Microsphere System Coloading Type I Collagen and Dexamethasone Sodium Phosphate for Osteoarthritis Therapy

Abstract

Knee osteoarthritis (KOA) has no effective nonsurgical treatment. Intra-articular therapy constitutes an effective localized intervention; however, persistent lymphatic drainage and synovial vascular exchange lead to rapid drug elimination. Encapsulating bioactive agents into microsphere delivery platforms enables localized intra-articular administration, with their scaffold-based spherical architecture serving as pharmaceutical and cellular carriers or tissue engineering scaffolds, representing a highly promising therapeutic avenue for KOA. Herein, PLGA was employed to prepare microspheres and a sustained-release microsphere formulation (MPs PLGA DSP@COLI) was prepared for KOA therapeutics, co-encapsulating type I collagen (COLI) and dexamethasone sodium phosphate (DSP). Characterization revealed uniform particle size distribution, favorable dispersibility, and extended-release kinetics. Functional assessments demonstrated robust anti-inflammatory efficacy and enhanced chondrocyte-mediated extracellular matrix deposition. Histopathological and immunohistochemical analyses demonstrated preserved cartilage matrix integrity, augmented COLII synthesis, diminished inflammatory cellular infiltration, and maintained homeostasis of cartilage matrix components, concomitant with reduced OARSI and synovitis scores. These effects collectively suppressed synovial hyperplasia and inflammation and suggested potential improvement in joint conditions. Overall, MPs PLGA DSP@COLI combines the pharmacological anti-inflammatory properties of DSP with the extracellular matrix-related biological functions of COLI, establishing an innovative “drug-device combination” therapeutic paradigm, which represents a promising therapeutic strategy for KOA.

1. Introduction

Knee osteoarthritis (KOA), characterized by degenerative articular cartilage alterations, demonstrates an escalating incidence as the population ages, substantially compromising patient quality of life and imposing significant socioeconomic burdens. − The pathological process encompasses the entire knee joint, manifesting predominantly as chondromalacia, fibrosis, and cartilage loss, accompanied by vascular invasion, synovial hyperemia with proliferation, and osteophyte formation, clinically presenting with progressive arthralgia and functional impairment. − Early stage KOA exhibits mechanically induced inflammatory mechanisms, wherein chondrocytes release TNF-α, IL-6, and catabolic enzymes including ADAMTS and MMPs. The innate immune system participates through complement activation and alternative pathway engagement. MMPs compromise cartilage structural integrity, while abnormal subchondral bone remodeling directly elicits nociception; mechanical abrasion exacerbates meniscal degeneration and induces synovial vascularization. , The pathological microenvironment centers on chronic synovitis and cartilage degeneration, with synovial inflammation frequently preceding cartilage deterioration. Synovial macrophages constitute the principal inflammatory cells; damage-associated molecular patterns (DAMPs) induce M1 macrophage polarization, aggravating cartilage degradation through signaling pathway modulation and establishing an “inflammation-cartilage injury” vicious cycle. − Articular cartilage possesses minimal reparative capacity; chondrocyte dysfunction directly compromises cartilaginous homeostasis. Hypoxia within the inflammatory microenvironment induces metabolic dysregulation, while activated macrophages accelerate joint destruction. Paracrine interactions between macrophages and chondrocytes further exacerbate pathological progression.

Currently, there is no clinical treatment that can completely reverse the course of KOA. Existing treatment methods mainly include oral nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular injections of glucocorticoids or hyaluronic acid, and end-stage total joint replacement surgery. , However, while oral NSAIDs can rapidly relieve pain symptoms, long-term use may lead to adverse reactions such as cardiovascular events; conventional intra-articular drug injections, affected by synovial lymphatic drainage and vascular exchange, are cleared quickly and have a short duration of efficacy, requiring repeated injections that increase the risk of infection; total joint replacement surgery involves significant trauma, high treatment costs, and a limited prosthesis lifespan, making it difficult to meet the treatment needs of patients with early and mid-stage KOA. Therefore, developing new therapeutic strategies that can provide long-acting release, local targeting, and safe and effective treatment has become a critical scientific issue that urgently needs to be addressed in the current field of KOA research. ,

The emergence of microsphere drug delivery systems has provided new approaches to address the aforementioned clinical treatment challenges. Among them, poly­(lactic-co-glycolic acid) (PLGA) microspheres have become a research focus for long-acting intra-articular drug delivery due to their excellent biocompatibility, controllable biodegradability, and flexible drug release regulation capabilities. PLGA is a biodegradable polymer approved by the FDA, and its safety was fully validated. By adjusting the monomer ratio, molecular weight, and microsphere preparation process of PLGA, precise control over drug release rates can be achieved, thereby significantly reducing injection frequency and lowering the risk of infection. In addition, as scaffold-type spherical carriers, PLGA microspheres can not only extend the residence time of drugs within the joint cavity but also provide joint lubrication and mechanical support, creating a favorable microenvironment for chondrocyte adhesion and growth, while functioning similarly to a bearing to reduce pain caused by friction during osteophyte formation. Microsphere delivery systems can achieve precise accumulation at lesion sites through surface modification and targeted design, enhancing local drug concentrations and reducing systemic adverse effects; they can also carry anti-inflammatory and immunomodulatory active substances to regulate the intra-articular inflammatory microenvironment and alleviate synovitis and cartilage damage. Furthermore, they can deliver growth factors, stem cells, and other agents to promote cartilage repair and regeneration. , Additionally, they can enable combination therapies with multiple drugs and mechanisms, providing an efficient delivery strategy for KOA treatment that integrates sustained release, targeting, anti-inflammation, repair, and combination therapy.

Studies have engineered a ROS/MMP13 dual-responsive organoselenium hydrogel microsphere (HSPHR) delivery system, enabling intelligent drug release in response to the osteoarthritic pathological microenvironment, effectively attenuating cartilage damage, synovial hyperplasia, and osteosclerosis while promoting neocartilage formation. Additionally, Wharton’s jelly mesenchymal stem cell-laden gelatin microsphere delivery systems provide favorable growth microenvironments, effectively mediating stem cell engraftment and functionalization at articular sites, thereby significantly facilitating cartilage defect repair. , Regarding clinical translation, Varian’s Embozene microspheres represent the first and only CE-approved embolic microspheres specifically indicated for genicular artery embolization (GAE), achieving effective, minimally invasive, and durable pain relief in KOA patients through occlusion of inflammatory regional blood supply and reduction of local inflammatory responses, bridging the therapeutic gap between conservative management and arthroplasty. Furthermore, NEXTBIOMEDICAL’s absorbable embolic microsphere Nexsphere-F has received FDA “Breakthrough Device” designation, utilizing absorbable microsphere technology for targeted delivery of rapid-acting microspheres to aberrant vasculature associated with osteoarthritic pain; rapid microsphere dissolution effectively alleviates OA pain in patients with inadequate conservative treatment response.

Based on the aforementioned research background and clinical needs, this study innovatively established a sustained-release PLGA microsphere delivery system coloaded with type I collagen (COLI) and dexamethasone sodium phosphate (DSP) with uniform particle size and good stability by optimizing preparation conditions such as homogenization speed, homogenization time, and stirring duration, for the treatment of KOA. The core design concept of this system lies in integrating the anti-inflammatory and analgesic pharmacological effects of DSP with the physicobiological supportive function of COLI, achieving a transition in KOA treatment from mere symptom control to microenvironmental remodeling and tissue repair through a “drug-device synergistic” strategy (Figure a). DSP, as a glucocorticoid drug, possesses significant anti-inflammatory and immunosuppressive effects; local application in the joint cavity can achieve effective anti-inflammatory concentrations with reduced dosage, thereby inhibiting synovial inflammation, alleviating synovial tissue hyperplasia, relieving joint pain, and improving joint function. , COLI, as a natural biomaterial, exhibits good biocompatibility, biodegradability, and low immunogenicity, supporting cell adhesion, improving synovial fluid viscosity, regulating the physicochemical microenvironment of the joint cavity such as pH, promoting chondrocyte adhesion and proliferation, and enhancing their viability, thus providing favorable matrix conditions for cartilage repair. , The synergistic action of the two can effectively regulate macrophage phenotypic switching, inhibit pro-inflammatory M1 macrophages from secreting inflammatory factors such as IL-6 and TNF-α, block the inflammatory cascade, reduce the loss of cartilage matrix components, and accelerate tissue repair and inflammation resolution. This overcomes the limitation of traditional single-drug treatments that only address symptom control, enabling proactive intervention in KOA pathological progression, and provides new ideas and strategies for the clinical treatment of KOA.

1.

