Chondrogenic medium in combination with a c-Jun N-terminal kinase inhibitor mediates engineered cartilage regeneration by regulating matrix metabolism and cell proliferation

Abstract Cartilage tissue engineering is a promising strategy for repairing cartilage defects. However, achieving satisfactory cartilage regeneration in vitro and maintaining its stability in vivo remains a challenge. The key to achieving this goal is establishing an efficient cartilage regeneration culture system to retain sufficient active cells with physiological functions, generate abundant cartilage extracellular matrix (ECM) and maintain a low level of cartilage ECM degradation. The current chondrogenic medium (CM) can effectively promote cartilage ECM production; however, it has a negative effect on cell proliferation. Meanwhile, the specific c-Jun N-terminal kinase pathway inhibitor SP600125 promotes chondrocyte proliferation but inhibits ECM synthesis. Here, we aimed to construct a three-dimensional cartilage regeneration model using a polyglycolic acid/polylactic acid scaffold in combination with chondrocytes to investigate the effect of different culture modes with CM and SP600125 on in vitro cartilage regeneration and their long-term outcomes in vivo systematically. Our results demonstrate that the long-term combination of CM and SP600125 made up for each other and maximized their respective advantages to obtain optimal cartilage regeneration in vitro. Moreover, the long-term combination achieved stable cartilage regeneration after implantation in vivo with a relatively low initial cell-seeding concentration. Therefore, the long-term combination of CM and SP600125 enhanced in vitro and in vivo cartilage regeneration stability with fewer initial seeding cells and thus optimized the cartilage regeneration culture system.


Introduction
Cartilage defects are a common refractory disease [1,2].Fortunately, tissue engineering technology has made it possible to address this challenge [3,4].The core principle of cartilage tissue engineering is to establish a cell-scaffold construct and implant it into the defect area, where it can regenerate functional tissues similar to native tissue [5,6].The key to successful engineered cartilage regeneration and stability after implantation into the in vivo environment is to retain sufficient active cells with physiological functions, generate abundant cartilage extracellular matrix (ECM) deposition and maintain a low level of cartilage ECM degradation.To construct such an efficient engineered cartilage regeneration culture system, it is insufficient to rely solely on the construction of cell-scaffold composites; some effective exogenous factors must also be introduced.
Chondrocytes are the most commonly used cells for cartilage tissue engineering, but they are inevitably associated with limited cell supplies [7,8] and are prone to lose their phenotype (dedifferentiate) [9].Moreover, the current chondrogenic medium (CM) effectively promotes cartilage ECM production and leads to the redifferentiation of dedifferentiated chondrocytes; however, it negatively affects chondrocyte survival [10], leading to the regenerated cartilage failing to maintain a high content of active cells after implantation in vivo and eventually achieving poor cartilage regeneration.Strategies targeting the c-Jun N-terminal kinase (JNK) pathway remarkably reduce chondrocyte apoptosis in osteoarthritis (OA) [11][12][13].Our previous studies have confirmed that the specific JNK inhibitor SP600125 can promote chondrocyte proliferation at the cellular and tissue levels in vitro and that the combined application of CM enhanced the synthesis and deposition of cartilage ECM [10].This may be a feasible way to achieve satisfactory cartilage regeneration with a relatively low initial cell-seeding concentration and overcome the lack of active cells after implantation to improve cartilage regeneration quality in vitro and in vivo.
During multicellular organism development, cells proliferate before commencing functional differentiation [14].During the differentiation of primordial cells into specific differentiated cells, their proliferation ability is limited [15,16].As cells differentiate and acquire specific physiological functions, they may enter the G0 phase and then lose their ability to divide, stopping cell proliferation [17,18].Our previous study demonstrated that SP600125 significantly promoted chondrocyte proliferation while inhibiting ECM secretion and cartilage differentiation.The current chondrogenic culture system promotes cartilage ECM production but impairs chondrocyte survival [10].Since cell proliferation and differentiation belong to two different cell cycles and stages, whether phased application of different acting factors (i.e.first applying SP600125 to promote chondrocyte proliferation in the early stage and then replacing it with CM to promote chondrocyte differentiation and cartilage ECM synthesis) will achieve satisfactory cartilage regeneration is still unknown.Several issues must be clarified to determine the optimal culture mode of CM and SP600125 for cartilage regeneration.First, will phase application of CM and SP600125 enhance in vitro cartilage regeneration?Second, what is the outcome of regenerated cartilage treated with SP600125 in vitro after in vivo implantation?Third, can SP600125 enhance cartilage regeneration in vitro and in vivo, with fewer initial seeding cells?Therefore, we aimed to establish a three-dimensional cartilage regeneration model with polyglycolic acid/polylactic acid (PGA/PLA) scaffolds in combination with chondrocytes to investigate the effect of different culture modes with CM and SP600125 on in vivo cartilage regeneration systematically.

