TA has limited long-term efficacy and causes chondrocyte cell cycle arrest in human OA
The knee cartilage of OA patients with IA and had received 3-4 TA treatments for over one year before testing was used to observe the long-term efficacy of TA in human OA patients. Histomorphological staining objectively proved the condition of tissue repair because the articular cartilage layer is defective after OA. As the disease progresses, the defect is filled with new cartilage or fibrous connective tissue. The H&E and Masson staining (Fig. 1B) showed that the surface fibrillation area was larger, and the chondrocytes were abnormally distributed in the OA and TA than the normal group.
The Safranin O Fast Green Staining showed that the cartilage polysaccharide level reduced, and the cartilage matrix was severely degraded in the OA and TA groups than the normal group. However, there was no substantial difference in cartilage damage between the OA and TA groups. Quantitative analysis using the OARSI scoring revealed high scores, which indicates poor tissue repair. The results showed that the OA and TA groups had significantly higher scores than the normal group, but the scores between OA and TA groups were not substantially different (Fig. 1C).
The SOX9 and MMP13 IH staining determined the proliferation, differentiation, and degradation of chondrocytes. The results showed that SOX9 expression in the cartilage layer significantly decreased, but MMP13 increased in the OA and TA than the normal group. However, both SOX9 and MMP13 expressions were not substantially different between the OA and TA groups (Fig. 1D, E). Cell cycle-related factors P21, P16 (negative cell cycle regulator), and ki67 (cell proliferation and regeneration marker) were detected to observe chondrocyte cell cycle arrest after TA treatment. The results showed that P21 and P16 expression significantly increased in the cartilage layer, but Ki67 considerably decreased in the OA and TA than the normal group. There was no significant difference in the expression of P21, P16, and Ki67 between the OA and TA groups (Fig. 1F-H). Altogether, these results revealed that in the long-term, TA treatment causes cartilage tissue structure disorder and chondrocyte cell cycle arrest in OA patients.
Extraction and activity identification of CD90-positive stem cells from human synovium
The human synovium IF staining results showed that the CD90 expression significantly increased in the normal than the OA group, whereas the expression decreased in the TA group (Fig. 2B). This observation shows that TA treatment continually reduced the number of CD90-positive cells in the synovium after OA. Thus, we infer that CD90+ MSCs from the synovium are related to TA-induced chondrocyte cell cycle arrest after OA. We extracted CD45 (FITC) negative and CD90 (PC7) positive cells from healthy human synovium via fluorescence-activated cell sorting based on adherent growth on the plate and spindle morphology (Fig. 2C). Next, the proliferation ability of the obtained cells (normal CD90+ MSCs) was tested through different generations (P5, P10, and P15). The DAPI stain results showed that the cells increased from ~300 to ~2500 within 72 hours, but the cell proliferation ability was indifferent between generations (Fig. 2D). In addition, normal CD90+ MSCs expressed CD44 (93.2%) and CD106 (99.3%), representing common MSCs surface markers (Fig. 2E). Inducing normal CD90+ MSCs in osteogenic, adipogenic, and chondrogenic conditioned media verified their differentiation ability. The results showed that Alizarin Red, oil-red-O, or Toluidine blue positively stained the differentiated normal CD90+ MSCs (Fig. 2F). Altogether, these results infer that CD90+ MSCs from the healthy human synovium have good proliferation and differentiation ability.
Characterization of [email protected] and its uptake ability by cartilage
The TEM was used to observe the structure of [email protected], which consists of [email protected] NP, prepared for a DDS. The TEM results showed that [email protected] had a significantly clear core and shell structure, indicating the biofilm coating on the NP surface (Fig. 3B). The [email protected] size was approximately 154.3 ± 7.5 nm (Fig. 3C), slightly larger than NP (103.3 ± 6.7 nm). We tested the Zeta potential (ζ) of [email protected] Zeta potential is the potential of the shear surface, an important indicator of biofilm stability. The results showed that the [email protected] ζ potential (approximately −33.1 ± 1.6 mV) was similar to [email protected] (natural cell membrane, approximately -52.67 ± 1.5 mV). The [email protected] was stable within one week with a little size change (Fig. 3D). [email protected] has good biocompatibility because it is derived from CD90+ MSCs in the synovium. However, NP has poor biocompatibility and causes blood clotting. Thus, the CD90NP biocompatibility was tested against CD90MV for investigating the [email protected] stability in the blood to reduce the NP-caused blood clotting. Briefly, [email protected] and NP were incubated with FBS, followed by detection of [email protected] and NP coagulation. Coagulation was detected by testing the turbidity change over time. The coagulation results showed that NP had higher opacity (560nm) within 20 minutes compared with [email protected], while the [email protected] group remained stable within 120 hours (Fig. 3E). The good biocompatibility and stability of [email protected] may be related to [email protected] being wrapped on the surface of CD90NP, acting as a shield.
