS) mapping confirmed
   the uniform distribution of carbon (C), oxygen (O), sodium (Na), and
   silicon (Si) throughout the hydrogel matrix (Fig. [90]2E), validating
   the homogeneous integration of the hydrogel components. X-ray
   photoelectron spectroscopy (XPS) further confirmed the successful
   incorporation of silicool and puerarin, as evidenced by characteristic
   peaks for C 1 s, O 1 s, N 1 s, and Si 2p (Fig. [91]2F). Rheological
   analysis revealed a sol–gel transition behavior, where the storage
   modulus (G′) surpassed the loss modulus (G″) around 37 °C
   (Fig. [92]2G), indicating the hydrogel’s temperature-responsive
   transformation from a sol to a gel state. This transition demonstrates
   that the hydrogel acquires solid-like mechanical properties under
   physiological temperature, which is critical for maintaining structural
   integrity in vivo. The hydrogel also exhibited a sustained and
   controlled puerarin release over 14 days (Fig. [93]2H), supporting its
   potential for long-term therapeutic applications. Notably, the hydrogel
   adhered strongly to various substrates, including skin, polymer, glass,
   and metal surfaces (Fig. [94]2I), showcasing its versatile adhesive
   capability for biomedical applications. Collectively, these results
   demonstrate that the Si-A/PUE@HA hydrogel integrates injectability,
   mechanical robustness, sustained drug release, and broad-spectrum
   adhesion, offering significant promise for applications in drug
   delivery, tissue engineering, and regenerative medicine.

Fig. 2.

   [95]Fig. 2
   [96]Open in a new tab

   Characterization and performance analysis of the Si-A/PUE@HA hydrogel.
   (A) Schematic illustration of the hydrogel formation through physical
   crosslinking and self-assembly of hyaluronic acid, siliconol, arginine,
   and puerarin. (B) Photographs showing the sol–gel transition behavior
   of the Si-A/PUE@HA hydrogel. (C) Demonstration of the injectability of
   the Si-A/PUE@HA hydrogel. (D) SEM images showing the porous
   microstructure of the hydrogel at different magnifications. (E) SEM and
   corresponding energy-dispersive X-ray spectroscopy (EDS) mapping images
   highlighting the distribution of elements: carbon (C, red), oxygen (O,
   green), sodium (Na, cyan), and silicon (Si, purple). (F) X-ray
   photoelectron spectroscopy (XPS) spectrum confirming the chemical
   composition of the hydrogel. (G) Rheological analysis illustrating the
   storage modulus (G’) and loss modulus (G’’) of the hydrogel over a
   range of strain percentages. (H) Cumulative release profile of puerarin
   (PUE) from the hydrogel over 14 days. (I) Adhesion performance of the
   hydrogel on various substrates: skin, polymer, glass, and metal

In vitro cytocompatibility

   The in vitro cytocompatibility of the Si-A/PUE@HA hydrogel was
   systematically evaluated using bone marrow mesenchymal stem cells
   (BMSCs). Live/dead staining results (Fig. [97]3A, B) showed that the
   Si-A/PUE@HA hydrogel supported high cell viability, with only a few
   dead cells (red fluorescence) observed, comparable to the control and
   NC@HA groups, indicating negligible cytotoxicity. Cell proliferation
   was further assessed using the BrdU incorporation assay and the CCK-8
   assay. BrdU staining (Fig. [98]3C, D) revealed a significant increase
   in BrdU-positive cells in the Si-A/PUE@HA group compared to the NC@HA
   group (p < 0.05), demonstrating enhanced DNA synthesis and cell
   proliferation. Similarly, CCK-8 analysis (Fig. [99]3E) showed no
   significant difference in cell viability between the Si-A/PUE@HA group
   and the control, confirming the hydrogel’s ability to support cell
   growth over time. The antioxidative capacity of the hydrogel was
   examined under oxidative stress induced by hydrogen peroxide (H₂O₂). As
   shown in Fig. [100]3F, BMSCs exposed to H₂O₂ exhibited intense green
   fluorescence, indicating elevated intracellular reactive oxygen species
   (ROS). In contrast, treatment with the Si-A/PUE@HA hydrogel markedly
   reduced ROS levels, as quantified by DCFH-DA fluorescence intensity
   (Fig. [101]3G), confirming its potent antioxidative effect. To
   investigate the hydrogel’s impact on cell migration and wound healing,
   a scratch assay was performed. Brightfield images (Fig. [102]3H) and
   quantitative analysis (Fig. [103]3I, J) demonstrated that the
   Si-A/PUE@HA hydrogel significantly promoted BMSC migration and wound
   closure at both 12 and 24 h compared to the NC@HA and control groups
   (p < 0.01), highlighting its strong pro-healing capability.
   Collectively, these results confirm that the Si-A/PUE@HA hydrogel
   exhibits excellent in vitro cytocompatibility by supporting cell
   viability and proliferation while providing significant antioxidative
   and pro-migratory effects. These properties position the Si-A/PUE@HA
   hydrogel as a promising candidate for applications in tissue
   engineering and regenerative medicine.

Fig. 3.

