The Adverse Effect And Mechanism Of Nicotine On The Chondrogenic Repair Of Bone Marrow Stem Cells In Rat Articular Cartilage Defects

Articular cartilage defect is a type of cartilage lesions which involves in cartilage surface or even in subchondral bone. It is usually caused by trauma, cartilage wear, degenreation and congenital malformation, etc. The traffic and sportive injuries are the main reasons of cartilage defect. In recent years, with the developing of public transport and competitive sport, and the increasing striking aging population, the incidence of cartilage defect is also gradually rising. As articular cartilage is a poorly vascularized fibrous connective tissue, these histological and biological properties limit its capacity of self-repair. Therefore, the treatment of articular cartilage defect is often a perplexed question for orthopedists.Currently, there is a lack of effective therapy for cartilage defect, especially for large area defect. With the development of medical technology and tissue engineering, the application of tissue engineered cartilage makes advances in the treatment of cartilage defect. Bone marrow derived mesenchymal stem cells (BMSCs) have a capacity for self-renewal, sternness maintenance, and a potential for differentiation into multiple cells, therefore, they are ideal seed cells. It is reported that autologous culture expanded BMSCs transplantation may be an effective approach to promote the repair of articular cartilage defects. While, the current researches are mainly focus on how to promote chondrogenic differentiation, how to improve the repair effects, there is less study focusing on adverse risk factors in the process of chondrogenic differentiation. China is already the largest tobacco producer now, the number of smoker occupies the first of the world, the prevalence and mortality of various diseases caused by smoking are increasing year by year. Epidemiological study indicated that nicotine abuse was a risk factor that influenced clinical outcome following autologous chondrocyte implantation for cartilage defects of the knee. While, whether nicotine had the same effect on BMSCs transplantation repair of articular cartilage defects is still unknown. Our recent work has demonstrated that nicotine suppresses chondrogenic differentiation potential of BMSCs, leading to a poorly differentiated cartilage. However, the mechanism of nicotine’s adverse effect is still unclear.SRY-type high mobility group box9 (Sox9) is an initial transcriptional factor in chondrogenesis, it promotes and maintain the property of articular chondrocytes by binding with the enhancer region of cartilage phenotype gene in the process of cartilage development. α7 nicotinic acetylcholine receptor (a7-nAChR) is a ligand-gated ion channel existing in MSCs which can mediate increases in Ca2+. Calcineurin is a calcium/calmodulin-activated, serine-threonine phosphatase that is activated by the binding of calcium and calmodulin and transmits signals to the nucleus through the dephosphorylation and translocation of nuclear factor of activated T cell (NFAT) transcription factors. Epigenetic modification is an important regulation of gene expression which not change the DNA sequence, the most common form is histone acetylation. The status of histone acetylation is regulated by histone acetylase and related to the gene expression. The dynamic balance between histone acetylase and deacetylase maintains the regular gene expression. From all of above, we speculate that nicotine regulates Ca2+/CaN/NFAT signaling through a7-nAChR to induce the epigenetic change of Sox9, and finally influence the chondrogenic differentiation of BMSCs.In the present study, we firstly verified the adverse effect of nicotine on the chondrogenic repair of BMSCs in rat articular cartilage defects, and propose that Sox9 plays a key role in nicotine’s effect. Then on the basis of our previous study, we confirmed the epigenetic mechanism of nicotine’s suppression on Sox9 expression and chondrogenic differentiation of BMSCs. Finally, we used the selective α7-nAChR inhibitor and Si-RNA to clarify the molecular target of nicotine’s adverse effect. This study provides the theoretical foundation for risk evaluation, establishing the personalized treatment plan, avoiding nicotine’s adverse effect and achieving an optimal clinical outcome in nicotine dependence individuals.Part ONEThe adverse effect of nicotine on the chondrogenic repair of BMSCs in rat articular cartilage defectsObjective To confirm nicotine’s adverse effect on the chondrogenic repair of BMSCs in rat articular cartilage defects through the suppression of Sox9.Methods Whole bone