The 3D printed biphasic scaffolds incorporating epimedin C promotes osteochondral regeneration in osteoarthritis rats
Xiangbo Meng, Le Chang, Huajun Wang, Jiming Li, Cuishan Huang, Yuxi Jiang, Yuting Zhang, Huijuan Cao, Ling Li, Wenyao Yang, Jiake Xu, Ling Qin, Xiaofei Zheng, Wenxiang Cheng, Xinluan Wang
TL;DR
A 3D printed scaffold with epimedin C improves joint repair in rats with osteoarthritis by reducing inflammation and boosting tissue regeneration.
Contribution
A novel biphasic scaffold incorporating epimedin C for enhanced osteochondral regeneration in osteoarthritis.
Findings
The PTP@Epi C scaffold significantly promoted subchondral bone regeneration in a rat OA-OCD model.
Epi C inhibited TNF-α expression in synovial tissue, reducing synovitis.
Epi C suppresses NLRP3 mRNA and IL-1β secretion by inhibiting NF-κB activity.
Abstract
The regeneration of osteochondral defect in osteoarthritis (OA-OCD) remains a significant clinical challenge, frequently accompanied by synovial inflammation and persistent joint pain. Tissue engineering scaffolds show promising application prospects; however, due to their lack of bioactive molecules, they often fail to effectively regulate extracellular matrix remodeling and inflammatory responses, resulting in poor repair outcomes. In our previous study, we identified epimedin C (Epi C) as one of the most bioactive compounds from Xianlinggubao (XLGB) through 3D human osteoarthritic chondrocyte pellet cultures, demonstrating significant anti-inflammatory and anabolic effects. In this study, we developed a novel biphasic scaffold incorporating Epi C to enhance osteochondral regeneration in an OA-like microenvironment. The biphasic scaffold consists of a cartilage phase [PLGA (50:50)]…
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Figure 9| PLGA (50:50) | PLGA (50:50) @Epi C | PLGA (75:25)/ β-TCP | PLGA (75:25)/ β-TCP@Epi C | |
|---|---|---|---|---|
| Macropore size (μm) | 213.36 ± 12.21 | 200.24 ± 8.25 | 438.60 ± 6.73 | 444.58 ± 7.90 |
| Micropores size (μm) | 0.35 ± 0.14 | 0.39 ± 0.11 | 4.49 ± 0.67 | 4.75 ± 1.30 |
| Porosity (%) | 45.16 ± 0.95 | 47.00 ± 2.32 | 65.19 ± 5.82 | 69.67 ± 4.39 |
| Stiffness (N/mm) | 493.23 ± 27.02 | 446.30 ± 49.21 | 258.33 ± 70.37 | 251.83 ± 57.85 |
| E-modulus (MPa) | 46.56 ± 3.05 | 43.87 ± 2.68 | 33.07 ± 2.97 | 30.38 ± 4.74 |
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Taxonomy
TopicsMedicinal Plant Pharmacodynamics Research · Osteoarthritis Treatment and Mechanisms · Bone Tissue Engineering Materials
Introduction
Osteochondral defect (OCD) primarily results from wear and accidental trauma, such as sports injuries, which can lead to osteoarthritis (OA) and significant joint pain [1, 2]. Articular cartilage is an avascular and aneural tissue, which severely limits its self-regenerative capacity [3]. Furthermore, defective regions are often associated with progressive synovitis and aberrant subchondral bone remodeling, which further impede the repair process [4, 5]. Current clinical therapies, such as microfracture and autologous chondrocyte implantation, exhibit limitations including inadequate mechanical properties of the regenerated tissue, poor integration rates and donor-site morbidity [6, 7]. Tissue engineering scaffolds offer a promising regenerative strategy [2]. However, due to the insufficient presence of bioactive molecules, tissue engineering scaffolds face challenges in actively guiding and regulating critical biological processes, including extracellular matrix (ECM) remodeling and the modulation of inflammatory responses [8, 9]. This limitation leads to low efficiency in tissue regeneration and suboptimal repair outcomes, significantly hindering their potential for clinical application. Therefore, the integration of bioactive molecules into scaffold materials to create bioactive-functionalized tissue engineering scaffolds represents a critical and promising strategy for overcoming these limitations and enhancing scaffold efficacy.
Three-dimensional (3D) printing technology has emerged as an ideal method for fabricating biological scaffolds due to its exceptional ability to accurately produce complex structures and allow for individual customization [10, 11]. Porous scaffolds are extensively employed in tissue regeneration research due to their remarkable properties that enhance cell migration and nutrient transport [12, 13]. Polylactic acid-co-glycolic acid (PLGA) and β-Tricalcium phosphate (β-TCP) composites are widely investigated biomaterials in bone tissue engineering due to their combined advantages of biocompatibility, degradability and osteoconductivity [14, 15]. PLGA, a synthetic copolymer, is approved by the Food and Drug Administration (FDA) for medical applications due to its biocompatibility and controllable degradation, which allows it to be tailored for various drug delivery and tissue regeneration [15, 16]. β-TCP, a bioceramic, is known for its osteoconductive properties, promoting bone integration and mineralization, making it suitable as a bone graft substitute [17, 18]. The combination of PLGA and β-TCP enhances the mechanical strength and biological activity of scaffolds, providing a favorable microenvironment for tissue regeneration [19, 20].
Traditional Chinese medicine (TCM) formulas are widely utilized in China for the prevention and treatment of OA and osteoporosis. Among these, Xianlinggubao (XLGB) capsule and Gushukang capsule stand out as representative Chinese medicines that have garnered substantial practical experience and research evidence in clinical practice [21, 22]. In our previous study, using a 3D pellet culture system established with primary chondrocytes from osteoarthritic patients, we screened 34 compounds from XLGB and identified epimedin C (Epi C) as the most promising bioactive molecule for treatment of OA [23, 24]. In those studies, Epi C significantly enhanced glycosaminoglycan (GAG) synthesis, upregulated the expression of COL2A1 and aggrecan (ACAN) and suppressed MMP activity, collectively indicating its protective role in cartilage matrix remodeling [23, 24]. Furthermore, we validated its efficacy in medial meniscus transection (MMT) induced OA rat model. The results indicated that Epi C significantly increased the expression of Type II collagen (COL II) and ACAN in the cartilage, while decreasing the expression of matrix metalloproteinase-13 (MMP-13) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), thereby effectively protecting articular cartilage [25]. Given these positive results, we speculate that the local delivery of Epi C may provide a more effective strategy for the treatment of OA. In this study, we incorporated Epi C into a biphasic scaffold, utilizing a layered controlled release design to synergistically enhance osteochondral regeneration and mitigate synovitis. This approach presents a novel paradigm for the advancement of functional tissue engineering scaffolds.