Group design
For the in vitro cartilage regeneration model, high (6.0Â 10 7 cells/ml) and low (3.0 Â 10 7 cells/ml) concentrations of cells were seeded onto the scaffolds.According to the period of application of 0.02 mg/ml SP600125 (a specific JNK pathway inhibitor, abbreviated as SP in grouping labels; AdooQ Bioscience, Irvine, CA) and CM (Dulbecco's modified eagle medium [DMEM] containing 10 ng/ml TGFb1 [R&D Systems, Minneapolis, MN], 40ng/ml dexamethasone [Sigma-Aldrich, St. Louis, MO], 100 ng/ml IGF-1 [R&D Systems] and other supplements; serumfree [7]), all samples were divided into six groups and cultured in vivo for 8 weeks.Chondrocytes from these six groups were obtained from the same rabbit.The overall group design of in vitro study is shown in Table 1.
To study the in vivo outcomes of in vitro cultured regenerated cartilage treated with SP600125, the optimal group of the in vitro study and simple CM groups with high and low cell-seeding concentrations were cultured in vitro for 8 weeks and then implanted into nude mice subcutaneously.The overall group design of the in vivo study is shown in Table 2.

Isolation and culture of auricular chondrocytes
Rabbit auricular chondrocytes were harvested as previously described [19].Chondrocytes from passage two were collected and used.The Ethical Committee of Shanghai Ninth People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine approved all animal study protocols.

Preparation of cell-scaffold constructs
PGA fibers (20 mg; National Tissue Engineering Center of China, Shanghai, China) were prepared to form cylinders by molds with a diameter of 9 mm and a thickness of 2 mm.Then, 1% PLA (Sigma, St. Louis, MO) solution was added dropwise to solidify the shape of the scaffolds [20].After sterilization with a 75% ethanol solution for 30 min, the scaffolds were washed three times with phosphate-buffered saline for the following experiments.Harvested chondrocytes with different concentrations of 6.0 Â 10 7 cells/ml and 3.0 Â 10 7 in a regular medium (RM; DMEM containing 10% fetal bovine serum) were seeded onto each scaffold, followed by a 4-h pre-incubation [21].Subsequently, different culture media were added according to group design (Tables 1 and 2).

Scanning electron microscopy (SEM)
The microstructure of the PGA/PLA scaffolds and ECM deposition on the surfaces of scaffolds after culturing in vitro for 12 h, 4 days and 7 days were examined using SEM (Philips XL-30, Amsterdam, Netherlands).After being fixed overnight in 2.5% glutaraldehyde at 4 C, all samples were dehydrated in a graded series of ethanol solutions and then examined using SEM [22].

Cell-seeding efficiency
Chondrocytes with a concentration of 3.0 Â 10 7 cells/ml were seeded onto PGA/PLA scaffolds and cultured in different culture systems for 24 h.The samples were transferred for subsequent experiments and the remaining cells in the culture dishes were collected and counted.The cell-seeding efficiencies of scaffolds in different culture systems were calculated based on the formula [23]: ðtotal cell number À remaining cell numberÞ=total cell number Â 100%

Cell proliferation evaluation
After 4 h, 1, 4 and 7 days of culture, the DNA content of samples in different culture systems was assessed using the DNA quantification assay (PicoGreen dsDNA assay; Invitrogen, Carlsbad, CA), as described previously [24], to evaluate the proliferation capacity of chondrocytes on scaffolds.

Cell viability evaluation
After 1, 4 and 7 days of culture, the viability of chondrocytes on scaffolds in different culture systems was assessed using the live and dead cell viability assay (Invitrogen, Carlsbad, CA), and the results were examined using a confocal microscope (Nikon, Japan) [25].