marrow adherence method was used to expand BMSCs from primary passage to third passage. Forty 12-weeks Wistar rats (male:female=1:1) were anesthetized using 10% chloral hydrate (3.5 ml/Kg), a full-thickness cylindrical cartilage defect of 3 mm diameter and 1.5 mm deep was created in the femoral trochlear. The rats were randomized into 4 groups:Non-treated group:no treatment in articular cartilage defect model; Alginate group:repair the cartilage defect with alginate gel only; BMSCs group:repair the cartilage defect with alginate gel and BMSCs; Nicotine group:nicotine 2.0 mg/kg·d was injected every day after repairing the cartilage defect with alginate gel and BMSCs. Rats were sacrificed under ether anesthesia 3 month after operation and distal part of the femur was extirpated. Portion of samples from each group were firstly examined and photographed for evaluation according to the International Cartilage Repair Society (ICRS) macroscopic assessment scale, then sections of these samples were used for pathological examination and evaluated the scale for cartilage repair. Immunohistological staining was used to analyze the expression of Col2A1 and Sox9. The repair tissues from the remaining samples in defect areas were scraped and extracted total RNA, detected the mRNA expression of Col2A1, Aggrecan and Sox9.Results Macroscopic and histological observations:In the non-treated group, newregenerated cartilage tissues almost in level with surrounding cartilage could be seen, but the surface of the defects were still concave or empty in the middle. HE and safranine staining showed the matrix in regenerated cartilage tissues was faintly stained. In Alginate group, more than 50% defects were covered by new regenerated cartilage tissues which were a little lower in level with surrounding cartilage, large fissures could be seen in the surface. HE and safranine staining showed that the defects were filled with fibrous tissue, no staining of matrix was observed. In BMSCs group, the original defect was filled of glossy white repaired tissue that appeared to be smooth and well-integrated with the surrounding tissues. HE and safranine staining showed that the defects were filled with hyaline-like cartilage with a regular surface that was well-integrated with the native cartilage, an abundance of cartilage matrix could be identified by safranine staining. In Nicotine group, the margin between the regenerated tissue and the original cartilage was not distinguishable but was still slightly concave. HE and safranine staining showed that larger amounts of cartilage-like tissue was observed, the safranine staining of matrix was light than native cartilage. The ICRS scores in BMSCs group were higher than the other groups (P<0.05), the histological scores in BMSCs group were lower than the other groups (P< 0.05). Immunohistological observations:Immunohistochemical staining of type Ⅱ collagen in BMSCs group was especially stronger than the other 3 groups, no positive staining was observed in Alginate group. The positive stained Sox9 cells in BMSCs group was also larger than the other 3 groups, no positive stained Sox9 cell was found in Alginate group. The mean optical density of Col2A1 and Sox9 in BMSCs group was higher than Nicotine group (P<0.01, P<0.05). Gene expression: The mRNA expression of Col2A1, Aggrecan and Sox9 in BMSCs group was higher than Nicotine group (P<0.05).Conclusion BMSCs with Alginate gel transplantation was an effective method to repair articular cartilage defects. Nicotine has an adverse effect on repair outcome through suppressing the expression of Sox9.Part TWOEpigenetic mechanism of nicotine’s suppression on chondrogenic differentiation of rat BMSCsObjective To investigate the epigenetic mechanism of nicotine’s suppression on chondrogenic differentiation of rat BMSCs in vitro.Methods The third passage rat BMSCs were suspended in alginate gel to make the Alginate beads, then cultured with chondrogenic differentiation medium. During the period of chondrogenic differentiation the culture medium with or without nicotine at concentrations of 0.1,1,10 and 100(?)M was replaced every other day. RT-PCR was used to detect the mRNA expressions of Col2A1, Aggrecan and Sox9 after 28 days. Total RNA and protein were extracted from the third passage BMSCs, the subunit of nAChR was identified by PCR and Western Blotting. BMSCs were incubated by Fluo-3AM, confocal microscopy was used to measure the Ca2+ concentrations after stimulating by nicotine (0.1,1,10,100(?)M), and then the enzyme activity of CaN was detected using chromogenic substrate method. BMSCs were treated by nicotine 8h, 16h,24h respectively to detect the change of