INTRODUCTION

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ChooseTop of pageABSTRACTINTRODUCTION <<RESULTSDISCUSSIONConclusionMETHODSSUPPLEMENTARY MATERIALArticular cartilage has a low chondrocyte density (1%–5% of the total volume)11. A. M. Bhosale and J. B. Richardson, “ Articular cartilage: Structure, injuries and review of management,” Br. Med. Bull. 87, 77–95 (2008). https://doi.org/10.1093/bmb/ldn025 and avascular nature,22. C. A. Poole, “ Articular cartilage chondrons: Form, function and failure,” J. Anat. 191(1), 1–13 (1997). https://doi.org/10.1046/j.1469-7580.1997.19110001.x and it lacks lymphatics;33. J. Shi, Q. Liang, M. Zuscik et al., “ Distribution and alteration of lymphatic vessels in knee joints of normal and osteoarthritic mice,” Arthritis Rheumatol. 66(3), 657–666 (2014). https://doi.org/10.1002/art.38278 therefore, it has poor regeneration capabilities. After injury and during aging, articular cartilage may be unable to repair itself. After damage due to injury or overuse, weight-bearing biomechanisms of articular cartilage become altered, leading to progressive worsening of the condition and inducing osteoarthritis (OA).44. F. Guilak, “ Biomechanical factors in osteoarthritis,” Best Pract. Res. Clin. Rheumatol. 25(6), 815–823 (2011). https://doi.org/10.1016/j.berh.2011.11.013 The pathophysiology of articular cartilage deterioration involves increase in its water content and alterations and eventual decreases in proteoglycan levels in the extracellular matrix (ECM).55. J. A. Buckwalter and H. J. Mankin, “ Articular cartilage: Tissue design and chondrocyte-matrix interactions,” Instr Course Lect. 47, 477–486 (1998).After the alteration of the cartilage texture, a joint begins degenerating; the underlying mechanism may involve inflammatory changes in the synovium followed by the development of synovial hyperplasia or hypervascularity, remodeling of the subchondral bone, the formation of lytic lesions with sclerotic edges, and the proliferation of osteophytes, gradually affecting the appearance of the joint with varus or valgus deformation.66. G. S. Man and G. Mologhianu, “ Osteoarthritis pathogenesis—A complex process that involves the entire joint,” J. Med. Life 7(1), 37–41 (2014). Surgical intervention is required when symptoms can no longer be controlled using medication and rehabilitation therapy at the advanced stage. In younger patients with degeneration in only a single compartment, a proximal tibial osteotomy can be performed for contrary correction, thus preventing painful loading.77. H. S. Kyung, “ High tibial osteotomy for medial knee osteoarthritis,” Knee Surg. Relat. Res. 28(4), 253–254 (2016). https://doi.org/10.5792/ksrr.16.253 In older patients with single-compartment degeneration, unicompartmental arthroplasty may be performed.88. E. C. Rodriguez-Merchan, “ Medial Unicompartmental Osteoarthritis (MUO) of the Knee: Unicompartmental Knee Replacement (UKR) or Total Knee Replacement (TKR),” Arch. Bone Jt. Surg. 2(3), 137–140 (2014). However, total knee arthroplasty is required for severe degeneration involving two or more compartments.99. M. E. Steinhaus, A. B. Christ, and M. B. Cross, “ Total knee arthroplasty for knee osteoarthritis: Support for a foregone conclusion?,” HSS J. 13(2), 207–210 (2017). https://doi.org/10.1007/s11420-017-9558-4 Tissue engineering for cartilage regeneration and repair and to avoid surgery has been a research focus.Chondrocytes remodel the ECM by synthesizing, maintaining, and degrading in response to signals from cytokines, inflammatory mediators, and matrix fragments.1010. Y. Gao, S. Liu, J. Huang et al., “ The ECM-cell interaction of cartilage extracellular matrix on chondrocytes,” Biomed. Res. Int. 2014, 648459. https://doi.org/10.1155/2014/648459 In a healthy condition, articular chondrocytes are typically quiescent and highly differentiated; they are phenotypically stable and normally synthesize and maintain resilient ECM constituents principally composed of type II collagen (COL II) and aggrecan. Phenotypically unstable chondrocytes undergo dedifferentiation, resulting in the production of a substantially different protein, which integrates into the ECM, leading to inferior mechanical properties.1111. P. Singh, K. B. Marcu, M. B. Goldring, and M. Otero, “ Phenotypic instability of chondrocytes in osteoarthritis: On a path to hypertrophy,” Ann. N. Y. Acad. Sci. 1442(1), 17–34 (2019). https://doi.org/10.1111/nyas.13930 Numerous changes occur in dedifferentiated chondrocytes, including a considerable distortion in cell morphology, decreased expression of chondrogenic transcription factor SOX9, and suppressed production of the cartilage-specific matrix proteins, such as COL II and aggrecan. Cell metabolism alteration considerably increases the synthesis of COL I and type X collagen (COL X). In chondrocytes, phenotypic instability or morphologic and volume changes, which occur before considerable cartilage degeneration, might be a main feature of OA development.11,1211. P. Singh, K. B. Marcu, M. B. Goldring, and M. Otero, “ Phenotypic instability of chondrocytes in osteoarthritis: On a path to hypertrophy,” Ann. N. Y. Acad. Sci. 1442(1), 17–34 (2019). https://doi.org/10.1111/nyas.1393012. A. C. Hall, “ The role of chondrocyte morphology and volume in controlling phenotype-implications for osteoarthritis, cartilage repair, and cartilage engineering,” Curr. Rheumatol. Rep. 21(8), 38 (2019). https://doi.org/10.1007/s11926-019-0837-6Autologous chondrocyte implantation (ACI) involves the implantation of autologous chondrocytes cultured in vitro over the cartilage defect. In 1994, Brittberg et al. described this technique as one of the first tissue engineering techniques for articular cartilage regeneration.1313. M. Brittberg, A. Lindahl, A. Nilsson, C. Ohlsson, O. Isaksson, and L. Peterson, “ Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation,” N. Engl. J. Med. 331(14), 889–895 (1994). https://doi.org/10.1056/NEJM199410063311401 Currently, ACI is performed in two stages. The first step is to take a cartilage biopsy (weight 200–300 mg) from an area of the affected joint that is not in contact with weight-bearing activity. In the laboratory, enzymatic digestion is used to isolate chondrocytes from cartilage tissue. Then, monolayer cultures are used to expand them. Despite promising results,1414. A. G. McNickle, D. R. L'Heureux, A. B. Yanke, and B. J. Cole, “ Outcomes of autologous chondrocyte implantation in a diverse patient population,” Am. J. Sports Med. 37(7), 1344–1350 (2009). https://doi.org/10.1177/0363546509332258 ACI has limitations due to the limited availability of cells and the possibility of dedifferentiation during the expansion of chondrocytes in vitro. Moreover, implanting and adhering to definite lesions is the most challenging aspect of this process. It is anticipated that an alternative treatment that does not require surgical placement and fixation of the defect will be the way of the future. Instead, the cells' own healing ability will be stimulated to achieve therapeutic results by intra-articular injection of agents. Nanoparticles (NPs), defined in size range of 1 ∼ 100 nm, have high loading capacity and specific targeting to cells due to their size effect and intracellular uptake. Nanoparticles used for drug delivery are sub micrometer colloidal particles prepared from biocompatible and biodegradable materials. HA, a natural glycosaminoglycan, is biodegradable, biocompatible, and hydrophilic. HA is also a component of the extracellular matrix in cartilage tissue. Recently, HA is used in various drug delivery methods/materials to encapsulate active drugs/compounds or genes. We have successfully fabricated nano-sized HA particles with drugs in an aqueous-phase environment with anti-tumor activities using a high-voltage electrostatic field system under experimentally controllable parameters.1515. K. Y. Hsiao, Y. J. Wu, Z. N. Liu, C. W. Chuang, H. H. Huang, and S. M. Kuo, “ Anticancer effects of sinulariolide-conjugated hyaluronan nanoparticles on lung adenocarcinoma cells,” Molecules 21(3), 297 (2016). https://doi.org/10.3390/molecules21030297 In this study, the farnesol was aggregated with HA nanoparticles to prepare Farn/HA nanoparticles using this system, and the prevention and reparative effects of pure farnesol and Farn/HA nanoparticles on surgical-induced OA were investigated.Farnesol is an organic 15-carbon sesquiterpene compound produced by Candida albicans; it exhibits antioxidant,1616. R. Khan and S. Sultana, “ Farnesol attenuates 1,2-dimethylhydrazine induced oxidative stress, inflammation and apoptotic responses in the colon of Wistar rats,” Chem.-Biol. Interact. 192(3), 193–200 (2011). https://doi.org/10.1016/j.cbi.2011.03.009 anti-inflammatory,1717. Y. Y. Jung, S. T. Hwang, G. Sethi, L. Fan, F. Arfuso, and K. S. Ahn, “ Potential anti-inflammatory and anti-cancer properties of farnesol,” Molecules 23(11), 2827 (2018). https://doi.org/10.3390/molecules23112827 antimicrobial,1818. N. Cerca, F. Gomes, J. C. Bento et al., “ Farnesol induces cell detachment from established S. epidermidis biofilms,” J Antibiot. 66(5), 255–258 (2013). https://doi.org/10.1038/ja.2013.11 and tumor-related apoptosis-inducing properties.1717. Y. Y. Jung, S. T. Hwang, G. Sethi, L. Fan, F. Arfuso, and K. S. Ahn, “ Potential anti-inflammatory and anti-cancer properties of farnesol,” Molecules 23(11), 2827 (2018). https://doi.org/10.3390/molecules23112827 Ku et al. demonstrated that farnesol downregulates essential inflammatory cytokines, such as interleukin (IL) 1β, IL-6, and tumor necrosis factor alpha (TNF-α), in vivo.1919. C. M. Ku and J. Y. Lin, “ Farnesol, a sesquiterpene alcohol in herbal plants, exerts anti-inflammatory and antiallergic effects on ovalbumin-sensitized and -challenged asthmatic mice,” Evidence Based Complementary Alternat. Med. 2015, 387357. https://doi.org/10.1155/2015/387357 Farnesol can modulate connective tissue and ECM synthesis, which is required for wound healing2020. Y. C. Wu, G. X. Wu, H. H. Huang, and S. M. Kuo, “ Liposome-encapsulated farnesol accelerated tissue repair in third-degree burns on a rat model,” Burns 45(5), 1139–1151 (2019). https://doi.org/10.1016/j.burns.2019.01.010 and rotator cuff repair.2121. Y. H. Lin, S. I. Lee, F. H. Lin, G. X. Wu, C. S. Wu, and S. M. Kuo, “ Enhancement of rotator cuff healing with farnesol-impregnated gellan gum/hyaluronic acid hydrogel membranes in a rabbit model,” Pharmaceutics 13(7), 944 (2021). https://doi.org/10.3390/pharmaceutics13070944 Cartilage reconstruction is challenging because cartilage has poor intrinsic defect-repairing abilities. Studies have identified that in chondrocytes, phenotypic instability occurs with changes in the cell volume and morphology before considerable cartilage degeneration and loss, which might be essential for indicating the early stages of cartilage loss.1212. A. C. Hall, “ The role of chondrocyte morphology and volume in controlling phenotype-implications for osteoarthritis, cartilage repair, and cartilage engineering,” Curr. Rheumatol. Rep. 21(8), 38 (2019). https://doi.org/10.1007/s11926-019-0837-6 Wu et al. investigated the effect of farnesol on IL-1β-induced dedifferentiated chondrocytes. The authors suggested that farnesol restores the phenotype of the chondrocytes and salvages their ECM COL II and glycosaminoglycan (GAG) production capacity in vitro.2222. G. X. Wu, C. Y. Chen, C. S. Wu, L. C. Hwang, S. W. Yang, and S. M. Kuo, “ Restoration of the phenotype of dedifferentiated rabbit chondrocytes by sesquiterpene farnesol,” Pharmaceutics 14(1), 186 (2022). https://doi.org/10.3390/pharmaceutics14010186 Therefore, this study investigated whether intraarticularly administered farnesol could prevent further cartilage degeneration in a rabbit model with anticipated cartilage degeneration, and further investigated if they have a therapeutic effect.