Subcutaneous implantation in nude mice
Male nude mice aged 6-8 weeks were used for the in vivo study.
After anesthetization with a small animal gas anesthetic and disinfection with 75% alcohol, their back skins were cut open, and the skin and muscle tissues were separated to the left, right and upper, respectively.Three groups of regenerated cartilage were placed under the skins of nude mice to ensure separation.The skin was intermittently sutured with 5-0 suture, and the wound was disinfected with 75% alcohol [26].After 8 weeks, the nude mice were sacrificed, and samples from the three groups were taken for subsequent evaluation (Supplementary Fig. S1).

Histological and immunohistochemical evaluations
Tissue samples were fixed in 4% paraformaldehyde for 48 h, embedded in paraffin and sectioned into 5-lm sections.The tissue sections were then stained with hematoxylin and eosin, safranin O and type II collagen (mouse anti-human type II collagen monoclonal antibody, 1:100; Santa Cruz Biotechnology, Dallas, TX), as previously described [27][28][29].

Biomechanical and biochemical evaluations
After in vitro and in vivo culture, all samples were harvested and weighed.The volume was measured using the water displacement method [30].The biomechanical properties of the samples in the different groups were measured using a biomechanical analyser (Instron-5542, Canton, MA), as previously described.Young's modulus was calculated based on the stress-strain curves [31].
After mechanical analysis, tissue samples were minced for quantification of total collagen, glycosaminoglycan (GAG) and DNA using the hydroxyproline assay, the dimethyl methylene blue assay method and the PicoGreen dsDNA assay, respectively, as described previously [32][33][34].

Quantitative real-time polymerase chain reaction analysis
Total RNA was extracted and reverse transcribed as previously described.The quantitative real-time polymerase chain reaction (qRT-PCR) was performed following the manufacturer's protocol (Thermo Fisher Scientific, Waltham, MA).Expression levels of chondrogenesis-related genes (ACAN, COLII A1 and Sox9) and catabolism-related genes (TNF-a, IL-1b and MMP13) were analyzed.Results were analyzed using the 2 -DD CT method and normalized to the endogenous reference gene b-actin [35,36].The primer sequences are listed in Supplementary Table S1.

Statistical analysis
Quantitative data were collected from at least three replicates and were presented as mean 6 standard deviation.Statistical significance was analyzed using a one-way analysis of variance followed by post hoc tests with the Student-Newman-Keuls method using IBM SPSS Statistics (v.25;IBM Corp., Armonk, NY).Statistical significance was set at P < 0.05.

Biocompatibility of cell-scaffold constructs
The PGA/PLA scaffold appeared as a porous cylindrical scaffold with a 9-mm diameter and 2-mm thickness (Fig. 1A).The SEM showed the ultrastructure of the interlaced PGA fiber and PLA coating (Fig. 1B).After seeding with 3.0 Â 10 7 cells/ml chondrocytes, the cell-scaffold constructs were cultured in different culture systems for biocompatibility evaluations (Fig. 1C).No significant difference was observed in the groups' cell-seeding efficiency and initial DNA content (Fig. 1D and E).With increasing culture time, the DNA content of all groups gradually increased.The groups treated with SP600125 showed an upward trend, suggesting more rapid cell proliferation.Meanwhile, the group treated with CM showed lower levels of DNA content, indicating a negative effect on cell proliferation.However, adding SP600125 weakened this adverse effect and achieved a higher DNA content in CM þ SP than in RM.These results confirmed that SP600125 promoted cell proliferation and reversed CM's inhibition of cell survival (Fig. 1E).
The SEM evaluated ECM deposition on the scaffolds' surface (Fig. 1F).After 12 h of cell-seeding, the chondrocytes still appeared round in shape and adhered to the PGA fibers.After 4 days, the chondrocytes secreted ECM, and ECM deposition in the CM was more abundant.SP600125 inhibited ECM secretion in RM þ SP, but adding CM significantly diminished this negative effect in CM þ SP.After 7 d, more ECM was observed in all groups, and the fiber interspaces were covered in CM and CM þ SP.SEM examination showed satisfactory biocompatibility of the scaffolds, and SP600125 reduced ECM synthesis, but adding CM reversed this.
Cell viability was evaluated using live/dead fluorescence staining (Fig. 2).The chondrocytes proliferated well with time in all groups, and few dead cells were observed, indicating the good biocompatibility of the scaffolds.More significant cell proliferation was observed in the RM þ SP and CM þ SP groups, indicating the cell proliferation-promoting effect of SP600125.