Materials and methods
Preparation of biphasic scaffolds
Biphasic scaffolds were fabricated using a low-temperature 3D printer (SUNP Alpha-BP21, SUNP Biotechnology, Beijing, China) as established in our previous research [26]. PLGA [with a lactide/glycolide ratio of 75:25, average molecular weight (Mw)=15 kDa, viscosity = 1.18 dL/g] was dissolved in 1,4-dioxane (Shanghai Ling Feng Chemical Reagent Co., Ltd., Shanghai, China) to prepare a PLGA (75:25) solution. In the subsequent step, β-TCP (Aladdin) was incorporated into the mixture at a weight ratio of 4:1 and mixed overnight using a magnetic stirrer to obtain a uniform slurry, referred to as PLGA (75:25)/β-TCP slurry. Epi C (dissolved in DMSO, Chengdu Ruifen Si Biotechnology Co., Ltd., Chengdu, China) was blended with the PLGA (75:25)/β-TCP slurry to achieve a final Epi C concentration of 1.0% (w/w), which was determined according to our prior research findings [25]. The mixture was then homogenized using a magnetic stirrer at 500 rpm for 2 h to obtain the PLGA (75:25)/β-TCP@Epi C slurry. A similar methodology was employed to prepare the PLGA (50:50) slurry (with a lactide/glycolide ratio of 50:50, average Mw = 6.3 kDa, viscosity = 0.71 dL/g) combined with 1.0% Epi C, referred to as PLGA (50:50)@Epi C slurry. The drug-loaded slurries were sequentially loaded into a low-temperature 3D printer, and a biphasic Epi C-incorporated scaffold (denoted as PTP@Epi C) was fabricated layer-by-layer at −30°C. The printing parameters were set as follows: for the lower PLGA (75:25)/β-TCP@Epi C layer, the parameters were a line distance of 1.0 mm, a layer height of 0.12 mm, a printing speed of 20 mm/s and an extrusion speed of 0.8 mm^3^/s, for the upper PLGA (50:50)@Epi C layer, the parameters were a line distance of 0.45 mm, a layer height of 0.12 mm, a printing speed of 15 mm/s and an extrusion speed of 1.0 mm^3^/s. By adjusting the line distance parameters (0.45 mm, 1.0 mm, 1.5 mm), drug-loaded scaffolds with different pore diameters were prepared. Subsequently, the PTP@Epi C scaffold underwent a freeze-drying process in a freeze dryer at 0.1 mbar for 72 h (Christ Alpha 2-4 LD Plus, Germany). Following the same protocol, an Epi C-free scaffold was produced and designated as a PTP scaffold.
Characterization of the porous biphasic scaffolds
Morphological characterization
The microstructure of the biphasic scaffold was analyzed using scanning electron microscopy (SEM) with a ZEISS Supra 55 instruments. Three samples, each measuring 5 × 5 × 1 mm^3^, were prepared and positioned on the sample holder for imaging purposes. To improve electrical conductivity, a thin gold layer was deposited onto the scaffold’s surface prior to SEM examination. Additionally, energy-dispersive spectroscopy (EDS) was employed to analyze the distribution of calcium and phosphorus elements of β-TCP in the biphasic scaffold, verifying the uniformity of β-TCP distribution within the biphasic scaffold. Given the irregular macro-porous morphology of the scaffold and nonstandard circular shape, we quantified its pore size by measuring both the maximum diameter (long axis) and the minimum diameter (short axis) of the macropores, taking the average of these measurements as the representative pore size of the scaffold.
Porosity
The porosity of the biphasic scaffold was determined using liquid displacement methods [27]. To begin the evaluation, the scaffolds with varying pore sizes were sectioned into uniform cubes measuring 10 × 10 × 10 mm^3^, with a total of three samples (n = 3) included in the analysis. The initial volume and weight of each scaffold were measured and recorded as V_s_ and W1, respectively. Following this, the scaffolds were immersed in ethanol to facilitate the measurement process. After sufficient immersion, the scaffolds were removed from the ethanol, ensuring that there was no residual bubbling present, which could affect the accuracy of the results. The weight of the scaffolds postimmersion was then recorded as W_2_. The porosity of the scaffolds was subsequently calculated using these measurements.
W 1: the scaffold weight; W2: the ethanol-saturated scaffold weight; ρ_e_: the density of ethanol; V_s_: the volume of the scaffold.
Mechanical properties
The scaffolds (n = 3) were shaped into cubes measuring 10 × 10 × 10 mm^3^. A mechanical testing machine (MX-0350, China) equipped with a 500 N load cell was employed to evaluate the mechanical properties. Initially, the scaffold was in an undeformed condition (zero displacement), with the loading point applying a slight preload of 0.1 N to secure the scaffold in position. Subsequently, confined compression testing occurred at a displacement rate of 1 mm/min until the sample’s thickness reduced to 70% of its original measurement. Throughout the compression process, the force values were recorded to generate load–displacement curves. These curves were then converted into stress–strain curves for the scaffolds following the guidelines outlined by the International Organization for Standardization (ISO 844:2021). The stiffness and elastic modulus (E-modulus) of each sample were calculated from the linear portion of each load–displacement and stress–strain curve.
Tensile testing
To evaluate the interface’s stability of the biphasic scaffold, the scaffold (30 mm × 10 mm × 5 mm) was fixed to a mechanical testing machine for tensile testing. The beam movement speed was set at 50 mm/min, and loading was maintained until the sample fractured, recording the corresponding force–displacement curve (n = 4).
X-ray diffraction analysis
To verify the crystallographic integrity of β-TCP in PLGA/β-TCP scaffold and PLGA/β-TCP@Epi C scaffold, X-ray diffraction (XRD) analysis was conducted using a D8 Advance 10301 X-ray diffractometer (Bruker-AXS, Germany). Data collection was performed in the 2θ range of 20° to 80°, with an angular step size of 0.0170° a scan step of 10.1600 s, allowing for the determination of the characteristic diffraction peaks of β-TCP.
Scaffold degradation and drug release in vitro
To investigate the degradation and drug release behavior of PTP@Epi C scaffolds, we devised a testing system simulating physiological conditions [28]. The scaffolds with different pore sizes were cut into cubes (weighing approximately 100 mg) and placed in glass bottles (n = 3). Phosphate-buffered saline (PBS) was added at a ratio of 1:20 (w/v). All samples were incubated in a constant temperature shaking incubator at 37°C. Throughout the experimental period, the degradation solution was collected and replaced with fresh PBS every 3 days. The degradation solution was used to test the pH value via a pH meter and to analyze the released drug concentration via high-performance liquid chromatography (HPLC). The samples were collected every 6 days and eliminated residual moisture via freeze-drying. The remaining mass was weighed using an analytical balance. The remaining mass rate was calculated using the formula:
where W0 was the initial mass of the scaffold and W_n_ was the remaining mass measured at the nth time point.
Animal models and experimental design
The adult male Lewis rats (body weight: 335.2 g ± 17.01 g, age: 14 weeks) were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Zhejiang, China), and were housed in a specific pathogen-free (SPF) environment. This study received approval for all animal experimental procedures from the Institutional Animal Care and Use Committee at the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (SIAT-IACUC-201024-YGS-WXL-A1477). The rats were randomly assigned to three groups: (1) OA-OCD group with an empty defect (n = 12); (2) PTP group with a PTP scaffold without Epi C (n = 12); and (3) PTP@Epi C group with a PTP scaffold containing Epi C (n = 12).
Surgical procedure and scaffold implantation
The rat OA-OCD model was established to evaluate the in vivo repair effects of biphasic scaffolds. Initially, rats were anesthetized with 5% isoflurane (Reward, Shenzhen, China) and maintained on 1.5% isoflurane. Following meticulous cleaning of the surgical site, an incision was made in the right knee joint of the rat to expose the femoral trochlear groove. An OCD (2.0 mm in diameter and 2.0 mm in depth) was created in the femoral trochlear groove using a drill. Throughout the drilling process, the defects were irrigated with saline solution to prevent local overheating. Subsequently, sterilized scaffolds (PTP and PTP@Epi C scaffold) were press-fit into the pre-drilled cavities at the defect sites. Medial meniscectomy was then performed on each rat using microsurgical scissors. The incisions were then sutured layer by layer using a degradable suture (4-0 silk suture, Jinhuan, Shanghai, China). Postoperatively, penicillin (50 000 IU/kg) and gentamicin (5 mg/kg) were administered intramuscularly for three days to prevent bacterial infections. Joint samples were harvested in 6 weeks and 12 weeks postsurgery.
