Adolescent idiopathic scoliosis (AIS) is a common spinal deformity that occurs during puberty in children aged 10 to 18 years, with an uncertain pathogenesis. The morbidity rate ranges from 1% to 4% in adolescence, as reported in the literature, and the female-to-male ratio varies from 1.5 to 11.[1-3] Most cases of AIS progress until epiphyseal plate fusion. Severe AIS (main Cobb angle >70°) may cause cardiac dysfunction, impaired respiratory function, and chronic back pain. Generally, corrective surgery is the only way to treat AIS (main Cobb angle >40°), which may be accompanied by a decreased quality of life and heavy economic burden.[4-7]
Scholars agree that AIS is a multifactorial disease that includes genetics, the central nervous system, bone metabolism, biomechanics, and other factors.[8-11] Abnormal bone mass in AIS has long been a concern, and numerous studies have documented abnormal skeletal growth and lower bone mineral density (BMD) in AIS patients.[12,13] Notably, it has been reported that approximately 20% to 38% of AIS patients have osteopenia, which rarely occurs in adolescence. Additionally, previous studies have indicated that an imbalance in bone growth in the vertebrae is involved in the development of AIS. AIS patients with osteopenia tend to progress more easily and develop a large curve.[14,15] However, the cause of AIS-related osteopenia is still unknown. Several studies have revealed that osteopenia is related to abnormal levels of some metabolic hormones, such as estrogen, leptin, adiponectin, and ghrelin.[16-19] Some researchers have also suggested that diet and microelement intake contribute to AIS-related osteopenia.[17,20]
Bone marrow stem cells (BMSCs) are progenitor cells for bone tissue and adipose tissue in marrow cavity formation. The balance between the osteogenic and adipogenic differentiation of BMSCs is crucial for maintaining bone homeostasis.[21,22] Reduced osteogenic differentiation and increased adipogenic differentiation of BMSCs are characteristics of elderly individuals that induce age-related osteopenia and osteoporosis.[23-25] However, in our previous studies, we found that the osteogenic ability of BMSCs from AIS-related osteopenia patients was lower than that of BMSCs from control patients, which suggested dysregulated differentiation of BMSCs from AIS patients.
Accordingly, we further explored the osteogenic and adipogenic differentiation of BMSCs from the AIS and control groups. In this study, primary BMSCs from patients were extracted and stimulated for differentiation. Alizarin red staining and oil red O staining were performed after differentiation. The expression levels of osteogenesis- and adipogenesis-related genes and proteins were measured to identify whether the differentiation of BMSCs from AIS patients was different from that of controls.
This study adhered to the principles of the Declaration of Helsinki II and was approved by the medical ethics committee of Xiangya Hospital, Central South University (No. 201703358). Written informed consent was acquired from each of the patients (or their parents or legal guardians) to authorize treatment, imaging, and photographic documentation.
All participants who were selected had undergone treatment during January 1, 2018, to September 31, 2019. Bone marrow blood was collected during screw placement for subsequent experiments, including BMSC extraction, RNA sequencing (RNA-seq), and cell experiments [Table 1]. All patients were screened according to a detailed questionnaire, medical history, physical examination, and BMD assessment. Imaging examinations performed included X-ray, computed tomography, and magnetic resonance imaging. A double-blinded diagnosis of the participants was achieved by three senior spinal surgeons. The inclusion criteria for AIS-related osteopenia patients were a minimum Cobb angle of 10° on X-ray examination and a BMD Z score of less than −1. The included controls were age-matched non-AIS patients, including those with lumbar herniation and spinal fracture. The exclusion criteria were endocrine or metabolic disorders, congenital skeletal or spinal dysplasia, scoliosis secondary to other etiologies, connective tissue abnormalities, and a history of spinal surgery. The general clinical data collected included age, sex, height, weight, Risser sign, body mass index (BMI), bone marrow density, and Z score. The Cobb angle and Lenke classification were specifically recorded for AIS patients. For the cell experiment, bone marrow blood was obtained from the apical vertebrae of six AIS-related osteopenia patients and six control patients. For RNA-seq, BMSCs were extracted from the bone marrow blood of three AIS-related osteopenia patients and three control patients. To verify the RNA-seq results, total RNA was obtained from BMSCs from 13 AIS-related osteopenia patients and nine control patients.