   [104]Fig. 3
   [105]Open in a new tab

   In vitro biocompatibility, antioxidative stress, and wound healing
   evaluation of Si-A/PUE@HA hydrogel. (A) Live/dead cell staining images
   of cells treated with Control, NC@HA, and Si-A/PUE@HA groups. Live
   cells are stained with Calcein-AM (green), and dead cells with PI
   (red). (B) Quantification of the percentage of live cells in each
   group. (C) Immunofluorescence images showing BrdU (green) and DAPI
   (blue) staining to assess cell proliferation in different groups. (D)
   Quantification of BrdU-positive cells relative to DAPI-stained nuclei.
   (E) CCK-8 assay evaluating cell viability across the Control, NC@HA,
   and Si-A/PUE@HA groups. (F) Fluorescence microscopy images of
   intracellular reactive oxygen species (ROS) detected by DCFH-DA
   staining under oxidative stress induced by H₂O₂, with and without
   hydrogel treatment. (G) Quantification of ROS levels based on relative
   fluorescence intensity. (H) Wound healing assay images at 0 h, 12 h,
   and 24 h showing cell migration in different treatment groups. (I)
   Quantification of wound healing percentage at 12 h. (J) Quantification
   of wound healing percentage at 24 h. *Statistical significance: ns (not
   significant), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Promotion of in vitro chondrogenesis in BMSCs

   To evaluate the chondrogenic differentiation potential of the
   Si-A/PUE@HA hydrogel, bone marrow mesenchymal stem cells (BMSCs) were
   cultured in chondrogenic medium for 21 days. All cell pellets developed
   a whitish, round morphology, indicating the formation of cartilage-like
   tissue. Histological analyses were conducted to assess cartilage matrix
   deposition. Hematoxylin and eosin (H&E) staining (Fig. [106]4A)
   revealed that all groups exhibited cartilage tissue formation, with the
   Si-A/PUE@HA group showing the most abundant extracellular matrix (ECM)
   deposition. Alcian Blue staining (Fig. [107]4B) and Safranin O staining
   (Fig. [108]4C) further confirmed the enhanced synthesis of
   glycosaminoglycans (GAGs) and proteoglycans in the Si-A/PUE@HA group
   compared to the control and NC@HA groups. Immunohistochemical staining
   for type II collagen (Col II), a key cartilage-specific ECM component,
   demonstrated the strongest Col II expression in the Si-A/PUE@HA group
   (Fig. [109]4D), suggesting superior cartilage matrix production. The
   Bern score, a histological grading system for cartilage formation, was
   significantly higher in the Si-A/PUE@HA group than in the other groups
   (Fig. [110]4E), indicating improved chondrogenic differentiation.
   Additionally, RT-qPCR analysis showed that the Si-A/PUE@HA hydrogel
   significantly upregulated the expression of key chondrogenic marker
   genes, including Acan (Fig. [111]4F), Sox9 (Fig. [112]4G), and Col2a1
   (Fig. [113]4H), compared to the control and NC@HA groups. In contrast,
   the expression of the fibrocartilage marker gene Col1a1 was
   significantly lower in the Si-A/PUE@HA group (Fig. [114]4I), indicating
   a reduced tendency toward fibrocartilage formation. These findings
   collectively demonstrate that the Si-A/PUE@HA hydrogel effectively
   promotes the chondrogenic differentiation of BMSCs by enhancing
   cartilage-specific matrix production and gene expression. This
   highlights its potential as a promising biomaterial for cartilage
   tissue engineering and regenerative medicine.

Fig. 4.

   [115]Fig. 4
   [116]Open in a new tab

   In vitro chondrogenesis of BMSCs induced by Si-A/PUE@HA hydrogel. (A)
   H&E staining showing the morphology of BMSC-derived cartilage tissue in
   the Control, NC@HA, and Si-A/PUE@HA groups. Scale bar: 50 μm. (B)
   Alcian blue staining indicating glycosaminoglycan (GAG) accumulation in
   the extracellular matrix. (C) Safranin O staining demonstrating
   enhanced proteoglycan deposition in the Si-A/PUE@HA group. (D)
   Immunohistochemical (IHC) staining of type II collagen (COL II),
   confirming cartilage-specific matrix formation. (E) Quantitative
   analysis of Bern scores evaluating cartilage tissue quality. (F–I)
   Relative mRNA expression levels of cartilage-related genes: Acan (F),
   Sox9 (G), Col2a1 (H), and Col1a1 (I), indicating enhanced chondrogenic
   differentiation in the Si-A/PUE@HA group. *Statistical significance: ns
   (not significant), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

In vitro modulation of macrophage polarization

   To evaluate the immunomodulatory properties of the Si-A/PUE@HA
   hydrogel, RAW 264.7 macrophages were cultured with different treatments
   and stimulated with lipopolysaccharide (LPS) for 24 h to mimic an
   inflammatory microenvironment (Fig. [117]5A). Immunofluorescence
   staining revealed that LPS stimulation significantly increased the
   expression of the M1 macrophage marker CD86 (Fig. [118]5B, D),
   indicating enhanced pro-inflammatory polarization. However, treatment
   with the Si-A/PUE@HA hydrogel markedly suppressed CD86 expression
   compared to both the LPS and LPS + NC@HA groups, demonstrating the
   hydrogel’s ability to inhibit M1 macrophage polarization. Conversely,
   the Si-A/PUE@HA hydrogel significantly promoted M2 macrophage
   polarization, as evidenced by the elevated expression of the
   anti-inflammatory marker CD163 (Fig. [119]5C, E). Flow cytometry
   analysis further confirmed these results, showing a substantial
   decrease in CD86⁺ M1 macrophages (Fig. [120]5F, G) and a significant
   increase in CD163⁺ M2 macrophages (Fig. [121]5H, I) in the Si-A/PUE@HA
   group compared to the LPS and LPS + NC@HA groups. Moreover,
   quantitative real-time PCR (qRT-PCR) analysis revealed that the
   Si-A/PUE@HA hydrogel significantly downregulated the expression of
   pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6
   (Fig. [122]5J), while markedly upregulating anti-inflammatory cytokines
   such as IL-4, IL-10, and IL-1RA (Fig. [123]5K). Collectively, these
   results demonstrate that the Si-A/PUE@HA hydrogel effectively modulates
   macrophage polarization by suppressing the pro-inflammatory M1
   phenotype and promoting the anti-inflammatory M2 phenotype. This
   immunoregulatory effect highlights the potential of Si-A/PUE@HA
   hydrogel as a promising therapeutic strategy for controlling
   inflammation and enhancing tissue regeneration.