Micro-computed tomography analysis
At 6 weeks and 12 weeks postsurgery, the knee samples were collected and preserved in a 4% (w/v) paraformaldehyde solution for 72 h and subsequently maintained in a 70% ethanol solution at 4°C. The samples were analyzed using a micro-computed tomography (micro-CT; SkyScan 1176, Bruker, Kontich, Belgium) operating at a voltage of 65 kV, a current of 383 μA and an integration time of 300 ms, along with a 1 mm aluminum filter and a pixel size of 9 μm. The acquisition and reconstruction of all scan data were conducted using skyscan software. To differentiate mature bone from soft tissue, the threshold was set between 80 and 255 HU, as established in our previous research [29]. For the analysis of the subchondral bone plate, a cylindrical region of interest (ROI) with a diameter of 2 mm and a thickness of 0.2 mm was selected, beginning from the upper boundary of the subchondral bone within the defect area. The bone volume/total volume (BV/TV), bone mineral density (BMD) and bone mineral content (BMC) were calculated for this ROI. Below the subchondral bone plate ROI within the defect area, a cylindrical trabecular bone region (2 mm diameter, 1.8 mm depth) was selected to analyze the subchondral trabecular bone structure. The newly formed bone within the defect area was evaluated to determine the BV/TV, BMD, BMC, trabecular bone thickness (Tb.Th), trabecular separation (Tb.Sp) and trabecular number (Tb.N).
Histological evaluation
Following the micro-CT analysis, the samples underwent decalcification in a 10% (w/v) solution of ethylene diamine tetra acetic acid. They were subsequently dehydrated through a series of ethanol treatments, embedded in paraffin and sectioned longitudinally to a thickness of approximately 5 μm using a paraffin microtome (Leica RM 2235, Germany). The knee joints harvested at 6 weeks were sagittally divided into medial and lateral compartments. The lateral compartment, which contained the trochlear OCD, was used to evaluate osteochondral regeneration by hematoxylin and eosin (H&E), Toluidine Blue (T&B), Masson’s trichrome, COL II and Type I collagen (COL I). In contrast, the medial compartment was used for immunohistochemical staining of TNF-α. The sections were then stained with T&B, H&E and Masson’s trichrome staining to evaluate the new tissue present at the defect site. The stained sections were scored using the International Cartilage Repair Society (ICRS) [30] and modified O’Driscoll (MOD) histological scoring systems [31] for assessment of osteochondral regeneration. The percentage of newly formed bone area at the defect site was measured using Image-Pro Plus 6.0. The bone formation area was calculated using the following equation: new bone formation area (%) = [(newly formed bone area)/(original defect area)] × 100.
Immunohistochemistry staining
The sections were deparaffinized in xylene and subsequently rehydrated through a graded series of ethanol washes. After deparaffinization and rehydration, the sections were subjected to antigen retrieval as follows: incubate the sections in retrieval solution and heat them using a microwave oven (high power for 5–10 min until boiling, then medium-low power to maintain gentle boiling for 10–15 min); after retrieval, allow the sections to cool to room temperature naturally. Subsequently, the sections were incubated at room temperature for 10 min in the dark in a 3% hydrogen peroxide solution to quench endogenous peroxidase activity. This was followed by a blocking step using 10% bovine serum albumin (BSA) at room temperature for 60 min to minimize nonspecific binding. After blocking, the sections were incubated overnight at 4°C with primary antibodies: anti-COL II (1:100 dilution, ab34712, Abcam), anti-COL I (1:100 dilution, ab254113, Abcam), anti-TNF-α (1:50 dilution, ab307164, Abcam) and anti-ACAN (1:50 dilution, EPR28034-86, Abcam). The following day, the sections were incubated with the secondary antibody (1:50 dilution, A0208, Beyotime) at room temperature for 60 min. This secondary antibody is conjugated with an enzyme, typically peroxidase, which will facilitate the detection step. After thorough washing to eliminate unbound antibodies, the sections were treated with 3,3’-diaminobenzidine (DAB)-peroxidase substrate (cat#34002, Thermo Fisher, USA). DAB reacts with the enzyme-antibody complex, resulting in a brown precipitate at the antigen sites. This staining step was closely monitored to ensure appropriate staining intensity. Finally, the sections were counterstained with hematoxylin solution for 30 s, followed by a series of ethanol washes for dehydration. The sections were then cleared in xylene before being permanently mounted with neutral resin. The percentage of the positive stained area of the new cartilage layer in the defect site was calculated using Image-pro plus 6.0 (Media Cybernetics, USA).
RNA sequencing analysis of synovium
To generate mRNA sequencing data, synovial tissues were analyzed using RNA sequencing techniques. Synovial tissue samples from OA-OCD rats were collected at 12 weeks postoperation. Total RNA was isolated using the TRIzol total RNA extraction kit (TianGen, Cat. No. DP424), which yielded > 2 μg of total RNA per sample. The RNA quality was assessed through 0.8% agarose gel electrophoresis and spectrophotometry. High-quality RNA, with a 260/280 absorbance ratio ranging from 1.8 to 2.2, was utilized for library construction and sequencing. Whole mRNA sequencing libraries were generated by ApexBio Technology LLC (Shanghai, China) in accordance with the manufacturer’s recommendations. Poly(A) RNA was purified from 1 μg of total RNA using Dynabeads Oligo (dT). The purified poly(A) RNA was then fragmented and reverse-transcribed to create complementary DNA (cDNA). The cDNA was amplified to synthesize the second strand of cDNA and subsequently purified using the AMPure XP system (Beckman Coulter, Beverly, USA). Following library construction, library fragments were enriched through PCR amplification and selected based on a fragment size of 350–550 bp. The quality of the library was assessed using an Agilent 4200 Bioanalyzer (Agilent, USA). Sequencing was performed on the Illumina X plus sequencing platform (PE150) to generate raw reads. Raw data were filtered using Fastp software to remove sequences containing adapters and low-quality bases. The processed data were aligned to the rat genome using HISAT2 [32], followed by transcriptome data assembly and gene expression quantification with StringTie. Differentially expressed genes (DEGs) were identified using DESeq2 with a cut-off of log_2_|fold-change| > 1 and adjusted P values < 0.05. Functional enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway categories for significant DEGs was conducted using Cluster Profiler [33], with functions exhibiting a *P *< 0.05 considered significant.
Cell culture
The human synovial fibroblast cell line MH7A was obtained from the RIKEN Cell Bank (Tsukuba, Japan). Cells were cultured in RPMI-1640 medium (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin/streptomycin (P/S; Hyclone, USA), maintained at 37°C in a humidified atmosphere containing 5% CO_2_. The culture medium was replaced with a fresh culture medium every 3 days.
The human monocyte leukemia cell line THP-1 was obtained from American Type Culture Collection (ATCC). The cells were cultured at a density of 5 × 10^5^ cells/ml in RPMI 1640 medium supplemented with 10% FBS and 1% P/S at 37°C in a 5% CO_2_ incubator. The culture medium was replaced with a fresh culture medium every 2 days. THP-1 monocytes (8 × 10^5^ cells/well) were cultured in 6-well plates treated with 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 h to transform into adherent macrophages. Subsequently, the cells were pretreated with Epi C (25 μM, 50 μM, 100 μM) for 3 h, and then, stimulated with LPS (100 ng/mL) for 3 h. Afterward, cells were collected for RNA and protein extraction.