Table 1 -
Clinical data of all the AIS and control groups.
||15.95 ± 1.00
||16.17 ± 1.10
||46.10 ± 6.27
||52.72 ± 5.30
||1.59 ± 0.08
||1.63 ± 0.09
||18.39 ± 2.58
||19.78 ± 0.89
||3.00 ± 0.82
||2.95 ± 0.78
| BMD (g/m2)
||0.77 ± 0.08
||0.95 ± 0.10
||−2.05 ± 0.58
||0.45 ± 0.89
AIS: Adolescent idiopathic scoliosis; BMD: Bone mineral density.
Anthropometric and BMD assessments
Age and sex were determined from national identification cards. Body weight and standing height were measured using a standard electronic weighing scale and scale plate, respectively. Two senior surgeons participated in the assessments of the Risser sign, main Cobb angle, and Lenke classification based on X-ray images. BMI was calculated by dividing weight (kg) by height squared (m2). Dual-energy X-ray absorptiometry was used to measure the lumbar spine BMD and femoral neck BMD of all participants.
Isolation and culture of human primary BMSCs
For human primary BMSC isolation, intramedullary blood was drawn out from the pedicle screw trajectory before screw setting. Bone marrow blood was stored and transferred to ethylenediaminetetraacetic acid (EDTA) tubes at 4°C. Under sterile conditions, the blood was diluted with an equal amount of F12 medium, after which 5 mL of diluted blood was layered gently over 4 mL of LSM (Yang Biological Manufacture, LTS1077006, China) in a 15-mL tube and centrifuged at 2500 r/min for 10 min at room temperature. The cells in the buffy coat were collected, washed, resuspended, and cultured in F12 medium containing 20% fetal bovine serum (FBS), Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Gibco) in an incubator (5% CO2 atmosphere at 37°C).
For the analysis of immunophenotypic markers, BMSCs were incubated with anti-CD105, anti-CD29, anti-CD44, anti-CD34, anti-CD14, and anti-CD45 antibodies (Cyagen Biosciences, HUXMX-09011, USA) at 4°C for 30 min and then analyzed via flow cytometry [Supplementary Figure 1, https://links.lww.com/CM9/B299].
Osteogenic and adipogenic differentiation
For the induction of osteogenesis and adipogenesis and staining of BMSCs, human BMSC differentiation kits (Cyagen Biosciences, HUXMA-90031, HUXMA-90021, USA) were used according to the manufacturer's instructions. In brief, for osteogenic differentiation, BMSCs were cultured in α-minimum essential medium (MEM) containing 10% FBS, 50 μg/mL ascorbate, 10 mmol/L β-glycerol phosphate and 0.1 μmol/L dexamethasone. After 21 days of culture, the cells were fixed with 10% paraformaldehyde and stained with 0.1% alizarin red solution. For adipogenic differentiation, BMSCs were cultured in α-MEM containing 10% FBS, 50 μmol/L isobutylmethylxanthine, 0.5 μmol/L dexamethasone, 50 μmol/L indomethacin, and 10 μg/mL insulin. After 21 days of culture, the cells were fixed with 10% paraformaldehyde and stained with 0.5% fresh oil red O solution. For quantitative image analysis, the average optical density was calculated by Image-Pro Plus (version 6.0, Medica Cybernetics, USA).
Real-time quantitative polymerase chain reaction (qPCR)
Total RNA was extracted from isolated BMSCs using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. cDNA was synthesized from total RNA using a HifiScript cDNA Synthesis Kit (CWBIO, China). The following primers were used for qPCR of osteogenesis-related markers: RUNX2, TGT TTG GCG ACC ATA TTG AA (forward) and GGC TGC AAG ATC ATG ACT GA (reverse); Col1a, AGG AAG GGC CAC GAC AAA G (forward) and CAG TTA CAC AAG GAA CAG AAC AGT CTC T (reverse); BMP2, GCA AAG AAA AGG AAC GGA CAT T (forward) and GGG AAG CAG CAA CGC TAG AA (reverse); and Osterix, GCT TAT CCA GCC CCC TTT AC (forward) and CAC TGG GCA GAC AGT CAG AA (reverse). The following primers were used for qPCR of adipogenesis-related markers: C/EBP-α, GTT AGC CAT GTG GTA GGA GAC A (forward) and CCC AGC CGT TAG TGA AGA GT (reverse); PPARγ2, GAG CCC AAG TTT GAG TTT GC (forward) and CTG TGA GGA CTC AGG GTG GT (reverse); FABP4, TGC AGC TTC CTT CTC ACC TT (forward) and CAT CCC CAT TCA CAC TGA TG (reverse); and LPL, GTG GCC