RESULTS

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ChooseTop of pageABSTRACTINTRODUCTIONRESULTS <<DISCUSSIONConclusionMETHODSSUPPLEMENTARY MATERIALTransmission electron microscopic (TEM) images revealed that the HA nanoparticles and Farn/HA were successfully fabricated under high electrostatic field strength at certain settings [Figs. 1(a) and 1(b)]. The HA nanoparticles, produced first in the preparation system, had a relatively homogeneous diameter of approximately 5 nm. As the treatment progressed, Farn/HA gradually formed because of hydrophobic and hydrophilic interactions between the HA nanoparticles and farnesol molecules in the electrostatic field environment, yielding HA nanoparticle-farnesol aggregates (i.e., Farn/HA).2222. G. X. Wu, C. Y. Chen, C. S. Wu, L. C. Hwang, S. W. Yang, and S. M. Kuo, “ Restoration of the phenotype of dedifferentiated rabbit chondrocytes by sesquiterpene farnesol,” Pharmaceutics 14(1), 186 (2022). https://doi.org/10.3390/pharmaceutics14010186 At the treatment temperature (20–25 °C), which enabled the mobility of HA nanoparticles and farnesol molecules, the structure of the formed Farn/HA was nonuniform; initially (i.e., in the short-term), it was relatively loose, and as the treatment progressed, it became denser. The experiments on and mechanisms underlying the preparation of Farn/HA are not covered in this study.The Farn/HA had a mean diameter of approximately 110.5 ± 27 nm. The EE of farnesol in Farn/HA was approximately 91%, and approximately 92% of the farnesol was released after incubation for 12 h in a phosphate-buffered solution [Fig. 1(c)]. The in vitro effects of farnesol and Farn/HA on chondrocyte viability were examined using the MTT assay [Fig. 1(d)]. We detected nonsignificant reductions in chondrocyte viability after 24 h of incubation with 50) of farnesol in chondrocytes was approximately 0.5 mM. Chondrocytes demonstrated a considerable reduction in viability only after exposure to Farn/HA at high concentrations [4 and 8 mM; Fig. 1(e)]. We previously demonstrated that encapsulation of a drug in HA nanoparticles can both reduce its IC50 and enable its slow release, thus enhancing its therapeutic effects.1919. C. M. Ku and J. Y. Lin, “ Farnesol, a sesquiterpene alcohol in herbal plants, exerts anti-inflammatory and antiallergic effects on ovalbumin-sensitized and -challenged asthmatic mice,” Evidence Based Complementary Alternat. Med. 2015, 387357. https://doi.org/10.1155/2015/387357The half-maximum inhibitory concentration (IC50) of farnesol on the primary-cultured chondrocytes was 0.5 mM in this study; thus, we chose 0.4 mM farnesol, in order not to exert more toxicity and impair the function of primary-cultured chondrocytes. The farnesol concentration increased to 0.8 mM in the Farn/Ha nanoparticles (2-fold increase in 0.4 mM farnesol) for the slow-release behavior of farnesol from Farn/HA nanoparticles to assure enough amount of farnesol; importantly, this 0.8 Farn/HA nanoparticles concentration did not significantly exert toxicity to the chondrocytes [Figs. 1(d) and 1(e)]. According to the result, we chose these two concentrations to survey the repair and prevention effects of farnesol on the OA in the animal study.