Triamcinolone acetonide-loaded [email protected] ([email protected]) was prepared as the following description. In brief, The [email protected] preparation was optimized by inputting TA in the PLGA core (10% w/w). The TA release from [email protected] was tested within 200 h to determine the TA capacity of [email protected] The TA release rate in [email protected] decreased compared with T-NP, which might be related to the [email protected] cover on the surface of [email protected] (Fig. 3F). The low TA release rate is beneficial for prolonging the TA efficacy for joint cavity treatment.
The protein maintained on the [email protected] surface (from [email protected]) is essential for its biological function, such as cartilage repair. Thus, the CD90+ MSCs, the [email protected], and the [email protected] protein composition were tested using SDS-PAGE. The results of SDS-PAGE showed that CD90+ MSCs, the membrane of CD90+ MSCs, [email protected], and [email protected] have similar protein compositions (both 80Da and 22kDa have similar bands). Thus, the important protein bands (in the red-dotted frame) such as CD90 (22 kDa) and CD44 (80 kDa) were found (Fig. 3G). CD90+ MSCs, the membrane of CD90+ MSCs, [email protected], and [email protected] were subjected to western blotting to identify further the two potential functional proteins, CD90 and CD44. CD90+ MSCs, the membrane of CD90+ MSCs, [email protected], and [email protected] maintained similar expressions of CD44 and CD90 (Fig. 3H).
We constructed a physical damage model of chondrocytes (scratch model) and used DID (red) to stain [email protected] and T-RNP and verify whether the damaged primary chondrocytes will take up [email protected] The damaged primary chondrocytes take up more [email protected] than the T-RNP group, probably because [email protected] inherits the properties of CD90+ MSCs extracellular vesicles (Fig. 3I). Moreover, several assays were conducted to test [email protected] cytotoxicity, including 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in vitro, hepatotoxicity, routine blood tests and HE staining of important organs. The results of MTT assays showed that IL-1β treatment decreased the viability of primary chondrocytes, and the cell activities of [email protected] and [email protected] were the highest at the concentration of 2 mg/mL (Fig. 4A, B). The serological ELISA test was used to observe the effect of [email protected] on liver function, kidney function and the circulatory system in rats. The results showed no abnormalities in alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), urea nitrogen (BUN), white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB) and platelet (PLT) levels (Fig. 4B-I). Similarly, the HE staining of the heart, liver, spleen, lung and kidney in all groups were normal (Fig. 4J).
[email protected] promote articular cartilage repair in OA model rabbit
The study employed micro-CT to observe the formation of osteophytes in the knee joint cavity and subchondral bone defects after OA. The micro-CT results showed that 6 months after treatment, the subchondral bone surface in the [email protected] group was smooth and similar to the normal group (Fig. 5B). However, the subchondral bone surface in the other groups was rough and displayed varying degrees of bone defect. Meanwhile, the subchondral bone in the OA and T-NP groups increasingly collapsed.
Moreover, severe damage induced numerous osteophytes, and numerous osteophytes (red cubes marked osteophyte, region of interest, ROI) appeared in each group of joint cavities after OA. Similarly, the volume of osteophytes in the [email protected] significantly decreased than in the other treatment groups (Fig. 5C). Likewise, the [email protected] group showed lower osteophyte scores than the other groups (Fig. 5D). Altogether, these findings indicate that at 6 months, T-[email protected] is beneficial for subchondral bone and osteophyte formation in the OA model rabbit.