Fig. 5.

   [124]Fig. 5
   [125]Open in a new tab

   Immunomodulatory effects of Si-A/PUE@HA hydrogel on macrophage
   polarization. (A) Schematic of the experimental design for evaluating
   macrophage responses. RAW 264.7 cells were treated with Control, LPS,
   LPS + NC@HA, or LPS + Si-A/PUE@HA for 24 h, followed by fluorescence
   microscopy, flow cytometry, and qRT-PCR analysis. (B)
   Immunofluorescence staining of CD86 (green, M1 marker), F4/80 (red,
   macrophage marker), and DAPI (blue, nuclei) showing macrophage
   polarization in different groups. (C) Immunofluorescence staining of
   CD163 (green, M2 marker), F4/80 (red), and DAPI (blue) across groups.
   (D) Quantification of relative fluorescence intensity of CD86
   expression. (E) Quantification of relative fluorescence intensity of
   CD163 expression. (F) Flow cytometry analysis of CD86 (M1) and F4/80
   double-positive cells. (G) Quantification of CD86-positive cells from
   flow cytometry data. (H) Flow cytometry analysis of CD163 (M2) and
   F4/80 double-positive cells. (I) Quantification of CD163-positive cells
   from flow cytometry data. (J) Relative mRNA expression of
   pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in different groups.
   (K) Relative mRNA expression of anti-inflammatory cytokines (IL-4,
   IL-10, IL-1RA) in different groups. *Statistical significance: ns (not
   significant), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Si-A/PUE@HA hydrogel enhances in vivo cartilage regeneration

   Building on the promising in vitro results of BMSC chondrogenic
   differentiation, we further investigated the in vivo cartilage repair
   potential of the Si-A/PUE@HA hydrogel using a rat osteochondral defect
   model (Fig. [126]6A). Micro-CT imaging at 6 and 12 weeks
   post-implantation revealed minimal tissue regeneration in the model
   group, with distinct boundaries between the defect and native
   cartilage. In contrast, the Si-A/PUE@HA group showed substantial new
   tissue formation that was more seamlessly integrated with the
   surrounding cartilage, especially at 12 weeks, where the defect was
   nearly filled with smooth, cartilage-like tissue resembling native
   cartilage (Fig. [127]6B). Quantitative analysis of Micro-CT data
   provided further confirmation of these findings. The defect area
   percentage was significantly reduced in the Si-A/PUE@HA group compared
   to the model and NC@HA groups at both 6 and 12 weeks (Supplementary
   Fig. [128]2 A). Notably, the Si-A/PUE@HA group demonstrated the most
   significant reduction in defect area, indicating superior tissue
   regeneration. Additionally, the bone volume/total volume (BV/TV) ratio,
   an indicator of bone regeneration, was markedly higher in the
   Si-A/PUE@HA group than in the other groups at both time points
   (Supplementary Fig. [129]2B), suggesting enhanced subchondral bone
   repair. Histological analyses further confirmed these findings.
   Hematoxylin and eosin (H&E) staining (Fig. [130]6C) and Safranin O/Fast
   Green staining (Fig. [131]6D) demonstrated limited tissue regeneration
   in the model group, characterized by fibrous and poorly integrated
   tissue. In contrast, the Si-A/PUE@HA group exhibited dense,
   cartilage-like tissue with enhanced matrix deposition and improved
   integration with native cartilage. Toluidine blue staining
   (Fig. [132]6E) and type II collagen immunohistochemistry (IHC) staining
   (Fig. [133]6F) revealed increased glycosaminoglycan content and higher
   type II collagen expression in the Si-A/PUE@HA group, indicating the
   formation of hyaline-like cartilage. Quantitative assessments further
   supported these findings. The International Cartilage Repair Society
   (ICRS) scores were significantly higher in the Si-A/PUE@HA group at
   both 6 and 12 weeks compared to the model and NC@HA groups
   (Fig. [134]6G). Similarly, the Modified O’Driscoll Histological Scores
   (MOHS) confirmed superior cartilage repair quality in the Si-A/PUE@HA
   group (Fig. [135]6H). Immunohistochemical quantification of type II
   collagen (Col2) also showed significantly higher mean optical density
   (IOD) in the Si-A/PUE@HA group at both time points (Fig. [136]6I),
   further indicating enhanced cartilage matrix formation. Collectively,
   these results demonstrate that the Si-A/PUE@HA hydrogel significantly
   promotes in vivo cartilage regeneration by enhancing new tissue
   formation, improving tissue integration, and supporting the
   regeneration of hyaline-like cartilage. These findings highlight the
   hydrogel’s strong potential for clinical translation in cartilage
   repair and regenerative medicine.

Fig. 6.