Gross morphology

After 2 weeks, macroscopically, the treated cartilage appeared glossy white and transparent with mostly well-integrated surfaces [Fig. 2(a)]. Clear depressions and scrapes were noted on the untreated lateral condylar cartilage surfaces; by contrast, only a slight color change, without any depressions or wear lesions, was noted on the treated lateral condylar cartilage surfaces. After 6 weeks, the transparency and brightness of the treated cartilage increased. However, the untreated lateral condyle exhibited wear, which caused flattening. The lateral condylar cartilages of the rabbits treated with 0.4 mM farnesol or 0.8 mM Farn/HA exhibited relatively small areas of wear. Notably, the lateral condylar cartilage surfaces treated with 0.8 mM Farn/HA exhibited a rounder surface and thus more repair than did those treated with 0.4 mM farnesol. The cartilage treated with HA nanoparticles exhibited less wear than those in the untreated group but more pronounced wear than did those treated with farnesol. Therefore, on the basis of our observation of gross appearance, treatment with 0.4 mM farnesol, 0.8 mM Farn/HA, or HA nanoparticles leads to reparative effects in lateral condylar cartilage.

MMP-1 and MMP-13 in the synovium

MMPs, including MMP-1 and MMP-13, are inflammatory factors expressed in OA-affected joint tissues.2424. Y. Yoshihara, H. Nakamura, K. Obata et al., “ Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial fluids from patients with rheumatoid arthritis or osteoarthritis,” Ann. Rheum. Dis. 59(6), 455–461 (2000). https://doi.org/10.1136/ard.59.6.455 A study indicated that both chondrocytes and synovial cells in a rabbit anterior cruciate ligament (ACL) transection model expressed MMP-1; this expression was correlated with cartilage degradation.2525. H. Wu, J. Du, and Q. Zheng, “ Expression of MMP-1 in cartilage and synovium of experimentally induced rabbit ACLT traumatic osteoarthritis: Immunohistochemical study,” Rheumatol. Int. 29(1), 31–36 (2008). https://doi.org/10.1007/s00296-008-0636-2 Therefore, synovitis may be indicative of cartilage tissue breakdown or degradation.We harvested the synovium behind the patellar tendon after 2 and 6 weeks of treatment. Total proteins were isolated from the synovium, and MMP-1 and MMP-13 levels were analyzed through Western blotting. At two weeks following treatment, MMP-1 and MMP-13 levels were higher in the untreated cartilage than in the OA-affected cartilage treated with 0.4 mM farnesol, 0.8 mM Farn/HA, or HA nanoparticles and in the normal (control) cartilage [Fig. 2(b)]. MMP-1 and MMP-13 levels decreased with prolongation of the treatment period to 6 weeks—demonstrating the anti-inflammatory effects of farnesol and HA. Treatment with 0.8 mM Farn/HA led to the lowest MMP-1 and MMP-13 levels.

MMP-13 is an indicator of initial joint degeneration and degradation as well as OA progression. Among all the treatment groups, 0.8 mM Farn/HA led to the largest decrease in MMP-13 level, which may be attributable to the slow release of encapsulated farnesol, prolonging the effects compared with 0.4 mM farnesol alone. HA nanoparticles also exerted an anti-inflammatory effect on OA-affected cartilage.