ELISA for cytokine measurements
MH7A cells were seeded into 12-well plates at a density of 5 × 10^4^ cells per well and incubated for 24 h. Subsequently, cells were pretreated with various concentrations of Epi C (25 μM, 50 μM) for 2 h, followed by stimulation with lipopolysaccharide (LPS, 2 μg/mL) for 24 h or TNF-α (20 ng/mL) for 48 h. The culture medium was collected and the cell culture medium was centrifuged using enzyme-linked immunosorbent assay (ELISA) kits (Novus Biologicals, USA) to determine the concentration of TNF-α, IL-6, IL-8 and IL-1β, according to the manufacturer’s protocols.
THP-1 monocytes (4 × 10^5^ cells/well) were cultured in 12-well plates treated with 100 nM PMA for 24 h to transform into adherent macrophages. Subsequently, the cells were pretreated with Epi C (25 μM, 50 μM, 100 μM) for 3 h, and then, stimulated with LPS (100 ng/mL) for 3 h. The culture medium was collected for ELISA analysis.
Quantitative real-time PCR
Total RNA was extracted from cultured cells with TRIzol reagent (Invitrogen, USA) and reverse transcribed into cDNA using a Takara Prime Script RT Reagent Kit (Takara, Japan). Quantitative real-time PCR (qRT-PCR) was performed using a TB Green PCR Kit (Takara, Japan) in conjunction with the Roche LightCycler 96 real-time fluorescence quantitative PCR system (Roche, Switzerland). The thermal cycling parameters for PCR were set as follows: 95°C for 30 s; 40 cycles of 95°C for 5 s, 60°C for 20 s and 65°C for 15 s. The GAPDH gene was used as an endogenous control. The GAPDH gene served as an internal control, and the relative expression levels of genes were quantitatively analyzed using the 2^-ΔΔCT^ method. The primer sequences employed for qRT-PCR detection are presented in Supplementary Table S1.
Western blot analysis
Total proteins were extracted with RIPA buffer reagent, and protein concentrations were quantified using a BCA kit. A total of 15 μg of protein was loaded into each well, followed by separation via 10% SDS-PAGE and subsequent transfer to a PVDF membrane. The membrane was blocked with 5% BSA at room temperature for 1 h, and then, incubated overnight at 4°C with the following primary antibodies: phospho-IKBα (s32; ET1609-78, Huabio), IKBα (ET1603-6, Huabio), phospho-NF-κB p65 (s536; HA723223, Huabio) and NF-κB p65 (bsm-33117M, Bioss), all diluted at 1:3000, while GAPDH (60004-1-lg, proteintech) served as the internal control. The membranes were washed 3 times with 0.1% TBST buffer for 10 min each time, followed by incubation at room temperature for 1 h with the secondary antibody (1:5000, Beyotime). Finally, the ECL chemiluminescence method was employed for detection, and the gray values of the bands were analyzed using Image J software (National Institutes of Health, USA). The relative expression level of the target protein was represented as the ratio of the gray value of the target band to that of the GAPDH band. All experiments were independently repeated three times.
NF-κB p65 translocation
The cells were fixed with 4% paraformaldehyde for 10 min, followed by washing with PBS and transfer to slides. The cells were then permeabilized using 0.1% Triton X-100 and subsequently blocked with a TBST solution containing 5% BSA. After two washes with PBS, the cells were incubated overnight at 4°C in NF-κB p65 mouse monoclonal antibody (1:500, bsm-33117M, Bioss). Following this, a Goat anti-mouse IgG H&L (Alexa Fluor^®^ 488) was added for incubation at room temperature for 1 h. The cell nuclei were stained with DAPI. Finally, the cells were washed twice with PBS and observed under a ZEISS confocal microscope. Image data were analyzed using ZEISS’s accompanying software.
NF-κB luciferase reporter assay
NF-κB transcriptional activity was measured using a luciferase reporter assay. The NF-κB reporter (Luc)-Raw 264.7 cell line was provided by Professor Xu (Shenzhen University of Advanced Technology). Cells were cultured in α-MEM medium supplemented with 10% FBS and 1% P/S. The NF-κB reporter (Luc)-Raw 264.7 cells were seeded into 24-well plates at a density of 5 × 10^4^ cells/mL. After the cells adhered and reached 80% confluence, they were treated with Epi C at gradient concentrations for 3 h, followed by stimulation with LPS (100 ng/mL) for an additional 3 h. Transcriptional activity was assessed using the Luciferase Reporter Assay Kit (Promega Corp, USA).
Statistical analysis
Data were presented as means ± standard error of the mean (SEM). Statistical analyses were conducted using GraphPad Prism 10.0 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was evaluated using two-way or one-way analysis of variance (ANOVA) followed by Fisher’s least significant difference (LSD) test. Statistical significance was defined as follows: **P *< 0.05, ***P *< 0.01, ****P *< 0.001 and *****P *< 0.0001.
Results
Fabrication and characterization of 3D printed biphasic scaffolds
The biphasic scaffold (PTP@Epi C) was produced using a low-temperature 3D printing system with PLGA (50:50) and PLGA (75:25)/β-TCP composite materials. As illustrated in Figure 1A, the SEM image reveals that the PLGA (50:50) layer exhibited a smooth porous architecture with interconnected pores, demonstrating a porosity of 45.16 ± 0.95% and mean pore diameter of 213.36 ± 12.21 μm (Table 1). Similarly, the PLGA (75:25)/β-TCP layer displayed an interconnected porous structure featuring β-TCP particles embedded on its surface, with a larger mean pore diameter (438.60 ± 6.73 μm) and higher porosity (65.19 ± 5.82%) compared to the PLGA layer (Table 1). Notably, numerous micropores on the scaffold framework walls were observed, indicating potential for increased surface area. The interface between the PLGA (50:50) layer and the PLGA (75:25)/β-TCP layer was tightly integrated, with no significant gaps observed at the junction (Figure 1B). EDS elemental mapping demonstrated that the PLGA (75:25)/β-TCP layer contained abundant phosphorus and calcium elements, which were uniformly distributed throughout this layer, whereas these elements were absent in the pure PLGA layer (Figure 1C). XRD analysis revealed that the characteristic diffraction peaks of the PLGA (75:25)/β-TCP composite matched exactly with those of pure β-TCP. As PLGA is amorphous and lacks prominent crystalline peaks, these results indicate that encapsulation within PLGA does not affect the crystal structure of β-TCP (Figure 1D). Tensile testing (Figure 1E and F and Supplementary Figure S1) showed that fracture occurred exclusively within the PLGA (75:25)/β-TCP layer in all samples, indicating excellent structural stability at the biphasic scaffold interface.