Gross and histological evaluation of in vitro cartilage regeneration
The shape of the regenerated cartilage in each group remained relatively complete, and the scaffold materials on the surface had been fully degraded and replaced with new cartilage-like tissues.Groups treated with RM (HRM, LRM and LRM þ SP-RM) had a light-yellow appearance and looser tissue texture.Groups treated with CM (LRM-CM, LRM þ SP-CM and LCM þ SP) formed a more mature cartilaginous tissue appearance.These results showed that the effects of different culture modes on the Regenerative Biomaterials, 2023, Vol. 10, rbad079 | 3 secretion and deposition of cartilage ECM led to general differences and that adding CM promoted cartilage regeneration (Fig. 3).
A histological examination was performed to study further the effects of each culture mode on the fine structure and composition of cartilage regeneration in vitro.After 8 weeks of culture in vitro, the scaffold materials of each group degraded completely.However, groups treated with CM formed more mature cartilaginous tissues, showing richer ECM deposition, more typical cartilage lacunae and stronger GAG and type II collagen staining.The tissue structures were disordered and loose in the RM group, indicating a relatively immature state of regenerated cartilage.
Since the initial cell-seeding concentration of HRM was higher than that of LRM, the histological results showed that HRM had more cells and more ECM secretion, probably because numerous active cells secreted cartilage-specific ECM.Compared to LRM, SP600125 was used to promote cell proliferation in the first 10 d in LRM þ SP-RM.Histological results showed that LRM þ SP-RM had more cells, but less ECM deposition than LRM.These results showed that the SP600125 inhibitory effect on cartilage ECM synthesis might persist after withdrawal.Moreover, chondrocytes with inhibited differentiation function still had function loss after returning to routine culture.Due to the lower cell content, LRM-CM formed a typical cartilage-like tissue, but the cartilage layer was thin.Only the sample's surface formed cartilaginous tissue, and the lower layer comprised scaffold materials.In the LRM þ SP-CM group, SP600125 was used to promote cell proliferation and increase active chondrocytes in the early stage, and CM was used to promote cell differentiation and cartilage ECM synthesis.Compared with LRM-CM, LRM þ SP-CM had a higher cell content, richer secretion of cartilage ECM and stronger GAG and type II collagen expressions.However, LCM þ SP had the most mature cartilaginous tissue formation and the strongest staining of GAG and type II collagen, accompanied by the highest active cells.
These results showed that the phased application of CM and SP600125 had an effect; however, the cartilage regeneration result was less mature than that of the long-term combined group, achieving optimal cartilage regeneration in vitro.This suggests that the concept that cell proliferation and differentiation belong to two different stages in our past cognition may be incomprehensive rather the stages are interrelated.

Quantitative determinations of in vitro cartilage regeneration
Quantitative determinations of samples in each group were performed to verify further the histological results.CM groups had higher wet weight, volume, biomechanical strength and total collagen and GAG contents.These results demonstrated that CM promoted cartilage ECM secretion and the differentiation and maturation of regenerated cartilage in vitro.The LCM þ SP group exhibited optimal cartilage regeneration in vitro (Fig. 4A-E).
There was no significant difference in wet weight and volume among the RM groups; however, the CM groups had significantly higher wet weight and volume than the RM groups.LRM þ SP-CM had higher wet weight and volume than LRM-CM; however, they were lower than LCM þ SP (Fig. 4A and B).There was a significant difference among RM groups in mechanical properties and related quantification of cartilage ECM components (total collagen and GAG quantification).HRM with the highest cell content showed the highest mechanical strength, total collagen and GAG contents.Owing to the continuation of SP600125 inhibitory effect on ECM synthesis, LRM þ SP-RM showed the lowest in these directions.CM groups showed higher levels of biochemical and biomechanical parameters.Consistent with the histological results, LCM þ SP with a long-term combination of CM and SP600125 resulted in optimal in vitro cartilage regeneration (Fig. 4C-E).
The results showed that groups treated with SP600125 had higher DNA content than other groups, consistent with the results of the JNK inhibitor promoting chondrocyte proliferation in a previous study.Similarly, due to CM's negative effect on cell survival, LRM-CM had the lowest DNA content.The JNK inhibitor effectively promoted cell proliferation during longterm treatment in LCM þ SP, leading to the highest DNA content.This also maintains long-term stability after implantation in vivo (Fig. 4F).
These results showed that LCM þ SP with a long-term combination of SP600125 and CM resulted in optimal cartilage regeneration in vitro.