Characterization of drug-loaded polymer microspheres. (a) Schematic diagram of the preparation process of drug-loaded polymer microspheres. ACLT was used to construct rat knee osteoarthritis models. In the OA joint, inflammatory cells undergo polarization toward a catabolic phenotype, triggering the secretion of pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6. These inflammatory mediators further upregulate the expression of matrix-degrading enzymes, MMP3 and MMP13, which accelerate cartilage extracellular matrix degradation and aggravate osteoarthritic lesions. Intra-articular injection of MPs PLGA DSP@COLI promoted the polarization of pro-inflammatory M1 macrophages to anti-inflammatory M2 phenotypes, relieved synovitis, enhanced extracellular matrix synthesis, and inhibited cartilage degradation. (b) Particle size and zeta potential of drug-loaded polymer microspheres, n = 3. (c) SEM images of drug-loaded polymer microspheres. (d) In vitro cumulative release curves of drug-loaded polymer microspheres, n = 3. The data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significant difference.

2. Results and Discussion

2.1. Characterization of Drug-Loaded Polymer Microspheres

PLGA, an FDA-approved biodegradable polymer with metabolically absorbable degradation products, is extensively utilized in KOA-microsphere-based drug delivery systems. Accordingly, this investigation employed PLGA as the carrier material and fabricated drug-loaded polymeric microspheres via a water-in-oil-in-water (W/O/W) double emulsion methodology. By modulating homogenization parameters including rotational speed and duration, alongside optimizing the poly­(vinyl alcohol) (PVA) surfactant concentration, uniformly sized drug-loaded polymeric microspheres were successfully prepared (Figure a).

After particle size and potential detection using a Malvern laser particle size analyzer, the particle size of MPs PLGA DSP@COLI was measured to be approximately 15.10 μm, with a span of about 1.55; MPs PLGA DSP had a particle size of approximately 8.87 μm, with a span of about 1.26; MPs PLGA COLI had a particle size of approximately 10.42 μm, with a span of about 1.6; and MPs PLGA Blank had a particle size of 7.32 μm, with a span of 1.14. Moreover, all microspheres exhibited negative potentials, ranging from approximately −10.93 to −23.20 mV (Figure b, Figure S1). After placing the prepared microspheres in the refrigerator for 2 weeks, the particle size was measured again. The result was similar to that of the first measurement. Based on existing research findings, the loss of glycosaminoglycans reduces surface negative charge density on injured cartilage. We therefore hypothesize that negatively charged MPs PLGA DSP@COLI may preferentially accumulate at GAG-deficient cartilage-damaged sites due to reduced electrostatic repulsion. The morphology of MPs was observed using scanning electron microscopy (SEM), revealing microspheres with a regular spherical shape, smooth and intact surfaces, and good dispersibility, with no adhesion observed. Under SEM observation, the particle sizes of the microspheres were similar to those measured by the laser particle size analyzer (Figure c).

After verification by full-wavelength scanning, the results showed that COLI and the blank microsphere extract had no interference with DSP content detection, indicating that the UV method can be used for DSP content determination (Figure S2 and Figure S3). Therefore, the drug loading of DSP and COLI was measured using a UV spectrophotometer and a BCA protein assay kit, respectively. The results showed that the DSP loading in MPs PLGA DSP@COLI was approximately 2.00%, and the COLI loading was approximately 19.32%; the DSP loading in MPs PLGA DSP was approximately 1.78%; and the COLI loading in MPs PLGA COLI was approximately 18.96%. Among these, the encapsulation efficiency of DSP in each type of microsphere ranged from approximately 56% to 70%, and the encapsulation efficiency of COLI was approximately 60%–70%.

The in vitro cumulative release curves indicated that neither COLI nor DSP exhibited burst release in release media of different pH values; the release during the first 7 days was relatively slow; in the early stage, the PLGA scaffold had not undergone significant degradation, and drug release was mainly controlled by free diffusion driven by the concentration gradient, showing a gradual release trend. In the later stage, as PLGA degraded, the diffusion rate of the drug from the microspheres increased; under both conditions, the in vitro release of DSP from microspheres was faster than that of COLI; both drugs exhibited a faster release rate in release media with a lower pH (Figure d). During OA occurrence, the joint cavity microenvironment is slightly acidic, so these microspheres can better release the drug during KOA occurrence, with a release period of over 20 days, enabling long-acting sustained release and helping to reduce the frequency of injections.

2.2. Isolation of Primary Chondrocytes with Very High Purity

The extracted primary cells can be identified as chondrocytes by using specific antibodies to label characteristic chondrocyte proteins, such as collagen II and aggrecan. If the cells express these markers, then specific fluorescent signals can be observed using a laser-scanning confocal microscope or a fluorescence microscope, indicating that the cells are chondrocytes. Generally, a marker positivity rate over 90% is required for the cells to be considered qualified chondrocytes. In this experiment, chondrocytes adhered well and grew well (Figure S4). After immunofluorescence identification of type II collagen and processing with ImageJ, the results showed that the purity of the extracted chondrocytes was over 99% (Figure a).

2.

In vitro study on cellular biocompatibility and pharmacological efficacy. (a) Immunofluorescence identification of chondrocyte purity. Cell nuclei: Hoechst 33342; Collagen II: type II collagen antibody; scale bar: 10 μm. (b) CCK-8 assay to detect the concentration- and time-dependent effects of MPs on the viability of mouse chondrocytes. (c) CCK-8 assay to detect the concentration- and time-dependent effects of MPs on the viability of RAW264.7 cells. (d) Effects of free drugs and various MPs on chondrocyte secretion of cartilage matrix components, type II collagen (COL II) and glycosaminoglycan (GAG). The red dashed line represents the level of the control group. Values above the red dashed line indicate higher secretion levels of cartilage matrix components than those in the control group. The data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significant difference.

2.3. Drug-Loaded Polymer Microspheres Exhibit Good In Vitro Cellular Biocompatibility

The effect of DSP on mouse chondrocyte activity decreases with prolonged incubation time and increased concentration. At 24 h, when the DSP concentration is 1.95–15.63 μg/mL, the effect on mouse chondrocyte activity is minimal, but by 48 h of incubation, cell activity decreases to varying degrees. COLI promotes the proliferation of mouse chondrocytes. Within 24–48 h, the proliferation rate increases with the extension of incubation time; by 72 h, the proliferation rate may decrease due to high cell density. When the COLI concentration is 0.25–1 mg/mL, its effect on chondrocyte proliferation is better than at other concentrations, with a proliferation rate of approximately 27.46%–58.77% (Figure S5a–b).

At 24 h of incubation, DSP has little effect on RAW264.7 cell activity and can be disregarded; after 48 h, cell activity decreases to varying degrees with increasing concentration. After 24 h of incubation, different concentrations of COLI have no effect on RAW264.7 cell activity, and there is no significant difference compared with the control group; after 48 h, different concentrations of COLI show no toxic effect on RAW264.7 cells and even help enhance cell activity (Figure S5c–d).

After co-incubation with drug-loaded microspheres at different concentrations for 24 or 48 h, the viability of mouse chondrocytes was greater than or equal to 85%, and the drug-loaded microspheres could increase chondrocyte viability to some extent. This indicates that within the concentration range of 0–1 mg/mL, the drug-loaded microspheres have good biocompatibility with mouse chondrocytes (Figure b). After coincubation with drug-loaded microspheres at different concentrations for 24 or 48 h, the viability of RAW264.7 cells was greater than or equal to 85%, indicating that within the concentration range of 0–1 mg/mL, the drug-loaded microspheres have good biocompatibility with RAW264.7 cells (Figure c).

2.4. Drug-Loaded Polymer Microspheres Can Promote the Secretion of Matrix Components by Chondrocytes

Chondrocytes are the primary functional cells of articular cartilage, distributed within the lacunae of the cartilage matrix. Their core function is to synthesize and secrete cartilage matrix components, including type II collagen (which forms the fibrous scaffold of cartilage) and glycosaminoglycans (which form a hydrophilic gel to maintain the cartilage elasticity and compressive resistance). These components together constitute the structural basis of the articular cartilage, ensuring joint cushioning and flexible movement. In this experiment, type II collagen (COLII) and glycosaminoglycan (GAG) kits were used to detect the secretion levels of these two cartilage matrix components in the supernatants after co-incubation with free drugs and MPs. The results showed that medical chitosan could increase the secretion of cartilage matrix components (GAG and COLII) by chondrocytes in the joint cavity, with statistically significant differences compared to the control group. All drug groups could promote chondrocyte secretion of COLII; at 48 h, the MPs PLGA DSP@COLI group significantly increased COLII secretion compared with the control group. Regarding GAG secretion, except for the DSP, MPs PLGA DSP, and MPs PLGA Blank groups, all drug groups could enhance chondrocyte secretion of GAG. At 48 h, COLI, MPs PLGA COLI, MPs PLGA DSP@COLI, and medical chitosan significantly promoted GAG secretion (Figure d).