Fabrication and characterization of 3D-printed biphasic scaffolds incorporating Epi C. (A) Morphological SEM observations of the PLGA (50:50) layer and PLGA (75:25)/β-TCP layer of the biphasic scaffold (white arrow indicates β-TCP particles). (B) SEM images of the interface between the PLGA (50:50) layer and the PLGA (75:25)/β-TCP layer. (C) EDS analysis showed the distribution of phosphorus and calcium elements in the PLGA (50:50) layer and the PLGA (75:25)/β-TCP layer, where dashed lines mark the interlayer boundaries. (D) XRD patterns of β-TCP, PLGA (75:25)/β-TCP, PLGA (75:25)/β-TCP@Epi C and PLGA (75:25) scaffolds tested at room temperature. (E) Schematic diagram of the tensile test for biphasic scaffolds. (F) The tensile force versus displacement curve of biphasic scaffolds. (G) Changes in the pH of scaffolds during in vitro degradation in PBS at 37°C. (H) The remaining mass ratio change curves during the scaffold’s degradation. (I) The cumulative Epi C release percentage (%) over time profiles from the PLGA (50:50)@Epi C scaffold and PLGA (75:25)/β-TCP @Epi C scaffold. Statistical significance was evaluated by one-way ANOVA followed by Fisher’s LSD test. Data are presented as mean ± SEM.
The stiffness and E-modulus of the PLGA (50:50) layer were measured at 493.23 ± 27.02 N/mm and 46.56 ± 3.05 MPa (Table 1 and Supplementary Figure S2), respectively, with no significant difference observed when compared to the PLGA (50:50)@Epi C scaffold. Meanwhile, the stiffness and E-modulus of the PLGA (75:25)/β-TCP layer were recorded as 258.33 ± 70.37 N/mm and 33.07 ± 2.97 MPa (Table 1), respectively, also showing no significant difference in comparison to the PLGA (75:25)/β-TCP@Epi C scaffold. Therefore, the incorporation of Epi C did not influence the structure, mechanical properties or porosity of either the PLGA or PLGA/β-TCP scaffolds.
To evaluate the degradation kinetics and drug-release profiles of the biphasic scaffolds, in vitro degradation assays were performed on PLGA (50:50)@Epi C and PLGA (75:25)/β-TCP@Epi C constructs in PBS (pH 7.40 ± 0.02, 37°C) for 24 days (Figure 1G–I). As illustrated in Figure 1G and H, the PLGA (50:50)@Epi C scaffold exhibited rapid degradation characteristics, losing approximately 10.2% of its initial mass within 24 days, while the pH of the PBS decreased from 7.40 to 4.13. In contrast, the degradation rate of the PLGA (75:25)/β-TCP@Epi C scaffold was slower, with no significant mass change over the 24-day period, and the pH of the PBS remained stable throughout the experiment. The notable differences in degradation behavior between the two scaffolds indicate that the degradation rate of PLGA (50:50)@Epi C is significantly higher than that of the PLGA (75:25)/β-TCP@Epi C scaffold. Furthermore, the drug release behavior of the scaffolds aligns with their degradation trends. As shown in Figure 1I, the PLGA (50:50)@Epi C scaffold displayed a rapid drug release rate over 24 days, with a cumulative release amount reaching approximately 40%. In contrast, the drug release from the PLGA (75:25)/β-TCP@Epi C scaffold was markedly slower, with a cumulative release amount of only about 10% during the same period. The results in Supplementary Figure S3 demonstrate that scaffold composition has a greater impact on drug release and degradation than pore size/porosity. The PLGA (50:50) scaffolds exhibited faster Epi C release and more rapid pH decline compared to PLGA (75:25)/β-TCP scaffolds. Within the same material system, however, larger pore sizes accelerated both drug release and pH changes, as expected from enhanced medium infiltration and diffusion.
PTP@Epi C scaffold promotes subchondral bone formation in rat OA-OCD models
To further evaluate the osteochondral regeneration potential of the biphasic scaffold, we implanted it into a rat OA-OCD model (Figure 2). New bone formation in the OCD was assessed using micro-CT at 6 weeks and 12 weeks postsurgery. As illustrated in Figure 3A, the pattern of new bone formation at the defect site exhibited significant alterations in the scaffold implantation groups (PTP group and PTP@Epi C group) compared to the OA-OCD group. In the OA-OCD group, the OCD regeneration was characterized by a prioritized formation of a subchondral bone plate, followed by the development of new trabecular bone extending from this plate into the defect. In contrast, the implantation of the scaffold led to the preferential formation of new trabecular bone in the deeper region of the defect, with the subchondral bone plate forming subsequently.
This schematic diagram illustrates the procedure for establishing a rat OA-OCD model and implanting the scaffold. The rats were anesthetized, and surgeries were performed in a sterile environment. A small incision was made to access the right knee joint cavity, from which the medial meniscus was excised. Subsequently, a standardized OCD (Ø 2 mm × H 2 mm) was created at the center of the femoral trochlea using a dental drill. Postoperatively, the recovery of the rats was closely monitored to ensure their health and normal function. The figure was created by Figdraw (ID: AIURO3aea4).
*Micro-CT images and micro-architectural analysis of new bone at defect sites treated with different composite scaffolds. (A) Representative macroscopic images and sagittal plane images in the defect area at 6-week and 12-week postsurgery for the OA-OCD group, PTP group and PTP@Epi C group; red box indicates the region of subchondral bone plate, whereas the blue box outlines the region of newly formed trabecular bone at the defect site. (B) 3D reconstructed images of the newly formed the subchondral bone plate in the defect area. (C) 3D reconstructed images of the newly formed trabecular bone in the defect site. (D–F) Quantitative analysis of the newly formed subchondral bone plate in the defect area, including BV/TV, BMD and BMC. (G–L) Quantitative analysis of the newly formed subchondral trabecular bone in the defect area, including BV/TV, BMD, BMC, Tb.Th, Tb.N and Tb.Sp. Statistical significance was evaluated by two-way ANOVA followed by Fisher’s LSD test. Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 and ***P < 0.0001.
Given these observed differences, we performed separate quantitative analyses for both the newly formed subchondral bone plate and trabecular bone within the defect area. As illustrated in Figure 3B and D–F, the quantitative analysis of the newly formed subchondral bone plate further revealed that both scaffold groups exhibited significantly lower values compared to the OA-OCD group for BV/TV, BMD and BMC at 12 weeks postsurgery. Specifically, BV/TV was reduced by 13% in the PTP group and by 47.83% in the PTP@Epi C group, BMD decreased by 13.03% in the PTP group and by 53.11% in the PTP@Epi C group, and BMC was reduced by 26.39% in the PTP group and by 53.11% in the PTP@Epi C group. However, the scaffold implantation groups (PTP and PTP@Epi C) demonstrated that the defect area was filled with newly formed trabecular bone tissue (Figure 3C). In contrast, the OA-OCD group displayed only minimal reparative tissue. This outcome confirms that the biphasic scaffold promotes in situ regeneration of OCD. Quantitative analysis of the newly formed trabecular bone further indicated (Figures 3G–L) that the BV/TV, Tb.Th, BMD and BMC in the PTP@Epi C group were significantly higher than those in the OA-OCD group at 12 weeks postsurgery. Furthermore, the BV/TV and Tb.Th in the PTP@Epi C group were also significantly greater than those in the PTP group alone.
Histological analysis of regenerated tissue
The histological evaluation of regenerated tissue sections from OCD was presented in Figure 4. The defect sites in the PTP@Epi C, PTP and OA-OCD groups were filled with neo-tissue; however, their surfaces were predominantly covered with fibrous tissue. Notably, the PTP@Epi C group exhibited the formation of hyaline-like cartilage tissue at the edges of the defect 12 weeks postoperatively. In contrast, the defect sites in the OA-OCD and PTP groups remained primarily covered by fibrous tissue (Figure 4A and B). Quantitative assessment utilizing the ICRS and MOD histological scoring systems indicated that the PTP@Epi C group demonstrated ICRS scores that were 50% higher than those of the OA-OCD group at 12 weeks postoperatively (Figure 4C). Simultaneously, this group exhibited MOD scores that were 5% higher than those of the PTP group and 18% higher than those of the OA-OCD group (Figure 4D). Quantitative histomorphometric analysis indicated that the percentage of new bone area in the PTP@Epi C group was increased by 48.2% and 36.4%, respectively, compared to the OA-OCD and PTP groups at 12 weeks postimplantation (Figure 4E).