mRNA of Sox9. The time of nicotine treatment was determined according to the expressive change of Sox9 to verify the epigenetic mechanism. Total and nucleic protein were extracted to detect the expression of NFATc2, cytoplasm protein was used to detect the expression of phosphorylated NFATc2. Chromatin immunoprecipitation (ChIP) was used to examine the acetylation of histone H3K9 and K14 (H3K9ac and H3K14ac) in Sox9 promoter, the bindings of NFATc2 to Sox9 promoter and Sox9 to Col2A1 enhancer. Co-immunoprecipitation (Co-IP) was used to detect the interaction of NFATc2 with HDAC1.Results α7-nAChR was found to be expressed in BMSCs. A stimulation of BMSCs by nicotine (a typical a7-nAChR agonist) increased intracellular Ca2+ concentration and CaN activities in concentration-dependent manner (P<0.05). Although no significant changes were observed in whole cells, the nucleic expressions of NFATc2 protein were elevated and the cytoplasm phosphorylated NFATc2 were reduced by nicotine (P <0.01, P<0.05). The bindings of NFATc2 to Sox9 promoter were increased and thus reduced H3K9ac and H3K14ac in Sox9 promoter (P<0.01, P<0.05), a decreased binding of Sox9 to Col2A1 was also observed (P<0.01, P<0.05).The interaction of NFATc2 with HDAC1 was enhanced. All of the above changes induced by nicotine were characterized by concentration-dependent manner.Conclusion The epigenetic mechanism of nicotine’s suppression on chondrogenic differentiation of BMSCs was that the stimulation of nicotine increased intracellular Ca2+ concentration and activated CaN through a7-nAChR, resulting in dephosphorylation and increased nucleic translocation of NFATc2, NFATc2 binded to Sox9 promoter and recruited HDAC1 to form the complex, which caused an reduced H3K9ac and H3K14ac in Sox9 promoter and decreased expression of Sox9. A low binding of Sox9 with Col2A1 further reduced the Col2A1 expression and finally inhibited BMSCs chondrogenic differentiation.Part THREEThe molecular target of nicotine’s suppression on chondrogenic differentiation of rat BMSCsObjective To verify the molecular target of nicotine’s suppression on chondrogenic differentiation of rat BMSCs in vitro by using methyllycaconitine (MLA) and Si-NFATc2.Method The third passage BMSCs suspended in alginate beads were cultured 28 days with medium for chondrogenic differentiation. The alginate beads were randomized into 4 groups:Control group:cultured with chondrogenic differentiation medium; Nicotine group:cultured with the medium containing 100(?)M nicotine; MLA group: cultured with medium containing 10(?)M MLA; Nicotine + MLA group:cultured with medium containing 10(?)M MLA 0.5 h first, then changed with the medium containing 100(?)M nicotine. After 4 weeks, the mRNA expressions of Col2A1, Aggrecan and Sox9 were examined; the beads were fixed and dehydrated through alcohols and embedded in paraffin. The sections were routinely stained with Alcian blue and Safranine-O. To verify the molecular target of nicotine’s effect, the third passage BMSCs were randomized into 4 groups:Control group:cultured with chondrogenic differentiation medium 24 h; Nicotine group:cultured with medium containing 100(?)M Nicotine 24 h; Si-NFATc2 group:cultured with medium containing Si-NFATc224 h, then with the medium containing 100(?)M nicotine 24 h; MLA group: cultured with medium containing 10(?)M MLA 0.5 h first, then with the medium containing 100(?)M nicotine 24 h. Examined intracellular Ca2+ concentration, the H3K9ac and H3K14ac in Sox9 promoter, the bindings of NFATc2 to Sox9 promoter and Sox9 to Col2A1 enhancer, and the interaction of NFATc2 with HDAC1 by using the method introduced in Part Two.Results After 28 days of chondrogenic differentiation, compared with the control group, the Alcian bule and safranine-O staining was obvious light in nicotine group, there was no change in MLA group andNicotine+MLA group. The mRNA expressions of Col2A1, Aggrecan and Sox9 was significantly reduced in Nicotine group (P<0.01), no changes were observed in MLA group andNicotine+MLA group. The result of verifying molecular target of nicotine’s effect showed that MLA conversely eliminated nicotine-induced calcium influx. Compared with the control group, reduced H3K9ac and H3K14ac in Sox9 promoter, decreased binding of Sox9 to Col2A1 (P<0.01) and enhanced interaction of NFATc2 with HDAC1 was detected in nicotine group, no significant change was observed in Si-NFATc2 and MLA groups.Conclusion a7-nAChR and NFATc2 are molecular targets of nicotine’s suppression on chondrogenic differentiation of BMSCs, interventions aimed at α7-nAChR and NFATc2 could reverse nicotine’s adverse effect on chondrogenic differentiation of BMSCs.