2.5. Drug-Loaded Polymer Microspheres Can Induce Macrophages to Polarize into an Anti-inflammatory Phenotype

As shown in the flowChart Figure a, in in vitro experiments, RAW264.7 cells at passage 3 were seeded in six-well plates. After 24 h of cell adhesion, cells were stimulated with LPS-containing medium for 24 h to induce polarization, followed by treatment with the corresponding drugs and drug-loaded polymer microspheres. Subsequently, the polarization of the macrophage subpopulations was analyzed. In flow cytometry, CD86 is a marker for M1 macrophages, and CD206 is a marker for M2 macrophages; CD86+CD206– cells represent M1 macrophages, while CD86-CD206+ cells represent M2 macrophages. Quantitative analysis by flow cytometry showed that the proportion of M1 macrophages in the LPS group (21.95%) was relatively high and significantly different compared with the blank group. In the treatment groups with COLI, DSP, MPs PLGA COLI, MPs PLGA DSP, MPs PLGA DSP@COLI, MPs PLGA blank, and medical chitosan, the proportion of M1 macrophages decreased and was significantly different compared to the LPS group. Regarding the proportion of M2 macrophages, all treatment groups showed an increase relative to that of the LPS group (5.09%). Except for the DSP group, the proportion of M1 macrophages in the MPs PLGA DSP@COLI group (3.50%) was lower than that in other drug-treated groups, while the proportion of M2 macrophages (22.8%) was higher than that in other groups, indicating that MPs PLGA DSP@COLI induced RAW264.7 cell polarization toward M2 macrophages at a level comparable to free DSP (Figure b–c, Figure S6).

3.

Investigation of anti-inflammatory capacity in vitro. (a) Schematic diagram of the in vitro macrophage polarization induction experiment; created in BioRender. (b) Representative flow cytometry plots of M1 (CD86+CD206−) macrophages and M2 (CD86–CD206+) macrophages. (c) Quantitative analysis showing the percentage of M1 (CD86+CD206−) macrophages and M2 (CD86–CD206+) macrophages. (d) In vitro anti-inflammatory ability of free drugs and MPs. The data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significant difference.

2.6. Drug-Loaded Polymer Microspheres Can Reduce the Secretion of Inflammatory Factors In Vitro

An in vitro inflammatory model of RAW264.7 cells was established to simulate the inflammatory state of chondrocytes in KOA. After coincubation with different drug-loaded polymer microspheres, the supernatants were collected to analyze the secretion levels of related inflammatory factors such as IL-6 and TNF-α, in order to determine whether the drug-loaded polymer microspheres can exert anti-inflammatory effects. The results showed that following LPS stimulation, the levels of inflammatory factors TNF-α and IL-6 were elevated, with significant differences compared to the Blank group. Except for the MPs PLGA Blank and Medical Chitosan groups, all other treatment groups were able to reduce the levels of inflammatory factors TNF-α and IL-6 to varying degrees. Among them, the DSP group, MPs PLGA DSP, and MPs PLGA DSP@COLI demonstrated a more pronounced ability to reduce inflammatory factor levels, showing significant differences compared to the control group. Moreover, MPs PLGA DSP@COLI and MPs PLGA DSP exerted comparable effects on reducing TNF-α secretion; however, MPs PLGA DSP@COLI significantly outperformed MPs PLGA DSP in suppressing IL-6 secretion (Figure d).

2.7. Monitoring of Body Weight and Condition of Rats during the Treatment Process

A rat KOA model was established using anterior cruciate ligament transection (ACLT) (Figure a). Within 1 week after performing ACLT surgery, the rats ate normally and were in good health; however, 2–4 weeks after modeling, the rats showed reduced weight-bearing on the right hind limb, difficulty moving, and decreased activity. Rats were selected from both the healthy group and the ACLT group for sampling in the second, third, and fourth weeks after surgery to observe the femur and tibial plateau. The results showed that the tibial plateau joint surface in the healthy group was smooth and shiny, and the joint capsule structure was normal. In the ACLT group, obvious damage appeared on the cartilage surface of the medial tibial plateau. The joint surface was uneven, the cartilage surface showed erosion, glossiness decreased, the damaged cartilage area was light gray-white, and proliferation was observed in the surrounding joint capsule and soft tissue. This indicates that the model was successfully established (Figure S7). After intra-articular drug injection for treatment, during the efficacy observation period, the rats’ weight gain was not significantly affected, indicating that the free drugs and MPs have good in vivo biocompatibility and no obvious in vivo toxicity (Figure b).

4.

(a) Schematic diagram of in vivo pharmacologic verification. (b) Changes in body weight of rats (n = 6). (c) Measurement of inflammatory factors TNF-α and IL-6 in rat serum (n = 3). (d) Measurement of inflammatory factors TNF-α and IL-6 in rat joint synovial lavage fluid (n = 3). The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significant difference.

2.8. Drug-Loaded Polymer Microspheres Can Reduce the Levels of Inflammatory Factors in Serum and Joint Synovial Lavage Fluid

The concentrations of pro-inflammatory cytokines TNF-α and IL-6 in the serum of each group of rats were measured using TNF-α and IL-6 ELISA kits. In the detection of the inflammatory factor TNF-α in serum samples, the level of the secreted inflammatory factor in the model group rats was approximately 13.92 pg/mL. Except for the COLI, DSP, and Medical Chitosan groups, the secretion of inflammatory factors in the serum of the remaining treatment groups was reduced, showing significant differences compared with the control group. Compared with the model group, the MPs PLGA DSP group could reduce TNF-α by 4.28 pg/mL, and the MPs PLGA DSP@COLI group could reduce TNF-α by 4.65 pg/mL. Among these, MPs PLGA DSP and MPs PLGA DSP@COLI showed a better ability to reduce the inflammatory factor TNF-α compared with free DSP, with significant differences. In the detection of the inflammatory factor IL-6 in serum, the level of the secreted inflammatory factor in the model group rats was approximately 166.88 pg/mL. Except for the DSP, MPs PLGA Blank, and Medical Chitosan groups, the secretion of inflammatory factors in the serum of the remaining treatment groups was reduced, showing significant differences compared with the control group. Compared with the model group, the MPs PLGA DSP group could reduce IL-6 by 99.38 pg/mL, and the MPs PLGA DSP@COLI group could reduce IL-6 by 87.00 pg/mL. Among these, MPs PLGA DSP and MPs PLGA DSP@COLI showed a better ability to reduce the inflammatory factor IL-6 compared with free DSP, with significant differences (Figure c).

In the detection of the inflammatory factor TNF-α in joint synovial lavage fluid, the model group rats secreted the highest level of inflammatory factors, approximately 53.73 pg/mL. Except for the COLI group, the secretion of inflammatory factors in the joint synovial lavage fluid of rats in the other treatment groups decreased, all showing significant differences compared with the control group. Compared with the model group, the DSP group could reduce TNF-α by 25.62 pg/mL, the MPs PLGA DSP group could reduce TNF-α by 34.78 pg/mL, and the MPs PLGA DSP@COLI group could reduce TNF-α by 35.51 pg/mL. Among these, MPs PLGA DSP and MPs PLGA DSP@COLI had a better ability to reduce the inflammatory factor TNF-α compared with free DSP. In the detection of the inflammatory factor IL-6 in joint synovial lavage fluid, the model group rats secreted inflammatory factors at a level of approximately 152.88 pg/mL. Except for the DSP group, the secretion of inflammatory factors in the joint synovial lavage fluid of rats in the other treatment groups decreased to varying degrees. Compared with the model group, the MPs PLGA DSP group could reduce IL-6 by 109.25 pg/mL, and the MPs PLGA DSP@COLI group could reduce IL-6 by 99.25 pg/mL. Among these, MPs PLGA DSP and MPs PLGA DSP@COLI had a better ability to reduce the inflammatory factor IL-6 compared with free DSP, with significant differences (Figure d).

In summary, intra-articular injection of drug-loaded polymer microspheres can significantly reduce the secretion of pro-inflammatory cytokines in rat serum and joint synovial lavage fluid, thereby slowing the progression of KOA.

2.9. Drug-Loaded Polymer Microspheres Can Delay Articular Cartilage Degeneration and Lower the OARSI Cartilage Score

Glycosaminoglycans (GAGs) and type II collagen are the two main components of the ECM. The higher the content of type II collagen and GAGs, the better the condition of the cartilage. The results of Safranin O-Fast Green staining showed that in the sham surgery group and the MPs PLGA DSP@COLI group, Safranin O staining in the articular cartilage was uniform and red, indicating normal cartilage matrix staining, sufficient glycosaminoglycan (GAG) content, without significant loss, good integrity of the cartilage matrix, basically intact tide lines, and no cartilage wear. In the PBS and Medical Chitosan groups, there was a large area of Safranin O staining loss, indicating significant loss of cartilage matrix components and cartilage wear, with the PBS group being more severe. The COLI and DSP groups showed lighter staining, uneven matrix staining, and meniscal damage. The MPs PLGA DSP group showed lighter cartilage matrix staining and meniscal damage. The MPs PLGA COLI and MPs PLGA Blank groups had large areas of matrix staining loss, indicating disruption of the cartilage surface integrity (Figure a). The degree of cartilage degeneration was assessed by three experienced members by using the OARSI scoring system. The results showed that the OARSI scores were 0.33 for the sham surgery group, 5.33 for the PBS group, 3.67 for the COLI group, 4.67 for the DSP group, 3 for the MPs PLGA COLI group, 0.5 for the MPs PLGA DSP group, 0.33 for the MPs PLGA DSP@COLI group, 2.33 for the MPs PLGA Blank group, and 4.33 for the Medical Chitosan group (Figure b).