Histological and histochemical evaluation of lateral femoral condylar cartilage

Figure 3(a) presents the H&E-stained sections of lateral condylar cartilage after 2 and 6 weeks of treatment. The normal (control) cartilage tissue exhibited a smooth surface, with the surrounding matrix and associated chondrocytes appropriately oriented in three well-defined zones but without any enlargement or distortion in the chondrons or any proliferative activity in the chondrocytes. In the untreated OA-affected cartilage, the surface had prominent clefts and depressions; its focal fibrillation extended into the midzone portion through the superficial zone, and matrix loss occurred in the midzone with cell death. Moreover, no tidemark was visible in the areas due to deep wear. The cartilage treated with 0.4 mM farnesol or 0.8 mM Farn/HA for 2 weeks exhibited a relatively smooth surface with an intact superficial zone and no apparent fibrillation. Compared with 0.8 mM Farn/HA, 0.4 mM farnesol led to significantly more hypertrophic and clustered chondrocytes, which occupied a large area of the cartilage layer. The cartilage treated with HA nanoparticles, although generally smooth in appearance, exhibited depressions on the surface, all concentrated and pronounced in one area. Also, the tidemark was not clearly visible, and the chondrocytes had shrunken and appeared disoriented, and their number had also decreased. After 6 weeks of treatment, the surface of the untreated OA-affected cartilage had pronounced multiple fibrillation through the superficial zone. By contrast, no fibrillation was noted on the surface of the cartilage treated with 0.4 mM farnesol. However, the chondrocytes in the deeper zone displayed significant hypertrophy, and the chondrocytes appeared sparse in the superficial zone. Compared with the other groups, OA-affected cartilage treated with 0.8 mM Farn/HA had a smoother surface, the entire layer of cartilage is filled with chondrocytes, which are denser than other treatments and have slight hypertrophy. In the cartilage treated with HA nanoparticles, the surface was generally smooth with some frayed areas. The tidemark was not clearly visible, and the subchondral bone had undergone degenerative changes. The chondrocytes appeared to be disorganized, unevenly distributed, and few in number.Safranin O staining is a cationic indicator dye, which can stain acidic proteoglycans within cartilage. After 2 weeks of treatment, Safranin O staining of the untreated cartilage revealed heterogeneity in the cartilage matrix and proteoglycan depletion in the lower two-thirds of the cartilage layer. One loading with fissure breaks in the untreated group indicated the cartilage matrix defect. The cartilage treated with HA nanoparticles exhibited similar mild heterogeneity and depletion of the cartilage matrix. By contrast, the cartilage treated with 0.4 mM farnesol or 0.8 mM Farn/HA did not exhibit obvious cartilage matrix loss [Fig. 3(b)]. After 6 weeks of treatment, similar cartilage matrix degradation was observed in the untreated and HA nanoparticle-treated cartilage; nevertheless, the cartilage treated with HA nanoparticles demonstrated less matrix degradation than did the untreated cartilage. By contrast, treatment with 0.4 mM farnesol or 0.8 mM Farn/HA enabled OA-affected cartilage repair and intact cartilage matrix retention.We next used Alcian blue staining to evaluate GAG content in the repaired tissue compared with the surrounding host cartilage. GAG content was relatively high in the cartilage treated with farnesol for 2 and 6 weeks [Fig. 3(c)]. On the surface of the untreated and HA nanoparticle-treated cartilage, most of the repaired tissue exhibited fibrocartilage formation and weak blue staining. After 2 and 6 weeks of treatment with 0.4 mM farnesol and 0.8 mM Farn/HA, blue staining of the cartilage surface became stronger, demonstrating the reparative effect of farnesol in the OA-affected cartilage. These histological and histochemical results indicate that 0.4 mM farnesol and 0.8 mM Farn/HA can restore the chondrocyte phenotype and repair OA-affected cartilage.As illustrated in Figs. 3(d) and 3(e), we determined Safranin O and Alcian blue staining intensity after 2 and 6 weeks of treatment semiquantitatively. The untreated cartilage demonstrated the lowest Safranin O staining intensity, revealing extensive degradation of the cartilage matrix. The degree of degeneration in the untreated group after 6 weeks of treatment was higher than that after 2 weeks of treatment. By contrast, the cartilage treated with 0.4 mM farnesol, 0.8 mM Farn/HA, and HA nanoparticles exhibited relatively high Safranin O staining intensity, indicating that the cartilage was repaired. Among all treatment groups, HA nanoparticles led to the least cartilage matrix reparative effect. The results for Alcian blue staining were consistent with those for Safranin O staining. Taken together, these findings demonstrate that 0.4 mM farnesol and 0.8 mM Farn/HA have considerable reparative effects in OA-affected cartilage.