*Histological analysis of regenerated tissue at 6 weeks and 12 weeks postoperation. (A) H&E staining of regenerated tissue in all groups; red arrow indicates the interface between the neo-tissue and the host tissue, while black arrow denotes the newly formed bone tissue. (B) T&B staining of regenerated tissue of each group. (C) ICRS visual histological scores of repaired tissues. (D) MOD score of repaired tissues. (E) The percentage of new bone formation area in defect region. Statistical significance was evaluated by two-way ANOVA followed by Fisher’s LSD test. Data are presented as mean ± SEM; *P < 0.05 and *P < 0.01.
To better evaluate subchondral bone regeneration in the defect area, Masson’s trichrome staining was performed at 6 weeks postoperation (Figure 5A). In the OA-OCD group, only a thin bone plate formed at the top of the defect, with abundant bone marrow tissue and no evident new bone formation within the defect interior. In contrast, both the PTP and PTP@Epi C groups showed new bone ingrowth into the scaffold pores, accompanied by limited cartilage regeneration. Notably, the PTP@Epi C group exhibited superior bone-scaffold integration at the interface and greater deposition of collagen-rich tissue (likely representing immature new bone) compared with the PTP group.
*Masson-trichrome staining and immunohistochemical staining of ACAN at 6 weeks postoperation. (A) Masson trichrome staining shows the morphology of newly formed bone tissue and the distribution of collagen fibers; HB: host bone, NB: new bone, CLT: collagen-like tissue, S: scaffold. (B) Immunohistochemical staining of the cartilage-specific matrix protein ACAN; red arrow indicates the interface between the neo-tissue and the host tissue. (C) The percentage of new bone formation area in defect regions. (D) Quantification of integrated optical density (IOD) of ACAN in new cartilage. Statistical significance was evaluated by one-way ANOVA followed by Fisher’s LSD test. Data are presented as mean ± SEM; **P < 0.01 and **P < 0.001.
Immunohistochemistry analysis of regenerated tissue
The cartilage-specific matrix ACAN and COL II can reflect the functional status of repaired cartilage [34]. As shown in Figure 5B and D, the immunohistochemical staining of ACAN reveals that there is no positive signal in the defect area of the OA-OCD group, while the PTP group shows weak positive expression. In contrast, the PTP@Epi C group exhibits a greater area of brownish-yellow positive staining compared to the other two groups, suggesting an enhanced ability for cartilage matrix synthesis. As illustrated in Figure 6A, a faint Col II-positive area was observed in the regenerated tissue of the PTP@Epi C group at 6 weeks postoperatively. In contrast, no significant COL II formation was detected in the PTP and OA-OCD groups (Figure 6B). As the repair progressed to 12 weeks, the COL II-positive area in the PTP@Epi C group was 20% and 30% greater than that of the PTP and OA-OCD groups, respectively (Figure 6C). However, COL I expression levels in the PTP@Epi C group did not show a significant reduction compared to the PTP and OA-OCD groups (Figure 6D–F).
*Immunohistochemical analysis of regenerated tissue at 6 weeks and 12 weeks postoperation. (A) Immunohistochemical staining for COL II in regenerated sites, presenting results of each group (OA-OCD, PTP, PTP@Epi C) at 6 weeks and 12 weeks postoperation; red arrow indicates the interface between the neo-tissue and the host tissue. Quantitative analysis of COL II-positive area in the regenerated cartilage region at (B) 6 weeks and (C) 12 weeks postoperation. (D) Immunohistochemical staining for COL I in regenerated sites, presenting results of each group at 6 weeks and 12 weeks postoperation. Quantitative analysis of COL I-positive area in the regenerated cartilage region at (E) 6 weeks and (F) 12 weeks postoperation. Statistical significance was evaluated by one-way ANOVA followed by Fisher’s LSD test. Data are presented as mean ± SEM; *P < 0.05 and *P < 0.01.
PTP@Epi C significantly reduced PTP-induced the inflammatory response
Synovial tissue is recognized as a crucial component of joint health, and alterations in its condition are closely associated with the OA progression [35]. Normal histology of articular synovium has 1–2 layers of synovial lines, with regular epithelial cells in the flat synovium, and no inflammatory infiltration or angiogenesis [36]. However, the MMT-induced OA rat model exhibits synovial fibrosis, resulting from an imbalance caused by ECM disruption and vascular proliferation [37]. This fibrosis correlates with increased expression of TNF-α, indicating an amplified local inflammatory response. Immunohistochemical staining reveals that there was minimal brown-positive staining for TNF-α in the regenerated cartilage and subchondral bone regions across all groups, indicating low local expression of this pro-inflammatory cytokine within the defect site itself. In contrast, prominent TNF-α positive staining was confined primarily to the synovial tissue (Supplementary Figure S4). As illustrated in Figure 7A and B, synovial tissue fibrosis, accompanied by synovial thickening, was observed in the MMT-induced OA rats 6 weeks postsurgery. In comparison to the OA-OCD group, the rats with PTP scaffold implantation demonstrated a significant increase in synovial thickening, which was associated with elevated TNF-α secretion, suggesting that PTP implantation may exacerbate the inflammatory response. Quantitative analysis indicated that, relative to the OA-OCD group, synovial thickness in the PTP group increased significantly by 72.5%, while the positive area of TNF-α in the synovium rose significantly by 72.9%. Moreover, compared with the PTP group, the PTP@Epi C group showed a significant reduction in synovial thickness (36.68%) and a marked decrease in the TNF-α–positive area in the synovium (40.89%) (Figure 7C and D).
*Analysis of the synovial tissue of the rat knee joints at 6 weeks after surgery. (A) H&E staining showing synovial morphology. (B) Immunohistochemical staining of TNF-α. Quantitative analysis of (C) synovial thickness and (D) the percentage of TNF-α positive area. Statistical significance was evaluated by one-way ANOVA followed by Fisher’s LSD test. Data are presented as mean ± SEM; *P < 0.05 and *P < 0.01.
Transcriptomic sequencing analysis of the synovial tissue and validation of the anti-inflammatory effects of Epi C in vitro
To investigate the anti-inflammatory mechanism of Epi C in synovial tissue, we performed RNA sequencing on synovial tissue samples obtained from rats at 12 weeks postoperation and identified DEGs between the PTP@Epi C and PTP groups. As shown in Figure 8A, volcano plot analysis revealed 377 DEGs (Log_2_ |FC| > 1 and *P *< 0.05), including 173 upregulated genes and 204 downregulated genes. Notably, the pro-inflammatory cytokines-related genes (TNFAIP6, TNFAIP8, IL-6 and IL-8) were significantly downregulated, while osteogenesis-related genes (Bmpr1b, Runx2) were markedly upregulated (Figure 8B). These results suggest that Epi C may exert its anti-inflammatory effects by regulating the expression of genes associated with inflammatory responses and osteogenic processes. To assess the biological significance of these DEGs, we performed KEGG enrichment analysis and GO enrichment analysis. KEGG enrichment results indicated that the DEGs were primarily enriched in pathways associated with rheumatoid arthritis, the IL-17 signaling pathway, the MAPK signaling pathway and the NOD-like receptor signaling pathway (Figure 8C). This suggests that Epi C may be involved in inflammatory response, immune regulation and processes such as cell proliferation and apoptosis through these pathways. Furthermore, GO analysis demonstrated significant enrichment of the DEGs in biological processes such as regulation of macrophage activation, positive regulation of osteoblast proliferation, positive regulation of BMP signaling pathway and negative regulation of inflammatory response (Figure 8D), indicating that Epi C may play a pivotal role in mediating bone formation and immune-inflammatory regulation. Furthermore, Epi C can significantly upregulate the expression of BMPR1B, Runx2, Ucma and Comp genes in the BMP signaling pathway (Supplementary Figure S5).