Characteristic gene expression of in vitro cartilage regeneration
Chondrogenesis-related and catabolism-related genes were examined by qRT-PCR (Fig. 5).The expression levels of chondrogenesis-related genes (ACAN, COLII A1 and Sox9), were downregulated in groups treated with SP600125.However, adding CM weakened this adverse effect.In addition, TNF-a, IL-1b and Regenerative Biomaterials, 2023, Vol. 10, rbad079 | 5 MMP13 expressions were also downregulated, indicating reduced inflammatory response and ECM degradation.LCM þ SP had lower levels of chondrogenesis-related genes than LRM-CM due to the inhibition of ECM synthesis by SP600125.However, LCM þ SP also had the lowest cartilage catabolism-related genes (TNF-a, IL-1b and MMP13), whereas LRM-CM had the highest expression.LCM þ SP has the greatest potential for cartilage regeneration in vitro because good cartilage regeneration depends on synthesis and degradation balance.

Gross view and histology of in vitro and in vivo cartilage regeneration
This study's results showed that LRM þ SP-CM with the phased application of CM and SP600125 had better cartilage regeneration; however, it was inferior to LCM þ SP with a long-term combination.Nevertheless, the combined application of CM and SP600125 resulted in optimal in vitro cartilage regeneration.Therefore, LCM þ SP was selected as the experimental group, and groups cultured in simple CM with high and low cell-seeding concentrations were used as the control groups to evaluate their cartilage regeneration effects in vitro and their outcomes in vivo.
After 8 weeks of in vitro culture, smooth and ivory-white cartilaginous tissues were formed in all groups.Histological examination showed that mature cartilage lacunae formed in all groups.The expression of GAG and type II collagen, specific components of cartilage ECM, were positive.Regarding cell content, HCM and LCM þ SP had more nuclei stained blue-black than LCM.After 8 weeks of implantation in vivo, the cartilage in all groups appeared similar to natural cartilage, and the shapes were well maintained.Histological examination revealed that the cartilage lacuna structures of all three groups were more mature than those in vitro, and the expression of GAG and type II collagen was positive.Cartilage tissue formation was uniform and homogeneous in the LCM þ SP group, while only cartilage islands were distributed in the HCM and LCM groups.These results showed that cartilage formation matured over time after transplantation in vivo.LCM þ SP achieved more homogeneous and stable cartilage regeneration in vivo, possibly due to the retention of sufficient active cells and a lower degradation activity (Fig. 6).
Figure 3. Gross view and histology of in vitro regenerated cartilages with different culture modes.Tissue samples showed different appearances and tissue structures.The regenerated tissue in groups treated with CM obtained a more mature cartilage-like appearance and structure.The LRM þ SP-CM group with the phased application of SP600125 had an effect; however, the cartilage regeneration result was less mature than that of LCM þ SP, obtaining the optimal cartilage regeneration in vitro with more mature cartilage lacunae and stronger staining for cartilage-specific ECM molecules.H, high cell-seeding density (6.0Â10 7 cells/ml); L, low cell-seeding density (3.0Â10 7 cells/ml); SP, SP600125 (a specific JNK pathway inhibitor).