IHC for MMP-13 in lateral femoral condylar cartilage

MMP-13 has a major role in the pathology of early OA because it can initiate the degradation of a wide range of downstream matrix and collagen components. Here, we detected the presence of MMP-13 in the harvested lateral femoral condylar cartilage through IHC. The untreated cartilage had an intense brown color dispersed throughout the cartilage layer, indicating the presence of MMP-13 throughout the cartilage [Fig. 4(a)]. All cartilage treated with farnesol in any form, particularly 0.8 mM Farn/HA, exhibited lighter or smaller brown areas. While the brown staining in the HA group was not as extensive as in the untreated group, significant staining was still observed in specific areas as compared to the farnesol group. These results indicate that the untreated cartilage had higher MMP-13 levels, indicating more OA-related degeneration and degradation. However, after 2 weeks of treatment with 0.4 mM farnesol or 0.8 mM Farn/HA, the levels of MMP-13 decreased, suggesting that farnesol effectively protects cartilage tissue from degradation.

IHC for COL II

We next used IHC to detect COL II production after treatment. A more intense brown color, indicating the presence of higher levels of COL II, observed in the cartilage treated with 0.4 mM farnesol or 0.8 mM Farn/HA than in the untreated and HA nanoparticle-treated cartilage [Fig. 4(b)]. The cartilage treated with 0.8 mM Farn/HA displayed stronger staining with more even distribution throughout than did the cartilage treated with 0.4 mM farnesol. By contrast, in the untreated and HA nanoparticle-treated cartilage, COL II production was sporadic. Therefore, farnesol improved COL II production in OA-affected cartilage, facilitating its repair. Among of them, treatment with 0.8 mM Farn/HA is more effective than 0.4 mM farnesol.As illustrated in Figs. 4(c) and 4(d), we semiquantitatively determined IHC MMP-13 and COL II staining intensity after 2 and 6 weeks of treatment. The control (normal) groups demonstrated the lowest MMP-13 and highest COL II staining intensity, revealing health cartilage. The untreated group exhibited mild inflammation after 6 weeks of treatment. The inflammation reaction was alleviated after 6 weeks of treatment with 0.4 mM farnesol, 0.8 mM Farn/HA exhibited relatively low MMP-13 staining intensity, indicating that farnesol exerted an anti-inflammatory activity on the cartilage. The results for COL II staining demonstrate that 0.4 mM farnesol and 0.8 mM Farn/HA have considerable capability to produce COL II in OA-affected cartilage after 6 weeks of treatment. However, a longer period of 6 weeks of treatment is needed to acquire the anti-inflammation response and COL II production to repair the OA-affected cartilage.

OA grading and staging

We used OARSI's system to evaluate OA progression on the basis of the histological features of our cartilage samples; it aided in understanding the severity of cartilage damage and the repair efficacy of the treatment with OA progression. The untreated cartilage, with a combined score of 5, demonstrated considerable deterioration after 2 weeks (Table I). After 6 weeks, the combined score increased to 9, indicating the progression of OA. By contrast, the cartilage treated with 0.4 mM farnesol or 0.8 mM Farn/HA exhibited much lower scores (1–3), with a more intact cartilage matrix and healthy cartilage surface after 2 and 6 weeks of treatment, indicating the reparative effect of farnesol. However, the HA nanoparticle-treated group exhibited cartilage degeneration and matrix loss, yielding a combined score of 5, after 6 weeks of treatment.

TABLE I. Analysis based on OARSI's system.

GradeStageScore(0–6.5)(0–4)(Grade × stage)Groups (2-w after treatment)Control000Untreated2.5250.4 mM Farnesol1.511.50.8 mM Farn/HA nanoparticles1.011HA nanoparticles2.012Groups (6-w after treatment)Control000Untreated3390.4 mM Farnesol1.5230.8 mM Farn/HA nanoparticles1.523HA nanoparticles2.525