   [137]Fig. 6
   [138]Open in a new tab

   In vivo evaluation of cartilage regeneration promoted by Si-A/PUE@HA
   hydrogel. (A) Schematic diagram of the experimental design for in vivo
   osteochondral defect repair in rats, including surgery, treatment, and
   analysis at 6 and 12 weeks. (B) Micro-CT images showing the repaired
   osteochondral in the Control, Model, NC@HA, and Si-A/PUE@HA groups at 6
   and 12 weeks. (C) Hematoxylin and eosin (H&E) staining displaying
   tissue morphology and cartilage repair in different groups at 6 and 12
   weeks. (D) Safranin O–Fast Green staining indicating proteoglycan
   deposition in regenerated cartilage. (E) Toluidine blue staining
   showing glycosaminoglycan (GAG) distribution in repaired cartilage. (F)
   Immunohistochemical (IHC) staining for type II collagen, confirming
   cartilage-specific matrix formation. (G) Quantitative analysis of ICRS
   scores at 6 and 12 weeks, evaluating cartilage repair quality. (H)
   Quantitative analysis of Modified O’Driscoll Histological Scores (MOHS)
   at 6 and 12 weeks. (I) Quantification of type II collagen (Col2)
   expression based on mean integrated optical density (IOD) at 6 and 12
   weeks. *Statistical significance: ns (not significant), *p < 0.05,
   **p < 0.01, ***p < 0.001, ****p < 0.0001

Si-A/PUE@HA hydrogel modulates immune cell populations and macrophage
polarization

   To investigate the immunoregulatory effects of the Si-A/PUE@HA hydrogel
   on the local tissue microenvironment, single-cell RNA sequencing
   (scRNA-seq) was performed on regenerated tissue from the Model and
   Si-A/PUE@HA groups. UMAP analysis revealed distinct clustering of
   immune and stromal cell populations, including macrophages,
   fibroblasts, chondrocytes, B cells, and T/NK cells, with the
   Si-A/PUE@HA group exhibiting a significant shift towards a more
   regenerative immune landscape compared to the Model group
   (Fig. [139]7A, C). Dot plot analysis showed differential expression of
   key marker genes, with pro-inflammatory markers such as Cd86 highly
   expressed in the Model group, while anti-inflammatory markers like
   Cd163 were markedly upregulated in the Si-A/PUE@HA group
   (Fig. [140]7B). To further elucidate the molecular mechanisms
   underlying the immunoregulatory and chondrogenic effects of the
   Si-A/PUE@HA hydrogel, functional enrichment analysis of differentially
   expressed genes (DEGs) was performed on monocytes/macrophages and
   chondrocytes. The heatmap analysis revealed distinct expression
   patterns of key DEGs across various cell types, including chondrocytes,
   fibroblasts, neutrophils, B cells, monocytes/macrophages, proliferating
   cells, neural progenitor cells (NPCs), plasmacytoid dendritic cells
   (pDCs), and endothelial cells (ECs) (Supplementary Fig. [141]3 A). Gene
   ontology (GO) term enrichment word clouds provided deeper insights into
   the functional roles of DEGs in monocytes/macrophages and chondrocytes
   (Supplementary Fig. [142]3B). In monocytes/macrophages, genes
   associated with inflammation regulation and immune modulation, such as
   Trem2 and Pycarr, were highly enriched. In chondrocytes, key genes
   involved in cartilage development and matrix synthesis, including Sox9,
   Vegfa, and Bmp2, showed prominent enrichment. The larger and
   warmer-colored gene names in the word clouds represent higher
   enrichment scores, emphasizing the critical roles of these genes in
   modulating inflammation and promoting chondrogenesis. Gene set
   enrichment analysis (GSEA) further highlighted that Si-A/PUE@HA
   treatment suppressed inflammatory pathways and promoted
   immune-regulatory and tissue repair pathways (Fig. [143]7D).
   Macrophage-specific analysis identified four subpopulations: C0 (C6⁺
   macrophages), C1 (Tiparp⁺ macrophages), C2 (Mmp12⁺ macrophages), and C3
   (Vcam1⁺ macrophages), with the Si-A/PUE@HA group displaying reduced
   pro-inflammatory C2 macrophages and increased anti-inflammatory C1 and
   C3 macrophages (Fig. [144]7E). Heatmap analysis confirmed elevated
   expression of anti-inflammatory and tissue-regenerative genes in the
   Si-A/PUE@HA group, alongside reduced pro-inflammatory gene expression
   (Fig. [145]7F, G). Finally, scatter plot analysis demonstrated
   transcriptional reprogramming of macrophages, with a shift from
   pro-inflammatory phenotypes to anti-inflammatory, tissue-repairing
   states induced by Si-A/PUE@HA treatment (Fig. [146]7H). These findings
   indicate that the Si-A/PUE@HA hydrogel effectively modulates immune
   cell composition and macrophage polarization, creating a favorable
   microenvironment for tissue regeneration.

Fig. 7.