Quantitative determinations of in vitro and in vivo cartilage regeneration
Quantitative determinations further confirmed the histological results.In the in vitro culture stage, LCM þ SP had a lower wet weight, volume, mechanical strength, total collagen and GAG contents than HCM, and there was no significant difference compared to LCM, except for wet weight (Fig. 7A-E).However, the DNA content of the LCM þ SP group was the highest due to the promotion effect of SP600125 on chondrocyte proliferation (Fig. 7F).These results were consistent with those of a previous study.
After 8 weeks of implantation in vivo, the wet weight and volume of HCM and LCM decreased, whereas those of LCM þ SP were well maintained (Fig. 7A and B).These results showed that in the enzyme-active environment in vivo, numerous active cells were retained and further performed their biological functions due to the inhibition of cartilage degradation and promotion of chondrocyte proliferation by SP600125, resulting in the good maintenance of the shape and size of in vitro regenerated cartilage.The mechanical properties and ECM component contents were reversed after 8 weeks of in vivo implantation.LCM þ SP in vitro with the lowest mechanical strength and total collagen and GAG contents obtained the highest level after in vivo implantation (Fig. 7C-E).The DNA content increased in all groups after implantation, but the LCM þ SP group had the highest active cells, the key reason for the reversal in vivo (Fig. 7F).
These results showed that the cartilage regeneration of LCM þ SP was inferior to that of HCM and LCM in vitro; however, it was reversed after 8 weeks of implantation due to its numerous active cells and reduced degradation of cartilage ECM.

Characteristic gene expression of in vitro and in vivo cartilage regeneration
Chondrogenesis-related and catabolism-related genes were analyzed using qRT-PCR (Fig. 8).Chondrogenesis-related genes were upregulated after implantation in vivo in all three groups, indicating that the in vivo environment is conducive to further maturation of regenerated cartilage.Expression of chondrogenesis-related genes of LCM þ SP was relatively lower than those of HCM and LCM in vitro and in vivo.In contrast, the levels of LCM þ SP degradationrelated genes, including TNF-a, IL-1b and MMP13, remained the lowest.Importantly, in the complex in vivo environment, due to the influence of body fluids, degradation enzymes and other inflammatory factors, the degradation level of regenerated cartilage was upregulated, and a new balance between cartilage synthesis and degradation needs to be established.Thus, lower levels of cartilage degradation are important for in vivo cartilage regeneration.The lower expression of degradation-related genes was critical to Figure 4. Quantitative evaluations of in vitro regenerated cartilages with different culture modes.Groups with CM had higher wet weight (A), volume (B), Young's modulus (C), total collagen (D) and GAG contents (E) than the RM groups.The LCM þ SP group had the optimal cartilage regeneration result in vitro.The DNA content (F) revealed that the cell number of groups treated with SP600125 was significantly higher.Owing to the inhibition of cell survival by CM, the LRM-CM group obtained the lowest DNA content; due to the chondrocyte proliferation-promoting effect of SP600125, the LCM þ SP group obtained the highest DNA content.H, high cell-seeding density (6.0 Â 10 7 cells/ml); L, low cell-seeding density (3.0 Â 10 7 cells/ml); SP, SP600125 (a specific JNK pathway inhibitor).maintaining the stability of regenerated cartilage in vivo when the expression of chondrogenesis-related genes is not low.Therefore, the LCM þ SP combination exhibited the strongest cartilage regeneration potential in vivo.