DISCUSSION

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ChooseTop of pageABSTRACTINTRODUCTIONRESULTSDISCUSSION <<ConclusionMETHODSSUPPLEMENTARY MATERIALArticular cartilage is a highly complex tissue that can withstand tremendous force over a long period but cannot heal itself, even after a minor injury. Arthritis is caused by the absence of articular cartilage in a joint; joint health and function are dependent on cartilage viability. Chondrocytes are formed from mesenchymal stem cells and account for approximately 2% of the total volume of articular cartilage. They are highly specialized, metabolically active cells that play a unique role in ECM formation, maintenance, and repair.2626. J. W. Alford and B. J. Cole, “ Cartilage restoration, part 1: Basic science, historical perspective, patient evaluation, and treatment options,” Am. J. Sports Med. 33(2), 295–306 (2005). https://doi.org/10.1177/0363546504273510 Chondrocytes rarely form cell–cell contact that transmit signals and communicate directly with each other. However, they respond to various stimuli, including those from growth factors, mechanical loads, piezoelectric forces, and hydrostatic pressures.55. J. A. Buckwalter and H. J. Mankin, “ Articular cartilage: Tissue design and chondrocyte-matrix interactions,” Instr Course Lect. 47, 477–486 (1998). Their synthetic activity increases during the early stages as they attempt to repair the damage. However, after disruption of their pericellular matrix, chondrocytes become exposed to factors and components that influence their phenotype and behavior. After the cartilage matrix is weakened by proteolytic activity, even non-injurious loads may be perceived as injurious by the chondrocytes, resulting in a cycle of chronic, mechanically induced matrix breakdown.Articular cartilage has a structure similar to the cartilage of the growth plate. Patients with OA and several animal OA models have been observed to exhibit numerous pathological features typically associated with endochondral ossification of the growth plates, and these features have been associated with disease severity.27,2827. H. P. Gerber, T. H. Vu, A. M. Ryan, J. Kowalski, Z. Werb, and N. Ferrara, “ VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation,” Nat. Med. 5(6), 623–628 (1999). https://doi.org/10.1038/946728. D. A. Krawczak, J. J. Westendorf, C. S. Carlson, and J. L. Lewis, “ Influence of bone morphogenetic protein-2 on the extracellular matrix, material properties, and gene expression of long-term articular chondrocyte cultures: Loss of chondrocyte stability,” Tissue Eng., Part A 15(6), 1247–1255 (2009). https://doi.org/10.1089/ten.tea.2008.0249 In addition to chondrocyte proliferation and hypertrophy, superficial hyaline cartilage thinning, calcium phosphate crystal deposition, osteochondral remodeling, and cartilage calcification,2929. S. Suri and D. A. Walsh, “ Osteochondral alterations in osteoarthritis,” Bone 51(2), 204–211 (2012). https://doi.org/10.1016/j.bone.2011.10.010 increased expression of ossification-related proteins such as runt-related transcription factor 2, MMP-13, osteopontin, vascular endothelial growth factor, and osteocalcin is observed during skeletogenesis; these proteins are silenced in adult articular cartilage but re-expressed during OA. Inhibiting articular chondrocyte proliferation, hypertrophy, vascular erosion, subchondral bone remodeling, and other key events in the process of endochondral ossification may enable effective OA treatment.Transcription factor SOX9 is essential for embryonic chondrogenesis. Jacques et al. reported that SOX9 is required postnatally to prevent growth plate closure and preosteoarthritic cartilage deterioration.3030. A. Haseeb, R. Kc, M. Angelozzi et al., “ SOX9 keeps growth plates and articular cartilage healthy by inhibiting chondrocyte dedifferentiation/osteoblastic redifferentiation,” Proc. Natl. Acad. Sci. U. S. A. 118(8), e2019152118 (2021). https://doi.org/10.1073/pnas.2019152118 SOX9 upregulation may suppress a disintegrant and metalloproteinase with thrombospondin motifs (ADAMTS) in the early stage of OA in humans. Zhang et al. assessed whether SOX9 mediates ADAMTS dysregulation during cartilage degeneration3131. Q. Zhang, Q. Ji, X. Wang et al., “ SOX9 is a regulator of ADAMTSs-induced cartilage degeneration at the early stage of human osteoarthritis,” Osteoarthritis Cartilage 23(12), 2259–2268 (2015). https://doi.org/10.1016/j.joca.2015.06.014 and reported that SOX9 expression was negatively correlated with ADAMTS production in 22 randomly selected patients with OA. In addition, the inflammatory cytokines TNF-α and IL-1β repress SOX9 activity and activate ADAMTS expression, promoting OA in humans during its early stages.In load-bearing areas, SOX9 deficiency can result in chondrocyte-to-osteoblast conversion during the progenitor stage. In this phase of cell lineage transition, SOX9 may play a role in controlling the activation of transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) signaling pathways.3030. A. Haseeb, R. Kc, M. Angelozzi et al., “ SOX9 keeps growth plates and articular cartilage healthy by inhibiting chondrocyte dedifferentiation/osteoblastic redifferentiation,” Proc. Natl. Acad. Sci. U. S. A. 