*Transcriptomic and functional analysis of synovial tissue and inflammatory response following Epi C treatment. (A) Volcano plot of differentially expressed genes (DEGs) between the PTP and PTP@Epi C groups (|log2FC| > 1, P < 0.05). Red and blue dots represent upregulated and downregulated genes, respectively. (B) Heatmap showing expression profiles of DEGs. (C) KEGG pathway enrichment analysis and (D) GO biological process enrichment in PTP@Epi C group than PTP group. ELISA quantification of pro-inflammatory cytokines expression of (E) TNF-α, (F) IL-6 and (G) IL-8 in LPS-induced MH7A cells for 24 h. (H) ELISA quantification of pro-inflammatory cytokines expression of IL-1β in TNF-α–induced MH7A cells for 48 h. Statistical significance was evaluated by one-way ANOVA followed by Fisher’s LSD test. Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 and ***P < 0.0001.
To further validate the anti-inflammatory effects of Epi C, LPS-stimulated MH7A cells were used to examine whether Epi C influences the accumulation of proinflammatory cytokines, including TNF-α, IL-6 and IL-8. The cells were pretreated with or without Epi C for 2 h, followed by stimulation with LPS (2 μg/mL) for 24 h. Cytokine levels in the culture supernatant were measured using ELISA. The results showed that LPS significantly induced the release of TNF-α, IL-6 and IL-8, increasing their levels to 9.79-fold, 26.33-fold and 4.93-fold, respectively, compared to the normal group. Treatment with Epi C (25 μM and 50 μM) dose-dependently and significantly inhibited the release of TNF-α to 11.57% and 19.93%, respectively. Similarly, the release of IL-6 was suppressed to 26.76% and 37.42%, and that of IL-8 was reduced to 12.70% and 26.27% (Figure 8E–G). In addition, TNF-α (20 ng/mL) significantly enhances the production of IL-1β in MH7A cells, whereas Epi C exhibits a dose-dependent inhibitory effect on this pro-inflammatory response (Figure 8H).
Epi C alleviated inflammatory responses via the NLRP3 signaling pathway
NLRP3, a core effector in the NOD-like receptor pathway, plays a pivotal role in inflammatory responses [38]. To further investigate the regulatory effects of Epi C on NLRP3-related signaling pathways, we employed in vitro experiments utilizing THP-1 cells and NF-κB reporter (Luc)-Raw 264.7 cells. As demonstrated in Figure 9, LPS stimulation significantly upregulated NLRP3 mRNA expression in THP-1 cells, as detected by qPCR. Treatment with Epi C (25, 50 and 100 μM) dose-dependently reduced NLRP3 mRNA levels (Figure 9A) and IL-1β secretion (Figure 9B). Since NF-κB is a critical regulator of NLRP3 inflammasome activation through transcriptional promotion of NLRP3 [39], we further examined NF-κB signaling. Western blot analysis (Figure 9C–E) revealed that LPS markedly increased phosphorylation of IκBα and p65, elevating the p-IκBα/IκBα and p-p65/p65 ratios. Epi C treatment dose-dependently attenuated these phosphorylation events and reduced the p-IκBα/IκBα and p-p65/p65 ratios. In addition, confocal microscopy (Figure 9F) and NF-κB luciferase reporter assays (Figure 9G) demonstrated that LPS strongly promoted p65 nuclear translocation and NF-κB transcriptional activity, both of which were progressively inhibited by increasing concentrations of Epi C. These findings indicate that Epi C alleviates inflammatory responses by suppressing NF-κB mediated activation of the NLRP3 inflammasome.
*Epi C alleviated LPS-induced inflammatory responses via the NF-κB/NLRP3 signaling pathway. (A) qPCR analysis of the relative expression of NLRP3 mRNA in LPS-induced THP-1 cells at different concentrations of Epi C. (B) ELISA quantification of IL-1βsecretion in the culture supernatant. (C) Representative Western blot images showing protein levels of p65, p-p65, IκBα and p-IκBα, with GAPDH as the loading control. (D, E) Quantitative analysis of the relative ratios of p-IκBα/IκBα and p-p65/p65, respectively. (F) Immunofluorescence staining of NF-κB p65 (green) and nuclei (DAPI, blue) in THP-1 cells; white arrows indicate cells exhibiting p65 nuclear translocation, while red arrows show that the nuclear translocation of p65 was inhibited by Epi C and the p65 remained in the cytoplasm. (G) NF-κB luciferase reporter assay measuring transcriptional activity in NF-κB reporter (Luc)-RAW 264.7 cells. Statistical significance was evaluated by one-way ANOVA followed by Fisher’s LSD test. Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 and ***P < 0.0001.
Discussion
OCD remain a clinical challenge due to the intricate hierarchical structure of osteochondral tissue and its limited intrinsic capacity for self-repair. In this study, we fabricated a biphasic 3D-printed scaffold (PTP@Epi C) by integrating Epi C into PLGA and PLGA/β-TCP composites, achieving layered controlled release of Epi C to synergistically enhance osteochondral regeneration and mitigate synovitis. The results collectively demonstrate that the PTP@Epi C scaffold combines structural advantages, biological activity and anti-inflammatory effects, providing a promising strategy for OCD treatment.
In clinical practice, OCD are often complicated by OA, frequently arising from meniscal injuries or joint instability [40]. To better replicate this challenging clinical scenario, in which OCD repair occurs within an inflammatory and degenerative joint microenvironment, we established a rat model combining MMT with OCD creation. Our previous studies validated the utility of similar combined OA-OCD models. One employed MMT-induced post-traumatic OA with concurrent OCD in rats [41]. Another used nonself-healing OCD in a papain-induced OA rabbit model [42]. These investigations consistently showed that the arthritic environment markedly impairs regeneration of both cartilage and subchondral bone. The combined model accelerates joint-wide degeneration and more closely mimics the complex pathological conditions seen in clinical OA-complicated OCD cases. This approach thereby increases the translational relevance of our findings when evaluating the regenerative performance of the PTP@Epi C scaffold.
The structural design and degradation characteristics of biphasic scaffolds
Tissue engineering scaffolds for osteochondral regeneration require structural and functional mimicry of the native osteochondral interface, which exhibits distinct mechanical properties, porosity and degradation kinetics between cartilage and subchondral bone [43]. The pore size and porosity of the scaffolds are key structural parameters that significantly affect cell behavior (such as adhesion, proliferation and differentiation), nutrient transport efficiency and ultimately the regeneration outcomes [44–46].