Discussion
Cartilage regeneration engineering is gaining increased attention owing to the promising results in cartilage injury repair.Moreover, developing an efficient engineered cartilage regeneration culture system and maintaining the stability of the regenerated cartilage after in vivo implantation remains challenging.Here, we combined the currently used CM and SP60015, a specific JNK inhibitor, to construct a new in vitro-engineered cartilage culture system.Our results demonstrated that the long-term combination of CM and SP600125 enhanced cartilage regeneration in vitro and in vivo with fewer initial seeding cells by increasing the number of active cells, improving ECM synthesis and reducing ECM degradation.
Chondrocytes are the only cells that exist in cartilage tissues [37].Hence, satisfactory cartilage regeneration lies in the active cell quantities.However, strategies using chondrocytes as a cell source for cartilage tissue engineering are associated with limited cell supplies [7,38].Moreover, the CM currently used negatively affects cell survival, hindering the long-term stability of regenerated cartilage.Inhibiting the JNK pathway decreases chondrocyte loss in OA [13].Our study confirmed the significant effect of the specific JNK inhibitor SP600125 on promoting chondrocyte proliferation.This may promote satisfactory cartilage regeneration with fewer initial cell seeding, reducing the demand for cartilage donors.Meanwhile, the constructed tissue-engineered cartilage with high cell content can be maintained after implantation in vivo.
Another decisive factor affecting the quality of cartilage regeneration is the metabolic balance between cartilage ECM synthesis and degradation [39,40].Satisfactory cartilage regeneration depends on sufficient ECM secretion and low-level degradation activity.Consistent with our previous report, our results demonstrated that SP600125 negatively affects cartilage ECM synthesis, as indicated by the inferiority of cartilage-related quantitative evaluations and the downregulation of chondrogenesis-related gene expression.Fortunately, the combination with CM reversed these negative effects and enhanced cartilage ECM production.Simultaneously, SP600125 decreased cartilage degradation, as evidenced by the downregulation of inflammation and degradation-related gene expression.Overall, the combination of Figure 5. Analysis of the chondrogenesis-related and catabolism-related genes of in vitro regenerated cartilages with different culture modes.Expression of chondrogenesis genes (ACAN, COLII A1 and Sox9) was downregulated in groups treated with SP600125, and adding CM diminished the negative effect.Expression of catabolism-related genes (TNF-a, IL-1b and MMP13) was also decreased in groups treated with SP600125, reducing the inflammatory response and cartilage ECM degradation.Chondrogenesis genes in the LCM þ SP group were downregulated; however, they were higher than that in RM groups, and the inflammatory and degradation-related genes in the LCM þ SP group were the lowest.H, high cell-seeding density (6.0Â10 7 cells/ml); L, low cell-seeding density (3.0Â10 7 cells/ml); SP, SP600125 (a specific JNK pathway inhibitor).
CM and SP600125 compensated for each other and achieved abundant cartilage ECM production and low-level cartilage degradation, leading to satisfactory cartilage regeneration.
Cells proliferate for a certain period before commencing functional differentiation.In the process of cell differentiation, meaning that cells acquire specific physiological functions; their proliferation ability will gradually weaken and the ability of cell division is lost, stopping cell proliferation [17].Since cell proliferation and differentiation belong to two different stages, whether the phased application of CM and SP600125, that is, promoting cell proliferation with SP600125 in the early stage and promoting cell differentiation with CM in the later stage, will obtain optimal cartilage regeneration is still unknown.Our results showed that the phased application of CM and SP600125 avoided the negative effects on cartilage regeneration; however, it was inferior to the long-term combination group in the number of active cells and the quality of regenerated cartilage.These results suggest that our previous understanding of cell proliferation and differentiation stages may be insufficient.Cell proliferation and differentiation may penetrate and affect each other in tissue formation.Qiu et al. discovered that preadipocytes could differentiate into adipocytes without DNA synthesis and mitotic clonal expansion [41], while Parker reported that activated B-lymphocytes simultaneously undergo numerous clonal expansions when they mature [42].The evidence showed that although cell proliferation and differentiation are interrelated, they are independent of each other: cell proliferation is not accompanied by stagnation of differentiation and cell differentiation does not depend on cells exiting the proliferation cycle.Therefore, the long-term combination of CM and SP600125 can maximize their advantages and compensate for each other.Additionally, it can promote chondrocyte proliferation and differentiation, retain numerous active cells and secrete abundant specific cartilage ECM to achieve the optimal effect of cartilage regeneration.Figure 6.Gross view and histology of in vitro and in vivo regenerated cartilages cartilage-like tissues were formed in all three groups in vitro.After 8 weeks of in vivo implantation, cartilage formation matured with time.However, the formation of cartilage tissue was more uniform in the LCM þ SP group, while only cartilage islands were distributed in HCM and LCM groups.H, high cell seeding density (6.0Â10 7 cells/ml); L, low cell seeding density (3.0Â10 7 cells/ml); SP, SP600125 (a specific JNK pathway inhibitor).
Regenerative Biomaterials, 2023, Vol. 10, rbad079 | 9 In vivo cartilage regeneration involves a more complex physiological environment than in vivo regeneration [43,44].The shape, volume and long-term outcomes of regenerated cartilage constructed in vitro are affected by uncertain factors such as the pH of the body fluid, the tension of the surrounding tissues and degrading enzymes in vivo.Our results showed that, compared with the simple application of CM, the combination of CM and SP600125 did not have a significant advantage in the quality of cartilage regeneration in the in vitro culture stage, except for the high cell content.However, under the complex physiological and mechanical environment in vivo, it was unclear whether LCM þ SP could have numerous active cells and better maintain the long-term stability of the regenerated cartilage.Our results demonstrated that the wet weight and volume of LCM þ SP were maintained after 8 weeks of implantation in vivo.Similarly, the mechanical properties and content of cartilage-specific ECM components increased significantly and exceeded those of the simple CM groups.These results indicated that the induction effect of exogenous chondrogenic factors was lost in the complex in vivo environment.Additionally, further cartilage maturation depends more on cells with activity and normal physiological function [45].SP600125 in in vitro culture promoted chondrocyte proliferation, resulting in the largest active cells after implantation in vivo, to reverse and obtain the optimal effect of cartilage regeneration in vivo.Chondrogenesis-related gene expression evaluations revealed that SP600125 inhibited ECM degradation.The degrading enzymes and inflammatory factors were low in the in vitro culture environment.However, there were numerous unstable factors in the in vivo environment; thus, inhibiting the degradation of cartilage ECM for cartilage regeneration is important.In general, regenerated cartilage cultured with the longterm combination of CM and SP600125 obtained sufficient active cells to secrete abundant cartilage-specific ECM and lower levels of degradation activity, to obtain more stable cartilage regeneration in vivo.
This study confirmed that the long-term combination of CM and SP600125 enhanced cartilage regeneration in vitro and in vivo with fewer initial seeding cells, thus optimizing the current cartilage regeneration culture system.However, downstream transcription factors of the JNK pathway are closely related to tumor occurrence and development [46,47].The dosage of the JNK inhibitor SP600125 used in this study was within safe doses; however, there was a lack of direct evidence to prove its biological safety in regulating cartilage regeneration.In future studies, we will comprehensively examine the biological safety of SP600125 and further determine the optimal application dose for regulating cartilage regeneration through karyotype analysis, gene mutation screening, proto-oncogene expression, tumorigenicity assay and other related aspects.In addition, a large animal articular cartilage defect model will be constructed as described previously in the future study [48], combined with a biodegradable scaffold loaded with SP600125 controlled-release microspheres, to further discuss the regulation effect of SP600125 on the regeneration of cartilage defects in large animals.