AAA TAG CAC ATC CT (forward) and CCG AAA GAT CCA GAA TTC CA (reverse). The following primers were used for qPCR based on RNA-seq results: DCLK1, CGC ACG GTT CTT TCT TCT TC (forward) and GGG GCG TCA TCA GTA CAT CT (reverse); TSPAN5, TTG TGG TGG GAG GAG TGA T (forward) and CTG GGT GAA GTC TAT GAG GTT (reverse); LRRC17, CAA GAA ACA AGA TCC GCA CA (forward) and AAG TAC AGT GCC AGG GGT TG (reverse); PCDH7, CAA TGC TCC CAC AGT TAC CCT (forward) and ACT GTC ATT CAC TTG CAC CAC (reverse); NHSL2, CAG AAG GGG TTC ATG GAA GA (forward) and ATT GCA AGA ACT GGG GAC AC (reverse); CPT1B, GAA TAC AGA CGT GCA GGG AG (forward) and GCT GTC TGA GAG GTG CTG TA (reverse). RNA levels were normalized to 18S, AGA AAC GGC TAC CAC ATC CA (forward), and CCC TCC AAT GGA TCC TCG TT (reverse). Real-time qPCR was performed using SYBR qPCR Super Mix Plus (Novo Start, China) and an Applied Biosystems 7500 instrument. The program was allowed to function at 95°C for 1 min, followed by 40 cycles of 95°C for 20 s and 60°C for 1 min.
Total protein was extracted with ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (CWBIO) containing phenylmethylsulfonyl fluoride (PMSF) and a phosphatase inhibitor (48:1:1) according to the manufacturer's instructions. Protein levels were quantified using the bicinchoninic acid protein assay reagent (Beyotime, China). Fifteen micrograms of protein was added to each well, separated by 10%/15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and transferred to a PVDF membrane. The membrane was blocked with skim milk for 1 h at room temperature, followed by incubation with a primary antibody at 4°C on a rocker overnight. Primary antibodies against BMP2 (Proteintech, 18933-1-AP, USA), COL1 (Proteintech, 14695-1-AP, USA), C/EBPα (Santa Cruz, sc-365318, China), FABP4 (Santa Cruz, sc-271529 SAMPLE, China), P62 (Abcam, ab109012, USA), LC3B (Abcam, ab192890, USA), and Beclin 1 (Abcam, ab207612, USA) were used. After incubation with secondary antibodies for 1 h at room temperature, a BeyoECL Plus Kit (Beyotime) was used for detection. Gray value analysis was used to analyze the Western blotting results; the target protein gray value was divided by the corresponding internal reference gray value to obtain the relative protein expression.
Mitochondrial membrane potential
Staining with a mitochondrial membrane potential assay kit (JC-1 C2006, Beyotime) was performed at 37°C for 20 min to determine the BMSC mitochondrial membrane potential.
Total RNA was extracted from isolated BMSCs using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. RNA-seq library construction and high-throughput RNA-seq were performed by Wuhan Genomic Institution (BGI HUADA, China) on a BGISEQ-500 high-throughput sequencer (BGI, Shenzhen, China). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed according to a previously described method.
The results were recorded and analyzed using SPSS software (version 24.0, SPSS, Inc., Chicago, IL, USA). Quantitative data were assessed by one-way analysis of variance (ANOVA) with Bonferroni's correction or t test, and the results are expressed as the mean ± standard deviation. A difference was considered significant if the P value was <0.05, and Bonferroni's correction was considered to indicate a significant difference when the P value was <0.0167. For convenient charting, all qPCR and Western blotting results were normalized to those of the control groups.
The results of this study are presented in three parts. All participants were divided into the AIS-related osteopenia or control group. In the first part, primary BMSCs were isolated from intramedullary blood, after which BMSCs were subject to osteogenic induction to investigate the differences in osteogenic differentiation ability between the two groups. In the second part, adipogenic induction was performed to investigate the differences in adipogenic differentiation ability between the two groups. In the third part, to further reveal the possible related genes, RNA-seq analysis was performed.