5.

(a) Safranin O-Fast Green staining of the knee joint, scale bar = 500 μm (black arrows indicate uneven or missing Safranin O staining). (b) Assessment of cartilage degradation using the OARSI scoring system (n = 6). (c) H&E-stained sections of knee joints from each group of rats; the dashed areas indicate synovial tissue; scale bar = 500 μm. (d) Synovitis scoring (n = 6). The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significant difference.

2.10. Drug-Loaded Polymer Microspheres Can Alleviate Synovitis and Reduce Synovitis Scores

Through H&E staining of the knee joint, the results can reflect the degree of synovitis in the knee joint of rats in each group, and synovitis scoring was conducted accordingly. The results showed that in the sham surgery group, the synovial lining cells were a single layer of flat epithelium arranged regularly without thickening; fibroblasts in the matrix were evenly distributed without proliferation, blood vessels were normal in morphology, and the structure of the matrix was intact; no inflammatory cell infiltration was observed throughout the synovium, and the tissue morphology was normal. In the PBS group, the synovium showed significant proliferation and thickening; there was a diffuse infiltration of a large number of inflammatory cells, accompanied by cartilage wear. In the COLI group, synovial proliferation was moderately thickened with a considerable amount of inflammatory cell infiltration and angiogenesis. In the DSP group, synovial cells showed mild proliferation, with a small number of inflammatory cells infiltrating and mild thickening of the synovium. In the MPs PLGA COLI group, the synovium was slightly proliferated and thickened but without obvious inflammatory cell infiltration or angiogenesis. In the MPs PLGA DSP group, the morphology of rat synovial tissue was basically normal, the lining cells were neatly arranged with almost no thickening, and there was no significant inflammatory cell infiltration. In the MPs PLGA DSP@COLI group, rat synovial tissue morphology was essentially normal, with almost no thickening and no obvious synovial proliferation or inflammatory cell infiltration, similar to the physiological state of the sham surgery group. In the MPs PLGA blank group, synovial cells proliferated, the synovium was moderately thickened, with inflammatory cell infiltration accompanied by angiogenesis. In the Medical Chitosan group, synovial proliferation was moderately thickened, accompanied by a small amount of inflammatory cell infiltration (Figure c). At the same time, the synovial tissues of rats in each group were scored by three trained personnel. The synovitis scores were: sham surgery group 0.33, PBS group 8.33, COLI group 6.67, DSP group 4.67, MPs PLGA COLI group 2.67, MPs PLGA DSP group 1.67, MPs PLGA DSP@COLI group 1.67, MPs PLGA Blank group 6.67, and Medical Chitosan group 5.67 (Figure d).

These results indicate that MPs PLGA DSP@COLI can improve joint cartilage lesions an alleviate synovitis and synovial proliferation, thereby delaying the progression of KOA.

2.11. Type II Collagen, CD86, and CD206 Immunohistochemical Staining

To assess the synthesis of the cartilage matrix, immunohistochemical staining for type II collagen was performed. Additionally, immunohistochemistry was used to detect the expression of CD86 (a marker of M1 macrophages) and CD206 (a marker of M2 macrophages) to investigate the state of the inflammatory cell infiltration in the synovium.

The results showed that cartilage matrix components were depleted in the joints of rats in the PBS group; in contrast, MPs PLGA DSP@COLI significantly increased the expression of the cartilage matrix marker COL II, preventing the loss of cartilage matrix components and maintaining cartilage structure (Figure a). The CD86-positive staining in the sham surgery group was lighter; in the PBS group, the CD86-positive staining was darker, indicating a greater infiltration of inflammatory cells into the synovial tissue, suggesting the development of synovitis. In all treatment groups, CD86 staining showed varying degrees of reduction compared with the PBS group. Among these, the MPs PLGA DSP group and MPs PLGA DSP@COLI group exhibited lighter CD86-positive staining, indicating that they could reduce the expression of the M1 macrophage marker CD86 and thereby alleviate synovitis (Figure b). Similarly, the CD206-positive staining in the PBS group was lighter, while CD206 staining in all treatment groups was deeper compared with the PBS group. Notably, the MPs PLGA DSP@COLI group showed the deepest CD206-positive staining, suggesting that it could increase the expression of the M2 macrophage marker CD206, thereby modulating the synovial inflammatory microenvironment and relieving synovial inflammation (Figure c).

6.

(a) Representative images of COLII immunohistochemical staining, scale bar: 100 μm. (b) Representative images of CD86 immunohistochemical staining. (c) Representative images of CD206 immunohistochemical staining. For CD86 and CD206, the upper images have a scale bar of 1000 μm, and the lower images have a scale bar of 200 μm. (d) Immunohistochemical staining scores of COLII. (e) Immunohistochemical staining scores of CD86. (f) Immunohistochemical staining scores of CD206. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significant difference.

Immunohistochemical scoring results indicated that MPs PLGA DSP@COLI could inhibit synovial inflammation in KOA rats and also enhance the synthesis of the cartilage matrix component COL II (Figure d–f).

2.12. In Vivo Biocompatibility Assessment

After KOA treatment via intra-articular drug injection, histopathological analysis of the heart, liver, spleen, lungs, and kidneys was performed using H&E staining to further evaluate the in vivo biocompatibility of the drug-loaded polymer microspheres. The results showed that no detectable damage or histopathological lesions were observed in tissue sections of the heart, liver, spleen, lungs, and kidneys, indicating that the drug-loaded polymer microspheres have good in vivo biocompatibility (Figure S8).

3. Methods

3.1. Materials

Dexamethasone sodium phosphate (DSP) and sorbitol (lyophilization protectant) were purchased from Beijing InnoCare Technology Co., Ltd. Yuelixin recombinant type I collagen (COL I) (SPA) was purchased from Zhuji Juyuan Biotechnology Co., Ltd., Zhejiang. Medical chitosan was purchased from Shanghai Qisheng Biopharmaceutical Co., Ltd. Poly­(lactic-co-glycolic acid) (PLGA, MW 50,000–75,000), poly­(vinyl alcohol) (PVA), sodium hydroxide, ethyl acetate, and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich. Tween-20 was purchased from Shenggong Bioengineering (Shanghai) Co., Ltd. Micro BCA Protein Assay Kit, Mouse IL-6 Uncoated ELISA, and Mouse TNF-α Uncoated ELISA were purchased from Thermo Fisher. Acetonitrile (ACN) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Type II collagenase was purchased from Absin (Shanghai) Biotechnology Co., Ltd. 4% paraformaldehyde was purchased from Biosharp. CCK-8 was purchased from Bijun Tian Biotechnology Co., Ltd. 0.4% trypan blue staining solution, Hoechst 33342, antifluorescence quencher, citrate antigen retrieval solution, BSA, rabbit serum, and DAB color development kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. FITC-conjugated anti-Rabbit IgG­(H+L)­(AS011) was purchased from Suzhou Zhongchu Zhiyuan Technology Co., Ltd. Mouse collagen II antibody was purchased from Novotec (Cat. No. 20251, immunogen: native type II collagen extracted from mouse. Host: rabbit). DMEM high-glucose medium, penicillin-streptomycin, fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were purchased from Procell. Trypsin solution was purchased from Yishan Bio. Mouse type II collagen (COL II) and mouse glycosaminoglycan (GAG) ELISA kits were purchased from Wuhan Beinlai Biotechnology Co., Ltd. Flow cytometry antibodies were purchased from BioLegend. Membrane perforation reagent was purchased from Yisheng Bio. Isoflurane was purchased from RWD. Rat TNF-α and Rat IL-6 ELISA kits were purchased from Youke Life Science & Technology (Hangzhou) Co., Ltd. n-Butanol, anhydrous ethanol, xylene, hydrochloric acid, ammonia, neutral resin, and 3% hydrogen peroxide were purchased from China National Pharmaceutical Group Chemical Reagents Co., Ltd. H&E staining solution was purchased from Shanghai Jingke Chemical Technology Co., Ltd. Van Gieson staining kit (bone tissue) was purchased from Wuhan Google Biotechnology Co., Ltd. Hematoxylin staining solution was purchased from Shanghai Jingke Chemical Technology Co., Ltd. Primary antibodies were purchased from Sanying Biotechnology Co., Ltd. Secondary antibodies were purchased from Genentech.

3.2. Cells

The RAW264.7 cell line was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Cells were cultured in high-glucose DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (P/S). The mice used for isolating primary mouse chondrocytes for cell culture and identification were 6–8-week-old C57BL/6J mice purchased from Shanghai Jihui Laboratory Animal Co., Ltd. All animal experiments complied with the Laboratory Animal Administration Regulations of Soochow University (approval number 202407A0953).