   [147]Fig. 7
   [148]Open in a new tab

   Single-cell RNA sequencing analysis reveals the immunoregulatory
   effects of Si-A/PUE@HA hydrogel. (A) UMAP plot showing the distribution
   of different cell populations in the Model and Si-A/PUE@HA groups,
   highlighting distinct immune cell clusters. (B) Dot plot displaying the
   expression levels of representative marker genes across various cell
   types and treatment groups. The size and color of the dots represent
   gene expression levels and the percentage of expressing cells,
   respectively. (C) Proportional distribution of cell populations between
   the Model and Si-A/PUE@HA groups. (D) Gene set enrichment analysis
   (GSEA) of differentially expressed genes (DEGs) between the Model and
   Si-A/PUE@HA groups, indicating enriched biological pathways. (E)
   Clustering of macrophage subpopulations, including C0
   (C6 + macrophages), C1 (Tgmp + macrophages), C2 (Ympt2 + macrophages),
   and C3 (Vcam1 + macrophages). (F) Heatmap showing the mean expression
   of immune-related genes across different macrophage subtypes in both
   groups. (G) Heatmap of differentially expressed genes related to immune
   modulation between the Model and Si-A/PUE@HA groups. (H) Scatter plots
   illustrating the gene expression correlations among distinct macrophage
   subtypes, highlighting transcriptional shifts induced by Si-A/PUE@HA
   treatment

Modulation of macrophage subtypes and polarization

   Single-cell RNA sequencing (scRNA-seq) and functional analyses were
   conducted to investigate the immunomodulatory effects of the
   Si-A/PUE@HA hydrogel on macrophage polarization and differentiation.
   Heatmap analysis revealed distinct distributions of macrophage
   subtypes—C0 (C6⁺ macrophages), C1 (Tiparp⁺ macrophages), C2 (Mmp12⁺
   macrophages), and C3 (Vcam1⁺ macrophages)—between the Model and
   Si-A/PUE@HA groups, with the Si-A/PUE@HA group showing a significant
   reduction in pro-inflammatory C2 (Mmp12⁺) macrophages and an increase
   in anti-inflammatory C1 (Tiparp⁺) and C3 (Vcam1⁺) subtypes
   (Fig. [149]8A). UMAP visualization confirmed that Si-A/PUE@HA treatment
   effectively suppressed M1 macrophage polarization while promoting M2
   macrophage phenotypes, as indicated by reduced pro-inflammatory gene
   expression and increased anti-inflammatory gene expression
   (Fig. [150]8B). Pseudotime trajectory analysis further demonstrated
   that macrophage differentiation in the Si-A/PUE@HA group favored
   anti-inflammatory lineages, contrasting with the pro-inflammatory
   trajectory in the Model group (Fig. [151]8C–F), and gene expression
   dynamics of key markers (C6, Tiparp, Mmp12, Vcam1) validated this shift
   (Fig. [152]8G). Further analysis revealed lower genomic instability in
   the Si-A/PUE@HA group, indicated by reduced CNVscore (Supplementary
   Fig. [153]4 A), along with enhanced stemness properties (higher
   Cell_Stemness_AUC score) and reduced macrophage proliferation, as shown
   by decreased G2/M and S phase cell cycle scores (Supplementary
   Fig. [154]4B–D). Additionally, gene expression profiling showed
   downregulation of M1 markers and pro-inflammatory genes, with
   upregulation of M2 markers and anti-inflammatory genes in the
   Si-A/PUE@HA group (Supplementary Fig. [155]5A–D). GO enrichment
   analysis further highlighted that immune regulation, cytokine
   signaling, leukocyte migration, apoptosis inhibition, and stress
   response pathways were enriched in anti-inflammatory macrophage
   subtypes (Supplementary Fig. [156]5E). Collectively, these findings
   demonstrate that the Si-A/PUE@HA hydrogel effectively reprograms
   macrophage polarization by suppressing pro-inflammatory M1 phenotypes
   and enhancing anti-inflammatory M2 differentiation, while also
   improving genomic stability, promoting stemness, and reducing
   proliferation, thereby fostering a regenerative microenvironment
   conducive to tissue repair.

Fig. 8.

   [157]Fig. 8
   [158]Open in a new tab

   Analysis of macrophage polarization and lineage trajectory influenced
   by Si-A/PUE@HA hydrogel. (A) Heatmap showing the distribution of
   macrophage subtypes (C0 C6+, C1 Tiparp+, C2 Mmp12+, C3 Vcam1+) in the
   Model and Si-A/PUE@HA groups. (B) UMAP visualization of M1/M2
   macrophage polarization and pro-/anti-inflammatory gene expression
   profiles. (C) Pseudotime trajectory analysis illustrating the
   differentiation paths of macrophage subtypes. (D) Predicted ordering of
   macrophage subtypes along the differentiation trajectory. (E)
   Proportional distribution of cell states in different groups during
   macrophage differentiation. (F) UMAP plot displaying the lineage
   trajectory of macrophage subpopulations, with Lineage 1 and Lineage 2
   marked. (G) Expression dynamics of marker genes (C6, Tiparp, Mmp12,
   Vcam1) along pseudotime across different lineages

Si-A/PUE@HA hydrogel enhances chondrocyte differentiation and cartilage
regeneration