BMSCs from patients with AIS-related osteopenia have a lower osteogenic ability
Six BMSC samples from AIS patients and six paired samples from controls were subjected to osteogenic induction [Supplementary Table 1, https://links.lww.com/CM9/B299]. After 21 days of osteogenic differentiation, alizarin red staining showed that the osteogenic capacity in the AIS-related osteopenia group was significantly lower than that in the control group [Figure 1A]. Moreover, the mRNA levels of BMP2, COL1, RUNX2, and Osterix were substantially increased after induction, with obviously higher levels in the control group than in the AIS group [Figure 1B and 1C and Supplementary Figure 2A and 2B, https://links.lww.com/CM9/B299]. The trend of osteogenic marker expression in the BMSCs was also assessed over time (3, 7, 10, 14, 17, and 21 days). In general, the mRNA and protein levels of markers in both the AIS and control groups increased in a time-dependent manner. However, the trend showed more rapid increases in the control group and levels that remained higher than those in the AIS group from the beginning of induction [Figure 1D–G and Supplementary Figure 2C and 2D, https://links.lww.com/CM9/B299].
BMSCs from patients with AIS-related osteopenia have a higher adipogenic ability
BMSC samples from six AIS patients and six paired controls were subjected to adipogenic induction [Supplementary Table 1, https://links.lww.com/CM9/B299]. After 21 days of adipogenic differentiation, the results of oil red O staining indicated that the adipogenic capacity in the AIS-related osteopenia group was significantly higher than that in the control group [Figure 2A and 2B]. Furthermore, the mRNA levels of C/EBPα, PPARγ2, FABP4, and LPL were markedly increased in the AIS-related osteopenia group compared with those in the control group [Figure 2C and 2D and Supplementary Figure 3A and 3B, https://links.lww.com/CM9/B299]. The trend of adipogenic marker expression in the BMSCs was also assessed over time (3, 7, 10, 14, 17, and 21 days). In general, the mRNA and protein levels of markers in both the AIS and control groups increased in a time-dependent manner. In contrast with osteogenic differentiation, the trend showed increases to higher levels in the AIS group than in the control group [Figure 2E–I and Supplementary Figure 3C and 3D, https://links.lww.com/CM9/B299].
BMSC autophagy impairment in AIS-related osteopenia
To determine the mechanism underlying the osteogenic and adipogenic differentiation of BMSCs, we performed RNA-seq to identify differences in mRNA expression levels between the AIS-related osteopenia group and the control group [Supplementary Table 2, https://links.lww.com/CM9/B299]. Heatmap analysis depicting several differentially expressed genes showed that LRRC17, DCLK1, PCDH7, TSPAN5, and NHSL2 were markedly upregulated in the AIS group, while CPT1B was obviously downregulated in the AIS group [Figure 3A]. Using KEGG and GO enrichment analyses of the differentially expressed genes, we identified several biological processes involved in the regulation of autophagy and mitophagy [Figure 3B and 3C]. The RNA-seq results were further confirmed by real time-qPCR analysis [Figure 3D] [Supplementary Table 3, https://links.lww.com/CM9/B299]. Subsequently, we detected the expression levels of autophagy markers and the mitochondrial membrane potential, and the results suggested that autophagic activity was impaired in BMSCs in the AIS-related osteopenia group [Figure 4].
The correlation between abnormal bone metabolism and AIS-related osteopenia and the underlying molecular mechanism that causes the development and progression of AIS have generated tremendous interest over the years. In our study, we further revealed the unusual differentiation ability of BMSCs from AIS-related osteopenia patients, wherein BMSCs have a lower osteogenic capacity and a higher adipogenic capacity. Additionally, these characteristics of their differentiation were maintained during prolonged in-vitro culture. Next, RNA-seq was performed, and KEGG analysis identified the enrichment of pathways related to the regulation of autophagy and mitophagy. Significant differences in the mRNA expression of LRRC17, DCLK1, PCDH7, TSPAN5, NHSL2, and CPT1B were found between the two groups. Moreover, autophagy marker analysis revealed reduced autophagic activity in BMSCs in the AIS-related osteopenia group. Taken together, our results demonstrate impaired autophagy in BMSCs from AIS-related osteopenia patients, which may affect bone metabolism in these patients.