3.3. Ethics of Animal Studies

For in vivo experiments, SD rats weighing 151–200 g were used to establish the KOA model. They were purchased from Cavens Laboratory Animal Co., Ltd. During the experiments, all rats were housed at the animal facility of Soochow University in accordance with the guidelines for the care and use of laboratory animals. All animal experiments were conducted in compliance with the Soochow University regulations for laboratory animal management. The animal ethics approval number was 202606A0329.

3.4. Preparation of DSP- and COLI-Coloaded Microspheres

The following solutions were prepared in advance: ultrapure water was sterilized by passing it through a 0.22 μm filter. A 30 mg/mL COLI solution and a 20 mg/mL DSP solution were prepared, placed them in 20 mL vials, sealed, labeled, and storeed at 4 °C for later use. 1 mL of 20 mg/mL (2% w/v) PLGA solution was prepared in an 8 mL vial as the oil phase and kept it at room temperature for later use. 50 mL of 5 mg/mL PVA (0.5% w/v) solution and 10 mL of 10 mg/mL PVA solution were prepared and stored at room temperature for later use.

The W/O/W double emulsion method was used to prepare MPs PLGA DSP@COLI microspheres. 0.4 mL of 30 mg/mL COLI solution and 0.4 mL of 20 mg/mL DSP solution were added into 1 mL of 20 mg/mL PLGA ethyl acetate solution, vortexed for 10 s, and then emulsified with a homogenizer at setting 2 for 20 s to obtain the primary emulsion; quickly, the primary emulsion was added into 10 mL of precooled 10 mg/mL PVA solution (placed in a 15 mL centrifuge tube), vortexed for 10 s, and homogenized at setting 2 for 20 s to obtain the double emulsion. It was transferred with a pipet into 50 mL of 5 mg/mL PVA (0.5% w/v) solution that was being stirred at 300 rpm, and the mixture was stirred at 300 rpm for 3 h to evaporate the organic solvent, ethyl acetate, and solidify the microspheres.

After 3 h, the solution was centrifuged at 2000 rpm for 15 min, the supernatant was discarded, and the solution was washed three times with 10 mL of deionized water. After each wash, the solution was centrifuged at 2000 rpm for 15 min and the supernatant was discarded; then 1 mL of 10 mg/mL (1% w/v) sorbitol solution (500 mg dissolved in 50 mL ultrapure water) was added to resuspend the microspheres so that the ratio of drug core to the lyoprotectant sorbitol was 4:1. The samples were transferred into vials and stored them overnight in a −20 °C freezer, then placed them in a −80 °C freezer and freezed for at least 8 h or longer. Subsequently, samples were freeze-dried using a tetracycline freeze-dryer for 2 days, and the freeze-dried microspheres were stored in a −80 °C freezer. After lyophilization, the particle size and drug loading of the drug-loaded polymer microspheres were measured using a laser particle size analyzer.

3.5. Preparation of Separately Loaded DSP or COLI Microspheres

The following solutions were prepared in advance: 30 mg/mL COLI solution and 20 mg/mL DSP solution were prepared by sterilizing the ultrapure water solvent through a 0.22 μm filter membrane, placed in 20 mL vials, sealed, and stored at 4 °C for later use. 1 mL of a 20 mg/mL (2% w/v) PLGA solution was prepared in an 8 mL vial, used it as the oil phase, and set aside at room temperature. 50 mL of 5 mg/mL PVA (0.5% w/v) solution and 10 mL of 10 mg/mL PVA solution were prepared and set aside at room temperature.

MPs PLGA DSP or MPs PLGA COLI were prepared by the W/O/W double emulsion method. 0.4 mL of 30 mg/mL COLI solution or 0.4 mL of 20 mg/mL DSP solution was added to 1 mL of 20 mg/mL PLGA ethyl acetate solution, and then the corresponding monodrug microspheres were prepared by the following treatment: vortex-mixed for 10 s, then stirred with a homogenizer in 2 stages for 20 s to obtain primary emulsion; primary emulsion was quickly added to precooled 10 mL of 10 mg/mL PVA solution (this solution was packed in a 15 mL centrifuge tube), vortexed for 10 s, stirred for 20 s with a homogenizer for 20 s to obtain a double emulsion, transferred to 50 mL of 5 mg/mL PVA (0.5% w/v) solution under stirring at 300 rpm with a pipet, continued to stir at 300 rpm for 3 h, volatilized the organic solvent, ethyl acetate, and cured the microspheres.

After 3 h, the solution was centrifuged at 2000 rpm for 15 min, the supernatant was discarded, washed with 10 mL of deionized water 3 times, centrifuged at 2000 rpm for 15 min after washing, and the supernatant was discarded. Then, 1 mL of 10 mg/mL (1% W/V) of sorbitol solution (500 mg dissolved in 50 mL of ultrapure water) was added to resuspend to make the ratio of the core part of the drug to the lyophilized protectant sorbitol 4:1. The samples were placed in vials and stored overnight in a refrigerator at −20 °C, then frozen in a refrigerator at −80 °C for at least 8 h or more, and then lyophilized in a four-ring freeze dryer for 2 days, and the freeze-dried microspheres were stored in a freezer at −80 °C. After lyophilization, the particle size and drug loading of the drug-loaded polymer microspheres were measured by a laser particle size analyzer.

3.6. Preparation of Blank Microspheres

The following solutions were prepared in advance: the solvent ultrapure water was sterilized through a 0.22 μm filter. a 20 mg/mL (2% w/v) PLGA solution, 1 mL in an 8 mL vial, was prepared to serve as the oil phase and kept at room temperature until needed. 50 mL of 5 mg/mL PVA (0.5% w/v) solution was prepared and kept at room temperature until needed.

Preparation of MPs PLGA blank: 1 mL of 20 mg/mL PLGA solution in ethyl acetate was added to 50 mL of 5 mg/mL PVA (0.5% w/v) solution and stirred at 1000 rpm. After addition, stirring was continued at 1000 rpm for 2 min to emulsify and form a primary emulsion. Then, the speed was adjusted to 300 rpm and stirred for 3 h until the organic solvent completely evaporated to solidify the microspheres.

After 3 h, the solution was centrifuged at 2000 rpm for 15 min, the supernatant was discarded, and the solution was washed three times with 10 mL of deionized water. The solution was centrifuged at 2000 rpm for 15 min after each wash, and the supernatant was discarded. Then, 1 mL of 10 mg/mL (1% w/v) sorbitol solution (500 mg dissolved in 50 mL ultrapure water) was added to resuspend the sample, placed it in a vial, and stored at −20 °C overnight. The sample was transferred to a −80 °C freezer to freeze for at least 8 h or longer. Subsequently, the microspheres wer lyophilized for 2 days using a freeze-dryer and stored the freeze-dried microspheres at −80 °C. After freeze-drying, the particle size of the blank polymer microspheres was measured using a laser particle size analyzer.

3.7. Detection of Particle Size and Potential by Laser Particle Size Analyzer

A BT-2001 laser particle size distribution analyzer was used to measure the particle size of drug-loaded polymer microspheres. A microsphere solution at a certain concentration was prepared with ultrapure water and transferred to the testing dish, which was then placed in the test chamber for detection. During the detection process, appropriate dilution should be performed according to the instrument’s prompts.

A Zetasizer Nano Malvern laser particle size analyzer was used to detect the potential of drug-loaded polymer microspheres. A dilute solution of drug-loaded polymer microspheres at a certain concentration was prepared with ultrapure water, and then the dilute solution was aspirated into a potential testing dish, making sure that the solution contacted the metal of the testing dish and then placed in the test chamber for detection.

3.8. Observation of Microsphere Size and Morphology by Scanning Electron Microscopy

The surface morphology and size of drug-loaded polymer microspheres were observed using a scanning electron microscope. The prepared drug-loaded polymer microspheres were made into 1 mL solution at a concentraion of 8 mg/mL. After centrifugation at 2000 rpm for 5 min, the supernatant was discarded and the wet microspheres were collected. They were resuspended in 50 μL of ultrapure water, then 2 μL of the wet microspheres was pipeted onto a conductive silicon wafer, allowing the liquid to spread evenly. The conductive silicon wafer was affixed to the scanning electron microscope sample stage and placed in a fume hood overnight, and after gold sputtering the next day, it was observed under a Hitachi S-4700 scanning electron microscope.

3.9. Drug Loading Detection of Drug-Loaded Polymer Microspheres

DSP Drug-Loading Detection: drug-loading detection was performed on polymer microspheres loaded with COLI and DSP or on polymer microspheres loaded with DSP alone. An appropriate amount of lyophilized drug-loaded polymer microspheres was taken (about 15 mg per sample, three samples), 1 mL (about 3 times the amount) of acetonitrile was added, (ultrasonically disrupted or) dissolved with vortexing, and, after high-speed centrifugation (15000 rpm, 20 min, 4 °C), the supernatant was collected. The total concentration W of DSP in the supernatant was measured using a UV spectrophotometer. In addition, an appropriate amount of lyophilized microspheres (about 15 mg per sample, three samples) was taken, 1 mL (about 3 times the amount) of ultrapure water was added, (ultrasonically disrupted or) dissolved with vortexing, centrifuged and the supernatant was collected, and then the free concentration W₀ of DSP in the supernatant using the UV method was measured. W₁ is the concentration of the carrier. The drug-loading content (DLC%) is calculated according to the following formula: DLC% = (W – W₀)/W₁ × 100%.