   Single-cell RNA sequencing (scRNA-seq) analysis was conducted to
   explore the chondrogenic regulatory effects of the Si-A/PUE@HA
   hydrogel. UMAP analysis revealed six distinct chondrocyte
   subpopulations: C0 (Btg2⁺ chondrocytes), C1 (Tiam2⁺ chondrocytes), C2
   (Ibsp⁺ chondrocytes), C3 (Col11a2⁺ chondrocytes), C4 (Lyz2⁺
   chondrocytes), and C5 (Cd74⁺ chondrocytes) (Fig. [159]9A). Feature
   plots demonstrated elevated expression of key chondrogenic markers
   (Fos, Lyz2, Tiam2, Cd74, Col11a2, and Ibsp) in the Si-A/PUE@HA group
   compared to the Model group, indicating enhanced chondrogenic activity
   (Fig. [160]9B). Hierarchical clustering and heatmap analyses further
   confirmed distinct gene expression profiles among these chondrocyte
   subtypes, with significant upregulation of cartilage-related genes in
   the Si-A/PUE@HA group (Fig. [161]9C–D). Additionally, the distribution
   of chondrocyte subpopulations showed an increased proportion of
   regenerative C3 (Col11a2⁺) and C5 (Cd74⁺) chondrocytes in the
   Si-A/PUE@HA group, suggesting enhanced cartilage formation
   (Fig. [162]9E). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and
   Genomes (KEGG) enrichment analyses revealed that differentially
   expressed genes (DEGs) in the Si-A/PUE@HA group were significantly
   enriched in pathways related to extracellular matrix (ECM)
   organization, wound healing, and cartilage development (Fig. [163]9F).
   Pseudotime trajectory analysis demonstrated accelerated chondrocyte
   differentiation and maturation in the Si-A/PUE@HA group, with dynamic
   gene expression changes supporting cartilage regeneration
   (Fig. [164]9G–H). Cell-cell interaction network analysis further
   highlighted stronger communication between chondrocytes and other
   regenerative cells in the Si-A/PUE@HA group, facilitating coordinated
   cartilage repair (Fig. [165]9I–J).

Fig. 9.

   [166]Fig. 9
   [167]Open in a new tab

   Single-cell RNA sequencing analysis reveals the chondrogenic regulatory
   effect of Si-A/PUE@HA hydrogel. (A) UMAP plot showing distinct
   chondrocyte subpopulations, including C0 Btg2 + chondrocytes, C1
   Tiam2 + chondrocytes, C2 Ibsp + chondrocytes, C3
   Col11a2 + chondrocytes, C4 Lyz2 + chondrocytes, and C5
   Cd74 + chondrocytes. (B) Feature plots displaying the expression of key
   marker genes (Fos, Lyz2, Tiam2, Cd74, Col11a2, and Ibsp) across
   different chondrocyte clusters. (C) Hierarchical clustering and
   correlation heatmap showing gene expression profiles across chondrocyte
   subtypes. (D) Heatmap of differentially expressed genes (DEGs) among
   chondrocyte subpopulations. (E) Heatmap illustrating the distribution
   of chondrocyte subpopulations between the Model and Si-A/PUE@HA groups.
   (F) GO (Gene Ontology) and KEGG pathway enrichment analysis of DEGs in
   different chondrocyte clusters, highlighting pathways involved in
   extracellular matrix organization, wound healing, and cartilage
   development. (G) Predicted ordering of chondrocyte subtypes during
   differentiation. (H) Pseudotime trajectory analysis revealing the
   dynamic progression of chondrocyte differentiation and gene expression
   changes over time. (I) Cell-cell interaction networks depicting
   communication between chondrocyte subtypes and other cell populations
   in the Model and Si-A/PUE@HA groups

Si-A/PUE@HA composite hydrogel promotes cartilage regeneration by activating
the PI3K-AKT pathway

   To investigate the mechanism underlying the cartilage repair promoted
   by the Si-A/PUE@HA hydrogel, we evaluated inflammatory markers,
   chondrogenic proteins, and the activation of the PI3K-AKT pathway in
   the regenerated cartilage. Western blot analysis of cartilage tissue
   extracts demonstrated a significant reduction in the pro-inflammatory
   markers iNOS and CD86 in the Si-A/PUE@HA group compared to the Model
   group, along with increased expression of CD163, a key M2 macrophage
   marker, indicating a shift toward an anti-inflammatory microenvironment
   (Fig. [168]10A, B). Immunofluorescence staining of the M1 marker CD86
   showed a significant reduction in pro-inflammatory macrophage
   infiltration in the Si-A/PUE@HA group at both 6 and 12 weeks compared
   to the Model and NC@HA groups (Fig. [169]10C, D), with quantitative
   analysis confirming this decrease (Supplementary Fig. [170]6 A, B).
   Additionally, Si-A/PUE@HA hydrogel treatment enhanced cartilage repair
   by upregulating the expression of critical chondrogenic markers.
   Western blot analysis revealed increased levels of SOX9, Col2a1 (type
   II collagen), and Aggrecan in the Si-A/PUE@HA group, reflecting
   improved chondrocyte differentiation and extracellular matrix
   synthesis. Simultaneously, the expression of cartilage matrix-degrading
   enzymes MMP-13 and Adamts-5 was significantly reduced, suggesting
   protection against cartilage degradation (Fig. [171]10E, F).
   Immunofluorescence staining confirmed enhanced expression of ACAN and
   SOX9 at both 6 and 12 weeks post-implantation, further demonstrating
   the hydrogel’s role in promoting cartilage matrix formation
   (Fig. [172]10G, H, Supplementary Fig. [173]7 A, B). To elucidate the
   molecular mechanism driving these effects, we examined the activation
   of the PI3K-AKT signaling pathway. Western blot analyses showed
   upregulation of PI3K, phosphorylated AKT (p-AKT), and mTOR in the
   Si-A/PUE@HA group compared to the Model group (Fig. [174]10I, J),
   indicating activation of this pathway. Given the well-established role
   of the PI3K-AKT pathway in regulating cell proliferation, survival, and
   chondrogenesis, these findings suggest that the Si-A/PUE@HA hydrogel
   promotes cartilage regeneration by modulating inflammation and
   activating PI3K-AKT signaling, thereby enhancing chondrogenic
   differentiation and matrix production while mitigating cartilage
   degradation.

Fig. 10.