The most damaged femoral trochlear cartilage was selected for pathological examination to observe the changes of cartilage structure 6 months after treating the OA model rabbit. The H&E staining and micro-CT results were similar. The chondrocytes in the repaired [email protected] tissue were neatly arranged, similar to the normal group. However, the repaired cartilage appeared as disorderly, fibrous, and loose tissue in the other treatment groups. The Safranin O fast green results showed that the [email protected] and normal groups had the best cartilage repair compared with the other treatment groups. Masson staining is specific for fibrous tissue. The Masson staining results showed that the other treatment groups had increased fibrous tissue, exhibiting a disordered tissue structure than the [email protected] group (Fig. 6A). Moreover, the OARSI scores (histomorphology scores) were consistent with the staining results. The OARSI scores of the [email protected] group were significantly lower than the other treatment groups but higher than the normal group (Fig. 6B).
[email protected] regulates joint cavity inflammation by promoting the macrophage polarization to the M2 phenotype in OA model rats
Regulating the inflammation of the joint cavity microenvironment after OA effectively promotes cartilage regeneration, and the synovium macrophages regulate the inflammation levels of the microenvironment. We injected [email protected] into the knee joint cavity in a rat OA model to verify whether the anti-inflammatory ability of [email protected] is similar to TA after OA. IF staining for the proinflammatory cytokines IL-6 and the anti-inflammatory cytokine IL-10 in the joint synovium reflects the inflammation level of the joint cavity microenvironment (Fig. 7A). Two weeks after treatment, the IL-10 expression in [email protected] significantly increased, similar to the TA group. However, the expression of IL-6 decreased compared with the other groups, a trend similar to the TA group. These results indicate that [email protected] and TA have similar anti-inflammatory abilities. Two weeks after OA, we performed IF staining for three macrophage polarization markers (CD68, CD206, and iNOS) to explore further the [email protected] mechanism of regulating inflammation. Both IL-6 and IL-10 induce the polarization of macrophages (Fig. 7B). CD68 and iNOS significantly decreased in the [email protected] and TA groups, while the expression of CD206 increased in the [email protected] and TA groups than the other treatment groups (Fig. 7C). These results indicated that after 2 weeks of treatment, [email protected] induces synovium macrophages to polarize to M2 phenotype in OA model rats.
[email protected] promotes cartilage regeneration and reduce cartilage apoptosis in OA model rats
Cartilage damage and apoptosis caused by OA are key yet difficult points of treatment. Therefore, methods to reduce cartilage apoptosis and promote cartilage regeneration need attention. In a rat OA model, we injected [email protected] into the knee joint cavity to detect cartilage regeneration and apoptosis after [email protected] treatment. Six months after OA, we observed cartilage apoptosis and cell cycle by IF and qPCR. The cell regeneration marker (EDU) was enhanced in the cartilage layer of [email protected] and [email protected] than the other treatment groups. There was little difference in EDU expression between [email protected] and [email protected] groups (Fig. 8A, B).
However, the cell apoptosis marker (TUNEL) expression decreased in the [email protected] than in the other treatment groups. There was a decrease in [email protected] compared with [email protected], probably due to the early anti-inflammatory ability of the enclosed TA (Fig. 8C). The qPCR detected key cell cycle factors to explore the impact of [email protected] on the chondrocyte cycle. The cell cycle promoter (Cyclin and CDK family) significantly increased in the [email protected] and [email protected] groups than the other groups. However, the cell cycle inhibitor (CDKN family) decreased in the [email protected] than the other treatment groups, similar to the [email protected] group (Fig. 8D). The ability of [email protected] to promote cartilage cell regeneration may be related to the [email protected] surface cover because synovial mesenchymal stem cells produce [email protected], which promotes cartilage proliferation.
Next, we used mRNA sequencing and bioinformatic analysis to explore the [email protected] molecular mechanism promoting cartilage proliferation and anti-inflammation in rats 6 months after OA. [email protected] substantially changed the expression of many genes in the cartilage of rats 6 months after OA (Fig. 9A-B). The KEGG pathway analysis showed that the top 100 high-abundant mRNAs enriched cellular processes, environmental information (Fig. 9C). The study focused on the genes for cell growth and death, considering the [email protected] biological test results. Indeed, the FOXO signaling pathway was enriched in the [email protected] cartilage after OA (Fig. S1). Both qPCR mRNA results showed that the IL-10, IGF1, cyclin B, PLK, and catalase expression increased, while the IRS, SGK, and FOXO1 decreased in the [email protected] than the OA group (Fig. 9D). The other KEGG enriched pathways include the JAK-STAT, insulin, PI3K-Akt, and FOXO signaling pathways. In summary, these enriched pathways possibly regulate cartilage regeneration (Fig. 9E).