COLI Drug Loading Detection: 1) Preparation of the dilution solution: 9.5 mL H₂O, 450 μL Tween-20, 50 μL 10 M NaOH (used to dissolve MPs and release COLI from the microspheres for concentration measurement). 2) 10 M NaOH: Dissolve 12 g of sodium hydroxide in 30 mL of H₂O. 3) Prepare a Micro BCA Protein Assay Kit (23235, Thermo Fisher); preheat an electric thermostat water bath and set the temperature to 90 °C. 4) Sample preparation: Lyse 15 mg of MPs in 1 mL of dilution solution, incubate in a 90 °C water bath for 2 h, centrifuge at 15000 rpm for 20 min, collect the supernatant, and measure the protein concentration using the Micro BCA kit according to the supplier’s instructions. 5) Finally, measure the OD value at 562 nm using a microplate reader. Create a standard curve, then use it to convert to concentration and calculate drug loading.

3.10. Detection of the In Vitro Cumulative Release Rate of Drug-Loaded Polymer Microspheres

The release of COLI and DSP from PLGA microspheres was studied in a phosphate-buffered saline (PBS) system (pH 7.4 PBS simulating the physiological conditions of synovial fluid in normal joint cavities, and pH 6.5 PBS simulating the slightly acidic environment of synovial fluid in inflamed joints). Two portions of approximately 60 mg of each polymer microsphere were weighed and placed in two 10 mL EP tubes, each dissolved in 7.2 mL of PBS at different pH values, and then divided equally into 6 portions of 1.2 mL each in 2 mL EP tubes, which were placed in a 37 °C incubator shaker at 100 rpm. At each time point of 1, 3, 7, 10, 14, and 21 days, the polymer microspheres in the EP tubes were separated from the release medium by centrifugation at 8000 rpm for 5 min, and 500 μL of the release medium was taken to measure the concentration and calculate the in vitro drug release. The release samples were analyzed using the BCA protein assay and the UV method to determine the concentration of COLI and DSP in order to detect the cumulative release.

3.11. Primary Mouse Chondrocyte Extraction and Culture

Mice were euthanized by cervical dislocation and immersed in 75% ethanol for 5 min. The leg bones of the mice were removed, first soaked in 75% ethanol for 2 min, and then placed in PBS. Next, in a biosafety cabinet, sterilized scissors were used to separate the transparent cartilage from the knee joint into a 15 mL centrifuge tube and the cartilage was washed twice with an appropriate amount of PBS (which can contain antibiotics). Sterilized scissors were used to mince the washed cartilage, which was transferred into a six-well plate. 0.2% mouse type II collagenase solution (10 mg of type II collagenase dissolved in 5 mL of DMEM basal medium) was added just enough to cover the cartilage pieces, and the samples were digested in a cell culture incubator for 1 h, taking out the six-well plate every 15 min and pipeting a few times to ensure the collagenase solution fully contacted the cartilage pieces, which facilitated digestion into single cells. About 6 mL of complete DMEM was added to terminate the digestion, followed by pipeting and filteration through a 200-mesh filter. The filtrate was collected into a 50 mL centrifuge tube and centrifuged at 1500 rpm for 5 min. The supernatant was discarded, 6 mL of complete DMEM was added to resuspend and then centrifuged again at 400 rpm for 5 min. The supernatant was cultured at 2 mL per well in a six-well plate and incubated for 24 h before changing the medium.

The cartilage pieces left on the filter and the pellet obtained from the previous centrifugation were placed in a six-well plate, a type II collagenase solution was added again to submerge the cartilage pieces, and digestion continued in the incubator for 1 h, pipeting a few times every 15 min to ensure adequate contact between the cartilage pieces and the collagenase solution. Then, 6 mL of complete DMEM was added to terminate digestion, filtered again, the filtrate was collected into a centrifuge tube and centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. 6 mL of complete DMEM was added to resuspend and centrifuged at 400 rpm for 5 min, and the supernatant was collected and cultured at 2 mL per well in a six-well plate. The remaining pellet was resuspended with 2 mL of complete DMEM and cultured in a six-well plate. The mixture was incubated in the incubator for 24 h before changing the medium.

The cells were observed under a microscope. When the cell density is about 80%–90%, the cells are digested and passaged. Generally, the medium is changed every 2–3 days. Chondrocytes passaged to the third generation are used for subsequent studies.

3.12. Identification of Primary Mouse Chondrocytes

1 mL of pre-equilibrated DMEM complete was added to the confocal dish, placed in the incubator for 15 min, then discarded; the above third-passage chondrocytes were collected, the cell density was adjusted to 4 × 10⁴/mL (cell density should be about 50%), and 0.5 mL per well was seeded and cultured for 24 h to allow cell adhesion; at the set time, the old medium was discarded and washed 3 times with PBS, 1 min each time; 300 μL of 4% paraformaldehyde was added to the dish and fix for 20 min, then wash 3 times with PBS, 1 min each time; permeabilize with 0.2% Triton X-100 at room temperature for 20 min, wash 3 times with PBS, 1 min each time; block with 5% BSA at room temperature for 30 min without washing (BSA solution is prepared in PBS: 0.05 g BSA in 1 mL PBS).

Primary antibody incubation: mouse Collagen II antibody (1:500) was added to the confocal dish (diluted specifically in 5% BSA according to the manual), and incubate overnight at 4 °C. During secondary antibody incubation, the samples were protected from light, washed 3 times with PBS, 5 min each time, and the fluorescent secondary antibody (diluted according to the manual) was added and incubated for 1 h in the dark.

Nuclear staining: wash 3 times with PBS, 5 min each time, stain nuclei with 300 μL of 5 μg/mL Hoechst 33342 at room temperature in the dark for 30 min, then wash 3 times with PBS, 5 min each time, and mount with antifade mounting medium; finally, observe and photograph using a laser confocal microscope.

3.13. Effects of Drug-Loaded Polymer Microspheres on the Viability of Mouse Chondrocytes and RAW264.7 Cells

Chondrocytes and RAW264.7 cells at passage 3 were seeded into their respective 96-well plates at approximately 10,000 cells per well in 100 μL of complete DMEM and incubated overnight in a cell culture incubator. After 24 h, the drugs were added. The experimental setup included a blank group containing only the medium without cells and drugs; a control group containing medium and cells but no drugs; and experimental groups containing medium, cells, and drugs, with the drugs being drug-loaded polymer microspheres at different concentrations. The microsphere samples were MPs PLGA DSP@COLI, MPs PLGA COLI, MPs PLGA DSP, and MPs PLGA Blank. The concentrations of the microspheres were set to 1, 0.8, 0.6, 0.4, 0.2, 0.1, and 0 mg/mL. The next day, the old medium was discarded, and 100 μL of complete DMEM containing different concentrations of the various microsphere solutions was added to each well, then incubated in the cell culture incubator for 24 and 48 h. At predetermined time points, the spent medium was removed, and all wells were gently rinsed with PBS to clear residual microspheres. Then, 100 μL of medium supplemented with 10% CCK-8 was added to each well. Following approximately 2 h of incubation, absorbance was detected at 450 nm with a microplate reader.

3.14. Investigation of the Anti-inflammatory Ability of Drug-Loaded Polymer Microspheres In Vitro

In this experiment, the previously described third-generation RAW264.7 cells were seeded in a 48-well plate at approximately 4 × 10⁴ cells/well, with 250 μL of complete DMEM per well, and cultured overnight in a cell incubator. After 24 h, the old medium was aspirated from each well, and 250 μL of complete DMEM containing 1 μg/mL LPS was added to stimulate the cells for 24 h. Then, different drugs were added according to the drug grouping and incubated for another 24 h. The supernatant was collected and centrifuged at 1000 rpm for 3 min to remove cell debris. The secretion of relevant inflammatory factors in the culture supernatant was then measured using different ELISA kits (TNF-α and IL-6) according to the manufacturer’s instructions.

The groups were as follows: the experimental group contained medium, cells, and drugs with LPS stimulation, including 12 μg/mL DSP, 120 μg/mL COLI, 0.6 mg/mL MPs PLGA Blank, MPs PLGA DSP, MPs PLGA COLI, MPs PLGA DSP@COLI, and 3 mg/mL medical chitosan; the control group contained medium and cells without drugs but with LPS stimulation; the blank group contained medium and cells without drugs and without LPS stimulation.