   [175]Fig. 10
   [176]Open in a new tab

   Si-A/PUE@HA composite hydrogel promotes cartilage regeneration by
   activating the PI3K-AKT pathway. (A) Western blot analysis of
   inflammatory markers in cartilage tissue. The expression of
   pro-inflammatory markers iNOS and CD86 was significantly reduced, while
   the M2 macrophage marker CD163 was increased in the Si-A/PUE@HA group
   compared to the Model group. (B) Quantification of relative protein
   expression levels of iNOS, CD86, and CD163, demonstrating a shift
   toward an anti-inflammatory microenvironment in the Si-A/PUE@HA group
   (*p < 0.05, **p < 0.01). (C) Representative images showing CD86
   expression in cartilage tissue at 6 weeks post-implantation.
   CD86-positive areas (red) were reduced in the Si-A/PUE@HA group
   compared to the Model group. (D) Immunofluorescence images of CD86 at
   12 weeks post-implantation, further confirming decreased CD86
   expression in the Si-A/PUE@HA group compared to the Control, Model, and
   NC@HA groups. Scale bars: 200 μm. (E) Western blot analysis of key
   chondrogenic proteins (SOX9, Col2a1, Aggrecan) and cartilage-degrading
   enzymes (MMP-13, Adamts-5). The Si-A/PUE@HA group exhibited increased
   SOX9, Col2a1, and Aggrecan expression, along with decreased MMP-13 and
   Adamts-5 levels. (F) Quantification of protein expression levels,
   confirming significant upregulation of chondrogenic markers and
   suppression of cartilage degradation enzymes in the Si-A/PUE@HA group
   (*p < 0.05, **p < 0.01). (G) ACAN expression (green) at 6 and 12 weeks
   post-implantation, demonstrating increased cartilage matrix formation
   in the Si-A/PUE@HA group compared to the Model and NC@HA groups. (H)
   SOX9 expression (green) at 6 and 12 weeks post-implantation, confirming
   enhanced chondrogenic differentiation in the Si-A/PUE@HA group. Scale
   bars: 200 μm. (I) Western blot analysis of PI3K, phosphorylated AKT
   (p-AKT), and mTOR in regenerated cartilage. The Si-A/PUE@HA group
   exhibited increased expression of PI3K, p-AKT, and mTOR compared to the
   Model group. (J) Quantification of protein expression levels,
   confirming significant activation of the PI3K-AKT signaling pathway in
   the Si-A/PUE@HA group (*p < 0.05, ***p < 0.001)

In vivo biocompatibility of Si-A/PUE@HA

   To evaluate the long-term in vivo biocompatibility of the Si-A/PUE@HA
   hydrogel, major organs—including the heart, liver, spleen, lungs, and
   kidneys—were examined 12 weeks post-implantation. Hematoxylin and eosin
   (HE) staining revealed no histopathological abnormalities or structural
   damage in any of the examined organs across all groups, indicating the
   absence of tissue inflammation or necrosis (Supplementary Fig. 8 A).
   Furthermore, Masson’s trichrome staining confirmed no significant
   collagen deposition or fibrosis in the tissues, further supporting the
   hydrogel’s non-fibrotic and biocompatible nature (Supplementary
   Fig. 8B). Immunofluorescence staining for F4/80 (a macrophage marker)
   and α-SMA (a fibrosis marker) showed minimal immune cell infiltration
   and no significant fibrotic response in the Si-A/PUE@HA group,
   comparable to the control group (Supplementary Fig. 8 C, D). These
   findings collectively demonstrate that the Si-A/PUE@HA hydrogel
   exhibits excellent in vivo biocompatibility, causing no adverse immune
   reactions or organ toxicity after long-term implantation.

Discussion

   Osteochondral defects, characterized by damage to both the cartilage
   and underlying bone, represent a significant challenge in orthopedic
   medicine. These injuries can arise from trauma, degenerative diseases,
   or conditions such as osteochondritis dissecans, often leading to
   debilitating joint pain and reduced mobility. The natural repair
   capacity of cartilage is limited due to its avascular nature, which
   compounds the difficulty of achieving effective healing [[177]31].
   Consequently, untreated osteochondral defects frequently progress to
   osteoarthritis, a leading cause of disability worldwide, underscoring
   the urgent need for optimized therapeutic strategies to restore
   cartilage function and joint integrity.

   In this study, we investigated the potential of Si-A/PUE@HA composite
   hydrogels as a novel scaffold for cartilage tissue engineering. By
   employing a multi-step synthesis approach, we developed hydrogels that
   exhibit excellent biocompatibility and mechanical properties conducive
   to supporting cell proliferation and differentiation. Our research
   aimed to assess the efficacy of these hydrogels in promoting
   chondrogenic differentiation of stem cells and facilitating cartilage
   repair in vivo. The findings from this study demonstrate that
   Si-A/PUE@HA hydrogels not only enhance cellular activity but also
   modulate the inflammatory response, thereby contributing to improved
   healing outcomes in osteochondral defects [[178]32, [179]33].

   The Si-A/PUE@HA composite hydrogel has demonstrated significant promise
   as a bioactive material for cartilage tissue engineering. Its
   biocompatibility is evidenced by high cell viability and proliferation
   rates, indicating that it provides a supportive environment for the
   growth of bone marrow mesenchymal stem cells (BMSCs) which are crucial
   for cartilage repair. Compared to other hydrogels, such as those
   derived from chitosan or gelatin, Si-A/PUE@HA hydrogels show superior
   properties in terms of mechanical strength and elasticity, allowing for
   better integration and function within the joint environment [[180]34].
   The mechanisms underlying the hydrogel’s cytocompatibility likely
   involve its unique structural composition, which promotes cell adhesion
   and minimizes inflammatory responses, a critical factor in regenerative
   medicine [[181]35].