Conclusion
In summary, the results of this study indicated that a long-term combination of CM and SP600125 obtained stable cartilage regeneration, both in vitro and in vivo, with fewer initial seeding cells.Therefore, few cartilage donors are required to achieve satisfactory cartilage regeneration, thus enhancing the efficiency and stability of the engineered cartilage regeneration system.

Figure 1 .
Figure 1.Preparation of in vitro cell-scaffold constructs.Gross observation (A), SEM (B, the arrow shows the PLA coating) of scaffolds.Immediate gross view of the cell-scaffold construct after cell seeding (C), cell-seeding efficiency (D) and DNA contents (E) of 4 h, 1 day, 4 days and 7 days after cell seeding.SEM images (F) of cell-scaffold construct in different culture systems after 12 h, 4 days and 7 days.SP, SP600125 (a specific JNK pathway inhibitor).

Figure 2 .
Figure 2. Cell viability of cells on scaffolds in different culture systems.Live/dead fluorescence staining revealed that the viable chondrocytes gradually proliferated with the increase in culture time in all groups.Adding SP600125 led to more significant cell proliferation in RM þ SP and CM þ SP groups.SP, SP600125 (a specific JNK pathway inhibitor).

Figure 7 .
Figure 7. Quantitative evaluations of in vitro and in vivo regenerated cartilages.In the in vitro culture stage, the LCM þ SP group obtained lower levels of wet weight (A), volume (B), mechanical strength (C), total collagen (D) and GAG contents (E) but the highest DNA content.After 8 weeks of implantation in vivo, the outcomes reversed and the LCM þ SP group obtained the highest level of all cartilage regeneration-related evaluations, indicating that the content of active cells and degradation activity were the main determinants of cartilage regeneration in vivo.H, high cell seeding density (6.0Â10 7 cells/ ml); L, low cell seeding density (3.0Â10 7 cells/ml); SP: SP600125 (a specific JNK pathway inhibitor).

Table 1 .
Group design of in vitro culture modes

Table 2 .
Group design of in vivo outcomes