118(8), e2019152118 (2021). https://doi.org/10.1073/pnas.2019152118 The TGF-β family of polypeptide growth factors control the development and homeostasis of many tissues, including articular cartilage. The TGF-β family has more than 30 members, including TGF-β, activins, BMPs, and growth-differentiation factors.3232. N. G. M. Thielen, P. M. van der Kraan, and A. P. M. van Caam, “ TGFβ/BMP signaling pathway in cartilage homeostasis,” Cells 8(9), 969 (2019). https://doi.org/10.3390/cells8090969 Each TGF-β family member plays a specific role in cartilage biology and OA development, including inflammation, ECM production and degradation, and chondrocyte proliferation and hypertrophy.3232. N. G. M. Thielen, P. M. van der Kraan, and A. P. M. van Caam, “ TGFβ/BMP signaling pathway in cartilage homeostasis,” Cells 8(9), 969 (2019). https://doi.org/10.3390/cells8090969 Mutual regulation between TGF and SOX9 is an essential part of ECM protein production. TGF-β regulates the phosphorylation and stabilization of SOX9 in chondrocytes through p38 and Smad-dependent mechanisms.3333. G. Coricor and R. Serra, “ TGF-β regulates phosphorylation and stabilization of SOX9 protein in chondrocytes through p38 and Smad dependent mechanisms,” Sci Rep 6, 38616 (2016). https://doi.org/10.1038/srep38616 In addition, SOX9 is required for regulation of PAPSS2 mRNA expression by TGF-β; PAPSS2 is an enzyme essential for proteoglycan sulfation.3434. R. D. Chavez, G. Coricor, J. Perez, H. S. Seo, and R. Serra, “ SOX9 protein is stabilized by TGF-β and regulates PAPSS2 mRNA expression in chondrocytes,” Osteoarthritis Cartilage 25(2), 332–340 (2017). https://doi.org/10.1016/j.joca.2016.10.007Chondrocytes are generally isolated from articular cartilage through collagenase reaction and proliferated through in vitro monolayer culture. However, after multiple passages, the phenotype, including morphology and matrix, of chondrocytes alters; this is referred to as dedifferentiation.3535. L. J. Sandell and T. Aigner, “ Articular cartilage and changes in arthritis. An introduction: Cell biology of osteoarthritis,” Arthritis Res. 3(2), 107–113 (2001). https://doi.org/10.1186/ar148 In dedifferentiated chondrocytes, the phenotype gradually changes from polygonal to fibroblastic, and the secreted collagen type changes from COL II to type I collagen and COL X; their proteoglycan secretion and chondrocyte proliferation rates also decrease. Dedifferentiation also refers to the loss of function and characteristics. During the dedifferentiation process, the signal from the Indian hedgehog protein (IHH) induces chondrocyte hypertrophy and upregulates COL X and MMP expression in OA chondrocytes. Thus, increased IHH expression is associated with OA severity and chondrocyte hypertrophy markers expression; the markers include COL X, MMP-13, and chondrocyte size.3636. F. Wei, J. Zhou, X. Wei et al., “ Activation of Indian hedgehog promotes chondrocyte hypertrophy and upregulation of MMP-13 in human osteoarthritic cartilage,” Osteoarthritis Cartilage 20(7), 755–763 (2012). https://doi.org/10.1016/j.joca.2012.03.010 IL-1β can stimulate nuclear factor kB, which regulates nitric oxide (NO) and prostaglandin E2 (PGE2) production, thus activating MMP-1 and MMP-13 secretion and modulating inflammation, ECM synthesis, and apoptosis.3737. Z. Li, B. Liu, D. Zhao, B. Wang, Y. Liu, Y. Zhang, F. Tian, and B. Li, “ Protective effects of nebivolol against interleukin-1beta (IL-1beta)-induced type II collagen destruction mediated by matrix metalloproteinase-13 (MMP-13),” Cell Stress Chaperones 22, 767–774 (2017). https://doi.org/10.1007/s12192-017-0805-x In addition, IL-1β inhibits proteoglycan and collagen synthesis. We previously investigated the dedifferentiation of IL-1β-stimulated chondrocytes in response to farnesol treatment by measuring MMP-1, inducible NO synthase (iNOS), and IL-6. We concluded that levels of these proteins were higher in the IL-1β-stimulated chondrocytes than in the normal chondrocytes. After treatment with farnesol, these dedifferentiation- and inflammation-related proteins decreased the IL-1β-stimulated chondrocytes to levels similar to those in normal chondrocytes.2222. G. X. Wu, C. Y. Chen, C. S. Wu, L. C. Hwang, S. W. Yang, and S. M. Kuo, “ Restoration of the phenotype of dedifferentiated rabbit chondrocytes by sesquiterpene farnesol,” Pharmaceutics 14(1), 186 (2022). https://doi.org/10.3390/pharmaceutics14010186In the present study, the untreated OA-affected cartilage had higher MMP-1 and MMP-13 levels after 2 weeks; these levels increased further after 6 weeks, indicating OA progression [Fig. 2(b)]. By contrast, treatment with 0.4 mM farnesol or 0.8 mM Farn/HA reduced MMP-1 and MMP-13 levels. These levels continued to decrease with the progression of the treatment, indicating the anti-inflammatory effects of farnesol, which inhibited inflammation and reduced degeneration in the OA-affected cartilage. HA nanoparticles not only demonstrated considerable anti-inflammatory effects but also reduced MMP levels. However, the anti-inflammatory effect of HA was weaker than that of farnesol. In addition, 0.8 mM Farn/HA exerted more prolonged anti-inflammatory effects than did 0.4 mM farnesol,