To our knowledge, the pathogenesis of AIS is affected by multiple factors, including genetic factors, the development of the central nervous system, hormone metabolism, biomechanics, and environmental factors. A systematic analysis indicated that there was a moderate correlation between a lower bone mass density and the development of AIS. Additionally, a lower BMD has been revealed to be a risk factor for the progression of AIS. During the normal growth and development period, adolescents in puberty should exhibit a rapid bone mass increase. However, a number of studies have documented that some AIS patients have osteopenia or osteoporosis.[12,13] These studies suggest that a lower BMD may play an important role in the pathogenesis of AIS. In previous studies, several hormones have been proven to be involved in AIS-related osteopenia.[16-19] Dysregulation between hormones and osteogenic cells leads to a lower osteogenic ability in AIS patients, which eventually results in bone mass loss. BMSCs are progenitor cells in skeletal tissue that have strong osteogenic and adipogenic abilities and play an important role in the balance of bone metabolism.[30,31] A previous study by our group also found abnormal osteogenesis in BMSCs from AIS-related osteopenia patients. To further investigate the differentiation status of BMSCs from AIS-related osteopenia patients, we isolated and cultured BMSCs from AIS-related osteopenia patients and normal control patients. After osteogenic and adipogenic induction, we found that osteogenic markers were expressed at lower levels and adipogenic markers were expressed at higher levels in the AIS-related osteopenia group than in the control group. These trends in the two groups were maintained after prolonged culture in vitro, which suggested a stronger adipogenic capacity and weaker osteogenic capacity in BMSCs from AIS-related osteopenia patients. These results indicate that the BMSCs in the AIS-related osteopenia group tended to undergo adipogenic differentiation and that their osteogenic ability was significantly reduced, which may contribute to the lower bone mass in AIS patients.
Furthermore, BMSC samples from three AIS patients and three paired controls were subjected to RNA-seq to determine the underlying reasons for the unusual differentiation results. KEGG pathway analysis suggested high enrichment of autophagy- and mitophagy-related pathways. Autophagy is an intracellular degradation system that acts through the lysosome pathway, which is closely related to cell metabolism, function, and homeostasis.[32,33] A wealth of evidence has shown that autophagy is correlated with the maintenance of cellular homeostasis in multiple ways, such as by regulating cellular metabolism, the cell lifespan, and cellular differentiation.[34,35] Moreover, scholars have reported that autophagic activity could influence estrogen-induced osteoporosis. Another study indicated that enhanced autophagy could improve the osteogenic capacity of MSCs from older osteoporosis patients. Several scholars have also revealed that promoting the autophagy of MSCs could significantly alleviate OVX-induced osteoporosis in mice.[36,38] In addition to autophagy, alterations in mitophagy influence mitochondrial functions. Mitochondrial impairment has a major negative effect on osteogenic capacity.[40,41] Therefore, we subsequently detected the autophagy levels and mitochondrial membrane potential of BMSCs in the AIS-related osteopenia group and the control group. The results showed that the expression of autophagy-related markers and the mitochondrial membrane potential were significantly decreased in the AIS group, which is consistent with the phenotype of BMSCs in AIS-related osteopenia, suggesting that decreased autophagic activity may result in abnormal osteogenic and adipogenic differentiation of BMSCs in AIS patients.
Additionally, the heatmap indicated several significantly differentially expressed mRNAs between the two groups, including DCLK1, TSPAN5, LRRC17, PCDH7, NHSL2, and CPT1B. We verified the aforementioned mRNAs in the total RNA of BMSCs in the two groups via qPCR. The results showed that CPT1B expression obviously decreased and that LRRC17, DCLK1, and PCDH7 expression was significantly increased in the AIS group. LRRC17 and DCLK1 have been confirmed to be related to autophagy in previous studies. Roy et al suggested that the expression of DCLK1, which is involved in the survival and progression of colon tumors, impairs the process of autophagy. A number of studies have indicated that LRRC17 overexpression induces reduced osteogenic ability and bone mass.[43-46] Liu et al further revealed that LRRC17 overexpression in BMSCs impairs autophagy, which decreases the osteogenic ability of BMSCs. It is worth investigating the underlying mechanisms through which these genes influence the osteogenic ability and bone mass of AIS-related osteopenia patients.
However, there are some limitations of this study that should be considered. One is the limited sample size of the recruited patients. Patients with lower Cobb angles were excluded because they did not need surgical therapy. The second limitation is that the primary cells were extracted from apical vertebrae, which may not represent the general trend. The third limitation is that all the experiments were performed in vitro, and in-vivo experiments may provide different results. In addition, the underlying mechanism needs to be further investigated.
In conclusion, our study revealed that BMSCs from AIS-related osteopenia patients have lower autophagic activity, which may be related to the lower osteogenic capacity and higher adipogenic capacity of BMSCs and consequently lead to reduced bone mass in AIS patients. Our study provides novel insight into AIS-related osteopenia. To our knowledge, these results are the first to assert the abnormal autophagy of BMSCs in AIS-related osteopenia.
This study was supported by grants from the National Natural Science Foundation of China (No. 82072390), and the Natural Science Foundation of Hunan, China (No. 2020JJ4873).
Conflicts of interest
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