We propose that tissue-specific microenvironment determines whether Epi C exerts chondrogenic or osteogenic effects. The PTP@Epi C scaffold has two phases to match the physiological needs of each tissue. The cartilage phase (PLGA 50:50) has a lower porosity (45.16 ± 0.95%) and smaller pore size (213.36 ± 12.21 μm), which mimics the dense ECM of articular cartilage and supports early cell adhesion for chondrogenesis [47, 48]. In contrast, the subchondral bone phase [PLGA (75:25)/β-TCP] features higher porosity (65.19 ± 5.82%) and larger pores (438.60 ± 6.73 μm), combined with β-TCP’s osteoconductive properties-consistent with previous reports that β-TCP enhances bone integration by facilitating osteoblast migration and mineralization [49]. This is supported by our earlier work on the structurally similar compound icariin, which induced no osteogenesis in neutral conditions but strongly promoted it in an osteogenic microenvironment [50]. We hypothesize that Epi C exerts micro-environmental-dependent effects, favoring cartilage protection in the dense, low-oxygen upper phase and bone formation in the porous, vascularized lower phase. The exact mechanisms of this switching require further study.
The degradation and drug release of tissue engineering scaffolds are critical for effective osteochondral repair and inflammatory microenvironment regulation [51, 52]. Our results demonstrate that scaffold composition has a greater impact on drug release and degradation than pore size/porosity. The PLGA (50:50) scaffolds exhibited faster Epi C release and more rapid pH decline compared to PLGA (75:25)/β-TCP scaffolds, consistent with the higher glycolic acid content in PLGA (50:50) promoting quicker hydrolysis. Within the same material system, however, larger pore sizes accelerated both drug release and pH changes, as expected from enhanced medium infiltration and diffusion. These findings explain why the bone phase [higher porosity but slower-degrading PLGA (75:25)/β-TCP] shows sustained Epi C release despite its larger pores, while the cartilage phase [lower porosity but faster-degrading PLGA (50:50)] releases Epi C more rapidly.
PTP@Epi C scaffold promotes osteochondral regeneration
In this study, local delivery of Epi C via the biphasic PTP scaffold enhanced osteochondral regeneration through combined structural and biological advantages. The scaffold’s 3D porous architecture facilitated cell migration, adhesion and tissue ingrowth, while the incorporation of β-TCP in the bone phase improved osteo-conductivity by mimicking the inorganic component of native bone and providing a favorable microenvironment for osteoblast proliferation and mineralization [53]. Compared with systemic administration, this localized delivery maintains therapeutic concentrations at the defect site [54]. The synergy between structural support and sustained Epi C bioactivity thus contributed to superior regeneration outcomes.
Transcriptome sequencing further provided mechanistic insights, revealing significant enrichment of the BMP signaling pathway and upregulation of key downstream osteogenic genes (BMPR1B and Runx2) in the Epi C-treated group (Figure 8D and Supplementary Figure S5). These findings are consistent with prior reports demonstrating that Epi C promotes osteogenic differentiation under inflammatory conditions by upregulating BMP-2 and Runx2 expression, thereby enhancing markers such as ALP, osteocalcin and COL I [55] or via activation of the PI3K/AKT/Runx2 pathway in pre-osteoblast cells [56]. As the BMP/Runx2 axis is a central regulator of osteoblast differentiation and bone formation [57], its modulation likely underlies the observed improvements in subchondral bone repair. In addition, Epi C inhibits the NF-κB signaling pathway, a master regulator of inflammation, thereby reducing pro-inflammatory cytokines such as TNF-α and IL-1β. These cytokines are known to suppress osteoblast differentiation and bone formation in inflammatory settings like OA [58–60]. By mitigating NF-κB activation and creating a less inflammatory microenvironment, Epi C removes key barriers to osteogenesis, directly enhancing osteogenic differentiation and mineralization. However, direct protein-level validation in regenerated tissue remains to be performed in future studies.
PTP exacerbate synovial inflammation in OA knee and the protective role of Epi C
Synovitis is a key pathological feature of OA progression, and persistent inflammation can impair osteochondral repair by disrupting the ECM balance and inhibiting regenerative cell activity [61, 62]. Notably, implantation of scaffold materials may exacerbate synovial inflammation in OA knees. For instance, PTP scaffold implantation increased synovial thickness and TNF-α expression, potentially due to a mild inflammatory response triggered by foreign materials. This may be associated with the acidic microenvironment produced during PLGA degradation, as hydrolytic degradation generates acidic by-products such as lactic acid and glycolic acid [63–65]. To address this issue, several strategies have been employed with improved logical progression focusing on modulating degradation kinetics. First, altering PLGA degradation rates, such as by enhancing crystallinity through crosslinking and recrystallization annealing, can slow hydrolysis and reduce acidic by-product generation [65]. Second, incorporating inorganic components like TCP into PLGA composites helps neutralize acidic degradation products and buffers local pH. Our in vitro degradation experiments further indicate that PLGA reduces pH in the early stages, whereas PLGA/TCP composites do not exhibit this effect, suggesting that the pH drop is primarily attributable to the PLGA layer, a mechanism warranting further exploration in future studies. Third, loading anti-inflammatory agents into PLGA scaffolds enables localized sustained release to suppress inflammatory responses [66–68]. In our study, incorporation of Epi C significantly mitigated PTP scaffold-induced inflammation, although TNF-α levels did not fully return to those observed in normal tissue. Notedly, the present study lacks systematic TNF-α immunohistochemical staining within the defect area across all experimental groups. Future studies will utilize a larger sample size and adjacent sections to specifically investigate the local immune microenvironment following scaffold implantation.
Limitations of this work
Despite the promising regenerative outcomes observed in this study, several limitations should be acknowledged. First, the evaluation was limited to short-term follow-up at 6- and 12-week postimplantation, precluding assessment of long-term efficacy, durability of the regenerated tissue or potential late complications. Additionally, only a single Epi C loading dose was tested, leaving the effects of varying dosages unexplored. Second, although the rat OA-OCD model is valuable for preliminary mechanistic studies, it has inherent limitations in fully recapitulating the chronic progressive nature, biomechanical loading and limited intrinsic regenerative capacity characteristic of human OA. Translation to larger animal models or clinical settings may yield different outcomes. Third, the absence of a positive control group represents a notable limitation. The study included only the untreated OA-OCD group, the blank PTP scaffold group, and the PTP@Epi C group. While these comparisons clearly demonstrate the superior osteochondral repair achieved with Epi C loading relative to the scaffold alone or untreated defects, the performance of the PTP@Epi C scaffold relative to established clinical interventions remains unknown. Future investigations should incorporate clinically relevant positive controls, such as microfracture, autologous chondrocyte implantation or systemic/local anti-inflammatory treatments, to more accurately assess translational potential. Addressing these limitations in future work will be essential to strengthen the evidence base and advance the clinical translation of this biphasic Epi C-loaded scaffold strategy.
Conclusion
In this study, a biphasic PTP@Epi C scaffold with hierarchical controlled release of Epi C was developed for OCD regeneration in OA. Epi C alleviates inflammatory responses by suppressing NF-κB mediated NLRP3 inflammasome activation. The scaffold demonstrates favorable structural, mechanical and biological properties, synergistically promoting subchondral bone formation and cartilage regeneration while attenuating synovitis through combined structural support and Epi C bioactivity. This work presents a novel strategy for integrating TCM-derived active ingredients into tissue engineering scaffolds and provides a promising approach for clinical treatment of OCD.
Supplementary Material
rbag021_Supplementary_Data
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