   In terms of promoting chondrogenic differentiation, Si-A/PUE@HA
   hydrogels significantly enhance the expression of key chondrogenic
   markers such as Acan, Sox9, and Col2a1. This is particularly relevant
   for addressing cartilage degeneration, as previous studies have
   identified that the effective promotion of chondrogenesis is vital for
   restoring cartilage integrity and function in conditions like
   osteoarthritis [[182]36]. The mechanical properties of hydrogels also
   play a pivotal role; hydrogels with tunable stiffness and elasticity
   have been shown to influence stem cell fate, guiding differentiation
   towards chondrocytes rather than fibrotic pathways [[183]37, [184]38].
   Thus, the ability of Si-A/PUE@HA hydrogels to optimize these properties
   positions them as advanced candidates for clinical applications in
   cartilage repair.

   The immunomodulatory properties of Si-A/PUE@HA hydrogels are
   noteworthy, as they effectively modulate macrophage polarization. This
   hydrogel reduces pro-inflammatory M1 macrophage presence while
   promoting the anti-inflammatory M2 phenotype, an essential mechanism
   for a favorable healing environment [[185]39]. Such polarization has
   been associated with improved outcomes in tissue engineering, as a
   balanced inflammatory response can enhance tissue integration and
   regeneration. The significance of this finding underscores the
   potential of Si-A/PUE@HA hydrogels not only as scaffolds for cell
   growth but also as active agents in managing inflammation during the
   healing process.

   In vivo studies further support the viability of Si-A/PUE@HA hydrogels
   for cartilage regeneration, with micro-CT analyses revealing
   significant new tissue formation and integration with surrounding
   cartilage. This aligns with findings from other research indicating
   that hydrogels can enhance cartilage repair by providing a structurally
   and biochemically favorable environment for tissue growth and
   integration [[186]40]. The reduction in defect area observed in treated
   groups illustrates the hydrogel’s effectiveness in promoting
   regeneration, which is critical for translating these findings into
   clinical practice for managing osteochondral defects.

   Long-term biocompatibility studies of the Si-A/PUE@HA hydrogel also
   indicate its safety for clinical applications, with minimal immune
   response and no significant histopathological abnormalities noted in
   major organs after prolonged exposure. This is crucial for any
   biomaterial intended for implantation, as it highlights the hydrogel’s
   potential to minimize adverse effects and enhance patient safety
   [[187]41]. Such positive outcomes from both in vitro and in vivo
   assessments point towards the hydrogel’s readiness for further
   evaluation in larger clinical trials aimed at assessing its efficacy in
   human patients. Overall, the findings from this study substantiate the
   Si-A/PUE@HA composite hydrogel as a transformative biomaterial in the
   field of cartilage repair and regenerative medicine.

   The limitations of this study warrant careful consideration in the
   context of its findings. Notably, the relatively small sample size may
   hinder the generalizability of the results, and the absence of clinical
   validation restricts the applicability of the Si-A/PUE@HA hydrogel in
   real-world scenarios. Additionally, potential batch effects in the data
   analysis could introduce variability, leading to discrepancies in the
   interpretation of the hydrogel’s performance across different
   experiments. Future investigations should aim to address these
   limitations by incorporating larger cohorts and clinical trials to
   substantiate the efficacy and safety of this innovative biomaterial in
   cartilage repair applications. Furthermore, this study did not directly
   evaluate the in vitro or in vivo degradation kinetics of the hydrogel,
   which is an important factor influencing long-term scaffold integration
   and tissue remodeling. While the inclusion of biodegradable components
   like hyaluronic acid and puerarin suggests favorable breakdown
   behavior, future research should incorporate systematic
   biodegradability assessments under physiological conditions. Future
   investigations should aim to address these limitations by incorporating
   larger cohorts and clinical trials to substantiate the efficacy and
   safety of this innovative biomaterial in cartilage repair applications.
   It is also important to acknowledge the limitations associated with the
   use of a rat model for in vivo cartilage repair evaluation. While rats
   offer advantages such as low cost, reproducibility, and suitability for
   early-stage biomaterial screening, their articular cartilage is
   significantly thinner and structurally different from that of larger
   animals and humans. These differences may limit the translational
   relevance of observed regenerative outcomes. Furthermore, the rat joint
   environment does not fully recapitulate the load-bearing and
   biomechanical complexity of human knees. Future studies should
   incorporate larger animal models, such as rabbits or goats, which
   possess thicker cartilage and more closely mimic human joint
   physiology, to validate the therapeutic efficacy and long-term
   integration of the Si-A/PUE@HA hydrogel.

   In summary, the Si-A/PUE@HA hydrogel demonstrates significant promise
   as a biomaterial for cartilage regeneration, showcasing robust
   cytocompatibility, enhanced chondrogenic differentiation, and effective
   immunomodulatory properties. The in vivo results further indicate its
   potential for substantial cartilage repair, supporting its viability
   for clinical translation in regenerative medicine. These findings pave
   the way for future research focused on optimizing the hydrogel
   formulation and validating its therapeutic benefits in larger clinical
   populations, ultimately contributing to improved outcomes for patients
   suffering from osteochondral defects.

Supplementary Information

   [188]Supplementary Material 1^ (2.1MB, docx)
   [189]Supplementary Material 2^ (19.9MB, mp4)

Acknowledgements