3.15. Analysis of the Regulatory Effect of Drug-Loaded Polymer Microspheres on Macrophage Polarization

RAW264.7 cells were cultured to the third generation, followed by cell counting. Cells were seeded at 1 × 10⁶ cells/well in six-well plates with 2 mL/well of complete DMEM. After 24 h of adhesion, the cells were stimulated with medium containing 1 μg/mL LPS for 24 h. Different drugs were then added to the cells and coincubated for another 24 h. After incubation, the cells were collected into corresponding 15 mL centrifuge tubes and centrifuged at 1200 rpm for 5 min to remove the supernatant. Each tube was resuspended in 1 mL PBS (live/dead staining can be performed simultaneously). The samples included 120 μg/mL COLI, 12 μg/mL DSP, 0.6 mg/mL MPs PLGA DSP@COLI, MPs PLGA COLI, MPs PLGA DSP, MPs PLGA Blank, and 3 mg/mL medical chitosan, with three replicates per sample.

First, the live/dead dye was diluted to 1 μL/1 mL PBS, and 1 mL was added to each sample tube, mixed well, and incubated on ice in the dark for 30 min, followed by centrifugation at 1200 rpm for 5 min and removal of the supernatant. The Fc block antibody was diluted to 1 μL/100 μL FACS buffer (PBS with 1% FBS), and 100 μL was added per tube. Fc block was used to block for 5 min on ice in the dark. The CD86 antibody was diluted to 1 μL/500 μL FACS buffer and 500 μL was added per tube, then incubated on ice in the dark for 30 min. After incubation, 500 μL of 4% PFA was added per tube to fix the cells, and the cells were fixed on ice in the dark for 40 min, followed by centrifugation at 1200 rpm for 5 min to remove the supernatant. 100 μL of cell permeabilization reagent was added per tube and incubated on ice for 30 min. The intracellular antibody CD206 was diluted to 1 μL/500 μL FACS buffer and 500 μL was added per tube and incubated on ice in the dark for 30 min. After incubation, 500 μL of 4% PFA was added per tube to fix the cells again, incubated on ice in the dark for 40 min, followed by centrifugation at 1200 rpm for 5 min to remove the supernatant. Cells were resuspended in 400 μL of FACS buffer per tube and passed through a 400-mesh filter for analysis.

3.16. Effect of Drug-Loaded Polymer Microspheres on the Secretion of Cartilage Matrix Components

Third-passage chondrocytes were seeded at 10,000 cells/well in a 96-well plate, with 100 μL of complete DMEM per well. After culturing in a cell incubator for 24 h, drugs were added. The groups were set as follows: a blank group containing only complete medium without cells and drugs, a control group containing complete medium and cells but no drugs, and an experimental group containing complete medium, cells, and drugs. The samples included 120 μg/mL COLI, 12 μg/mL DSP, 0.6 mg/mL MPs PLGA DSP@COLI, MPs PLGA COLI, MPs PLGA DSP, MPs PLGA Blank, and 3 mg/mL medical chitosan, each replicated in 6 wells. The next day, 100 μL of complete DMEM containing different concentrations of different drugs was added to each well and incubated in the incubator for 24 and 48 h. At 24 and 48 h, the culture medium was collected and centrifuged according to the kit instructions to obtain the supernatant for detecting the secretion of cartilage matrix components, glycosaminoglycan (GAG) and type II collagen (COLII). Finally, the absorbance was measured at 450 nm using a microplate reader, with the blank well as a reference.

3.17. Construction of a Rat Knee Osteoarthritis Model Using the ACLT Method

After purchasing SD rats and allowing them to acclimate to the animal facility for 1 week, the experimental group rats were anesthetized using a small animal anesthesia machine with isoflurane. Once the rats were anesthetized and fixed, the hair on the right hind limb was shaved. The skin over the joint was incised, scrubbed with iodine, and a small longitudinal incision was made beside the patellar ligament at the knee joint using a scalpel. The rat’s leg was bent to expose the anterior cruciate ligament. The knee joint was exposed, and the anterior cruciate ligament was cut with microsurgical scissors. A drawer test was performed to confirm anterior cruciate ligament rupture, ensuring that the cartilage surface was not damaged during surgery. Afterward, the joint was thoroughly rinsed with sterile saline, and the joint capsule and skin were sutured with absorbable suture material, with the joint cavity being closed in layers. Iodine was applied to the surgical site for disinfection; for 3 days postoperatively, iodine was applied daily around the wound to prevent infection.

In the sham surgery group, the joints were opened in the same manner, but the ligaments were not cut. The patella was repositioned, the joint cavity was sutured, and iodine was applied to disinfect the surgical site. No immobilization measures were taken for the rats postoperatively, and iodine was applied daily to the surgical site for three consecutive days.

After model construction in all groups, the rats were observed for one month to allow the development of a mid-stage rat KOA model. Following this period, drug treatment was administered to the rats, with health monitoring conducted during the waiting period.

3.18. Methods of Drug Administration in Animal Experiments

After the model is established, the rats’ dietary conditions and health status are observed daily; one month after model establishment, the corresponding drugs are injected into the joint cavity of each group of animals for treatment. It is planned that each group will receive a joint cavity injection of the drug once, with a treatment course of one month. Among them, the dosage of DSP is 0.5 mg/kg. Based on the body weight of each rat being 200 g, the dosage of DSP is 0.1 mg per rat.

The administration is as follows: sham surgery group: no drug treatment; ACLT surgery PBS group: 100 μL PBS injected into the joint cavity for treatment; ACLT surgery COLI group: 1 mg/100 μL COLI injected into the joint cavity; ACLT surgery DSP group: 0.1 mg/100 μL injected into the joint cavity; ACLT surgery MPs PLGA DSP@COLI group: according to the drug loading, the loading of dexamethasone sodium phosphate in the microspheres is about 2%. With a dosage of 0.1 mg per rat, 5 mg of microspheres is required. Therefore, 5 mg/100 μL MPs PLGA DSP@COLI is injected into the joint cavity. The administered doses of MPs PLGA DSP and MPs PLGA COL I were calculated in the same manner as MPs PLGA DSP@COL I. ACLT surgery MPs PLGA Blank group: 5 mg/100 μL MPs PLGA Blank injected into the joint cavity; ACLT surgery medical chitosan: the instructions indicate that the maximum dose per repeated use is 40 mg. When converted to a dose per unit body weight, the equivalent dose for rats is 6.3 times that of humans, so the equivalent dose for rats is 40 mg/60 kg × 6.3 = 4.2 mg/kg, and 4.2 mg/kg × 200 g = 0.84 mg. Therefore, 0.84 mg/100 μL medical chitosan is injected into the joint cavity for administration.

3.19. Monitoring of Rat Body Weight and Condition During the Treatment Process

During the treatment process, it is necessary to weigh the rats every 3 days to assess the in vivo safety of the drug.

3.20. Detection of Inflammatory Factors in Joint Synovial Lavage Fluid and Serum

Serum and joint synovial lavage fluid were collected to detect changes in relevant cytokine levels, such as IL-10, IL-6, IL-1β, TNF-α, etc; Blood (serum) extraction method: isoflurane was administered to partially anesthetize the rats, then blood was collected from the eyeball using an orbital capillary tube. The blood was allowed to stand at room temperature for 2 h, then centrifuged at 4 °C, 3000 rpm for 20 min. The upper pale yellow liquid was collected and stored in a −80 °C freezer until testing, avoiding repeated freeze–thaw cycles; Joint synovial lavage fluid extraction method: Sequentially the knee ligaments and synovium were cut open to expose the knee joint cavity; a pipet was used to draw 10 μL of PBS and inject it into the knee joint cavity, pipeted up and down 2–3 times, then recovered into a new Eppendorf tube. Another 10 μL of PBS was withdrawn and the operation was repeated, continuing until the required amount for the experiment was collected (typically 100 μL). Finally, the Eppendorf tubes were labeled and stored them in a −80 °C freezer until testing. Subsequent detection of inflammatory factors TNF-α and IL-6 will follow the manufacturer’s kit instructions.

3.21. Safranin O-Fast Green Staining and OARSI Cartilage Scoring

Safranin O is a cationic dye that belongs to the category of basic dyes, which bind to multiple anions. In cartilage tissue, the extracellular matrix secreted by chondrocytes is rich in proteoglycans, which contain a large number of anionic groups such as chondroitin sulfate and keratan sulfate. The cations in Safranin O bind electrostatically to these anionic groups, causing the proteoglycans in cartilage tissue to stain red. Therefore, the intensity of Safranin O staining can reflect the content and distribution of proteoglycans in the cartilage matrix. When cartilage is damaged, the content or distribution of proteoglycans changes, and the staining effect of Safranin O will correspondingly change, such as becoming faint or not staining. Fast Green mainly binds to acidic components such as collagen fibers in the subchondral bone. Structures like collagen fibers have certain acidic properties, and they bind chemically with the acidic Fast Green dye, staining collagen fibers and other structures green or blue, which helps to clearly display structures such as subchondral bone. Through combined Safranin O and Fast Green staining, cartilage tissue (red) and subchondral bone or other tissues (green or blue) can be clearly distinguished, facilitating the observation and analysis of tissue morphology, pathological changes, and other characteristics.

In this study, to observe the pathological condition of the knee joints of rats in each group, we completely decalcified and stained the knee joints of rats from each group with Safranin O-Fast Green. The procedure was used to observe cartilage defects in the knee joints of SD rats, assess cartilage thickness, and determine whether matrix staining was normal in order to evaluate the effects of drug treatments in each group. Subsequently, OARSI scoring was performed based on the collected images to assess joint cartilage damage.

The specific staining procedure is as follows: first, paraffin sections were sequentially placed in xylene I for deparaffinization for 20 min, followed by xylene II for another 20 min. Next, the sections were dehydrated in absolute ethanol I and II for 5 min each, then treated in 75% ethanol for 5 min, and finally rinsed with distilled water. After deparaffinization and water washing, the sections were stained in Fast Green solution for 1–5 min and thoroughly rinsed with water. Then, they were rapidly differentiated with hydrochloric acid alcohol for several seconds and were gently rinsed with water. For Safranin O staining, the sections were incubated in Safranin O solution for 15–30 s, followed by graded dehydration through two baths of absolute ethanol. After dehydration, the sections were cleared sequentially with n-butyl alcohol and xylene, air-dried briefly, and mounted with a neutral resin. Finally, the sections were observed under an optical microscope, and image acquisition and analysis were completed.

3.22. H&E Staining and Synovitis Scoring

H&E-stained sections of the heart, liver, spleen, lungs, and kidneys from each group of SD rats were prepared to evaluate whether intra-articular injection caused fewer side effects than systemic administration and whether drug-loaded polymer microspheres had good in vivo biocompatibility, low toxicity, and did not damage organs. Simultaneously, to observe pathological changes in the joint regions of each group of SD rats, the entire knee joints were collected, decalcified, and subjected to H&E staining to assess the degree of synovitis. The detailed staining procedure is as follows: first, paraffin sections were sequentially deparaffinized in xylene I and xylene II for 20 min each, followed by graded dehydration in absolute ethanol I, absolute ethanol II, and 75% ethanol for 5 min each, then rinsed with water; next, sections were stained in hematoxylin for 3–5 min, washed with water, differentiated in 1% hydrochloric acid solution for a few seconds, rinsed with water, blued in 0.6%–0.7% ammonia solution, and briefly rinsed with running water; subsequently, sections were dehydrated with 85% and 95% ethanol and then stained in eosin for 5 min; after staining, sections were sequentially dehydrated in absolute ethanol I, II, and III for 5 min each, treated with n-butanol for 5 min, cleared in xylene I and II for 5 min each, air-dried slightly, and mounted with neutral resin. After staining, cell nuclei typically appear blue, and the cytoplasm appears red.

3.23. Immunohistochemical Staining of Type II Collagen, CD86, and CD206

Type II collagen is the main structural component of the extracellular matrix of articular cartilage and is a key molecule for maintaining normal cartilage structure and function. By detecting the expression level of type II collagen, the promoting effect of drug-loaded polymer microspheres on cartilage matrix synthesis metabolism and cartilage repair can be evaluated. CD86 is a classic surface marker of M1 macrophages, and its high expression indicates pro-inflammatory phenotypic activation, which can drive synovial inflammation and cartilage matrix degradation by secreting pro-inflammatory factors such as TNF-α, IL-6, and IL-1β. CD206 is a characteristic surface marker of M2 macrophages, and its upregulated expression indicates an anti-inflammatory phenotypic transformation, which can secrete anti-inflammatory factors such as IL-10 and TGF-β to promote tissue repair and inflammation resolution. Therefore, immunohistochemical staining of type II collagen, CD86, and CD206 can systematically evaluate the effects and mechanisms of drug-loaded polymer microsphere therapy for knee osteoarthritis in two dimensions: cartilage matrix metabolism and immune-inflammation regulation. This serves as a key histological indicator to verify the “drug-device synergy” treatment strategy.

At the end of the experiment, SD rats were overdosed with isoflurane to euthanize them. Completely decalcified knee joints were collected for immunohistochemical staining of type II collagen, CD86, and CD206. The staining steps were consistent with conventional immunohistochemical procedures. Finally, the stained sections were observed with a panoramic scanning microscope. Hematoxylin stained cell nuclei blue, and DAB-positive expression appeared brownish yellow. Based on widely adopted scoring protocols for knee joint histological staining, the evaluation incorporates two parameters: the percentage of positive cells and staining intensity. Using ImageJ software, regions expressing type II collagen, CD86 (M1 macrophage marker), and CD206 (M2 macrophage marker) were selected for analysis. The proportion of positive cells and staining intensity were quantified separately to calculate the immunohistochemical score. The final score was obtained by multiplying the positive cell percentage score by the staining intensity score, with a scoring range of 0–12.

3.24. Statistical Analysis

Statistical analysis of data was performed using GraphPad Prism 9.5 software, and flow cytometry data were analyzed using FlowJo 10 software. Values are presented as mean ± SD, and analysis was conducted using one-way ANOVA and Tukey’s post hoc test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

4. Conclusion

KOA is a common degenerative joint disease, accounting for approximately 85% of OA cases, with a very high prevalence worldwide. Currently, there is no definitive cure, and existing treatments are mostly symptomatic interventions, making it a significant public health challenge. In this study, MPs PLGA DSP@COLI microspheres were prepared using PLGA polymer as the carrier material. These microspheres have a relatively narrow particle size distribution, appear complete and regularly spherical, and exhibit good dispersibility. Prepared with PVA as a surfactant, they possess low negative charge. In follow-up research, fluorescent dye-labeled microparticles will be utilized to verify whether the negatively charged microparticles fabricated in this study can preferentially accumulate at cartilage injury sites with glycosaminoglycan deficiency based on existing research findings. In vitro release results indicate that MPs PLGA DSP@COLI has a relatively slow release rate, with a release period lasting more than 20 days. In in vitro pharmacological studies, MPs PLGA DSP@COLI could partially enhance chondrocyte viability, demonstrating good biocompatibility. Moreover, MPs PLGA DSP@COLI can inhibit LPS-stimulated RAW264.7 cells polarized to the M1 phenotype from secreting inflammatory factors such as IL-6 and TNF-α in vitro, thereby suppressing inflammation. By establishing an LPS-stimulated RAW264.7 inflammatory model to mimic the inflammatory microenvironment of knee osteoarthritis, our results suggest that drug-loaded polymer microspheres may trigger macrophage phenotypic alteration. The data indicates a potential shift in the M1/M2 balance, which could contribute to the alleviation of inflammation. In addition, collagen I-loaded polymer microspheres can partially enhance the secretion of cartilage matrix components by healthy chondrocytes, thereby improving the joint cavity microenvironment.

To further verify the anti-inflammatory efficacy, this study employed anterior cruciate ligament transection (ACLT) to establish a rat model of knee osteoarthritis. After intra-articular injection treatment, the results indicated that MPs PLGA DSP@COLI effectively inhibited the loss of cartilage matrix components and promoted the synthesis of type II collagen and glycosaminoglycans in the cartilage matrix. At the same time, it reduced synovitis scores and OARSI scores, decreased the infiltration of inflammatory cells into synovial tissues, and maintained the integrity of the cartilage structure.

In summary, MPs PLGA DSP@COLI integrates the anti-inflammatory activity of DSP with the biological functions of COLI. This system is capable of regulating the physicochemical microenvironment in the joint cavity and favors the adhesion and proliferation of chondrocytes. This achieves the synergistic effect of a “drug-device combination.” Its local sustained-release properties for DSP and COLI can inhibit joint inflammation, enhance the secretion of cartilage matrix components, promote cartilage repair, and subsequently accomplish precise linkage and complementary effects from anti-inflammation to repair. This improves the joint cavity microenvironment and delays disease progression, providing a new strategy for the treatment of KOA.

Nevertheless, this study still has certain limitations, and further systematic investigations, including more comprehensive materials characterization and in vivo functional verification, are required to fully elaborate the translational potential of this microsphere system.

The data supporting the findings of this study are included in the paper and its extended data. All other relevant data are available from the corresponding author upon reasonable request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.6c00389.

• Microsphere characterization (size, zeta potential); UV method specificity for the DSP assay; standard curves; primary mouse chondrocyte adhesion and growth images; CCK-8 assay results of free DSP and COLI cocultured with chondrocytes and RAW264.7 cells; flow cytometry gating strategy for M1/M2 macrophages; rat ACLT model construction; H&E staining histopathology of major organs (heart, liver, spleen, lung, kidney) (PDF)

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X.C. and L.Z. contributed equally to this study. M.L. and X.C. conceived and designed the study. X.C. conducted microsphere preparation, X.C., L.Z., H.C., and R.Z. conducted in vitro studies and animal studies. X.C., L.Z., K.H., and J.Z. performed flow cytometry studies. M.L., X.C., and K.H. analyzed the data and drew the figures. M.L. provided the resources for this study. X.C. wrote the draft and M.L. revised the manuscript. All authors reviewed and approved the final version.

The research was funded by Suzhou Ersheng Biopharmaceutical Co., Ltd., Suzhou, Jiangsu, People’s Republic of China, and Darwin Nuogao (Beijing) Biotechnology Co., Ltd., Beijing 100006, People’s Republic of China.

The authors declare no competing financial interest.