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Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_1 | Pages 2 - 2
23 Jan 2023
Newton Ede M Pearson MJ Philp AM Cooke ME Nicholson T Grover LM Jones SW
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To determine whether spinal facet osteoblasts at the curve apex display a different phenotype to osteoblasts from outside the curve in patients with adolescent idiopathic scoliosis (AIS). Intrinsic differences in the phenotype of spinal facet bone tissue and in spinal osteoblasts have been implicated in the pathogenesis of AIS. However, no study has compared the phenotype of facet osteoblasts at the curve apex with the facet osteoblasts from outside the curve in patients with AIS. Facet bone tissue was collected from three sites, the concave and convex side at the curve apex and from outside the curve from three female patients with AIS (aged 13–16 years). Micro-CT analysis was used to determine the density and trabecular structure. Osteoblasts were then cultured from the sampled bone. Osteoblast phenotype was investigated by assessing cellular proliferation (MTS assay), cellular metabolism (alkaline phosphatase and Seahorse Analyser), bone nodule mineralisation (Alizarin red assay), and the mRNA expression of Wnt signalling genes (quantitative RT-PCR). Convex bone showed greater bone mineral density and trabecular thickness than did concave bone. The convex side of the curve apex exhibited a significantly higher proliferative and metabolic phenotype and a greater capacity to form mineralised bone nodules than did concave osteoblasts. mRNA expression of SKP2 was significantly greater in both concave and convex osteoblasts than in non-curve osteoblasts. The expression of SFRP1 was significantly downregulated in convex osteoblasts compared with either concave or non-curve. Intrinsic differences that affect osteoblast function are exhibited by spinal facet osteoblasts at the curve apex in patients with AIS


Orthopaedic Proceedings
Vol. 104-B, Issue SUPP_8 | Pages 1 - 1
1 Aug 2022
Nicholson T Foster N Haj AE Ede MN Jones S
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We previously reported that osteoblasts at the curve apex in adolescent idiopathic scoliosis (AIS) exhibit a differential phenotype, compared to non-curve osteoblasts(1). However, the Hueter-Volkmann principle on vertebral body growth in spinal deformities (2) suggests this could be secondary to altered biomechanics. This study examined whether non-curve osteoblasts subjected to mechanical strain resemble the transcriptomic phenotype of curve apex osteoblasts. Facet spinal tissue was collected perioperatively from three sites, (i) the concave and (ii) convex side at the curve apex and (iii) from outside the curve (non-curve) from six AIS female patients (age 13–18 years; NRES 19/WM/0083). Non-curve osteoblasts were subjected to strain using a 4-point bending device. Osteoblast phenotype was determined by RNA sequencing and bioinformatic pathway analysis. RNAseq revealed that curve apex osteoblasts exhibited a differential transcriptome, with 1014 and 1301 differentially expressed genes (DEGs; p<0.05, fold-change >1.5) between convex/non-curve and concave/non-curve sites respectively. Non-curve osteoblasts subjected to strain showed increased protein expression of the mechanoresponsive biomarkers COX2 and C-Fos. Comparing unstimulated vs strain-induced non-curve osteoblasts, 423 DEGs were identified (p<0.05, fold-change >1.5). Of these DEGs, only 5% and 6% were common to the DEGs found at either side of the curve apex, compared to non-curve cells. Bioinformatic analysis of these strain-induced DEGs revealed a different array of canonical signalling pathways and cellular processes, to those significantly affected in cells at the curve apex. Mechanical strain of AIS osteoblasts in vitro did not induce the differential transcriptomic phenotype of AIS osteoblasts at the curve apex


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXVII | Pages 42 - 42
1 Jun 2012
Fendri K Patten S Zaouter C Parent S Labelle H Edery P Moldovan F
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Introduction. Adolescent idiopathic scoliosis (AIS) is the most common form of spinal deformity. It occurs mainly in girls and progresses during pre-pubertal and pubertal growth, which is a crucial period for bone mass acquisition. The cause and molecular mechanisms of AIS are not clear; at present the consensus is that AIS has a multifactor cause, with many genetic factors. During the past 5 years, considerable effort has been devoted to identify a gene or genes that cause a predisposition to AIS. Many loci for this disorder have been mapped to different chromosome regions, but no genes have been clearly identified as being responsible for AIS, and, most importantly, the resulting protein defects remain to be shown. We aimed to identify the gene(s) that could be involved in AIS and to validate their involvement by both genetic and functional analyses. Methods. A large multiplex AIS French family was chosen for this study on the basis of clinical and radiological data. Whole genome genotyping of the 20 members of this family led to the mapping of a dominant disease-causing gene to two critical genomic intervals (Edery and colleagues, Eur J Hum Genet, accepted [2011]), but the causative mutation remains to be identified. In parallel, gene expression profiling was investigated by microarray analysis in RNA samples isolated from osteoblasts derived from healthy individuals and those with AIS. RNA samples were extracted from osteoblasts, purified, fluorescently labelled, and then hybridised to gene expression microarrays with the Illumina expression BeadChips technology containing more than 46 000 probes for the human genome (HumanHT-12). Data analysis in R version 2.10.1 (Bioconductor packages oligo and limma) was done, and genes that had at least 1·5-fold change in expression were considered differentially regulated relative to controls. AIS candidate genes within the critical intervals were selected on the basis of their mRNA expression in AIS individuals and by their known functions. The coding regions of these candidate genes were then sequenced to identify potential mutations. The biological activity of mutant proteins is under evaluation by in-vivo functional studies in zebrafish. Results. In the AIS family, a maximum LOD score of 3·01 was reached on two specific chromosomal regions. The interval lengths of these regions were 7cM and 12cM. These two regions contain several genes that might be responsible for AIS. Microarray analysis showed many genes that are differentially regulated in AIS osteoblasts compared with control osteoblasts. We recorded that 2·6% of the 24000 genes examined were upregulated in AIS osteoblasts, whereas 2·16% of them were downregulated. We observed a roughly 3-fold increase or decrease in the transcripts of many genes in AIS osteoblasts. Some of the differentially regulated genes are located within the two chromosomal candidate regions. The sequencing of the candidate genes' coding sequences was done on the family members. Sequence analysis showed two rare SNPs located on the coding regions of a gene that we called CH5G1. These two SNPs are located on the C-terminal region of the CH5G1 protein and affect its structure and probably its cellular activity and biological process leading to the disease. The C-terminal region of this protein interacts with the mRNA of a gene whose defects cause scoliosis as a secondary phenotype. The pathogenic nature of these SNPs is being investigated in the zebrafish model. The results suggest that CH5G1 gene's defects could be associated with AIS in this family. Conclusions. Identification of susceptibility genes for AIS will facilitate the understanding of underlying biochemical pathways (functional studies) and ultimately the development of specific therapies (pharmacological studies). This is likely to have important implications, since the cause of AIS is unknown. Acknowledgments. This study is supported by the Fondation Yves Cotrel, Institut de France


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXVII | Pages 51 - 51
1 Jun 2012
Machida M Dubousset J
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Introduction. Although the association between osteoporosis and adolescent idiopathic scoliosis (AIS) has become widely accepted, the mechanism behind the development of osteoporosis and AIS remains unknown. To elucidate this relationship, we investigated the radiological and histological changes in a model of scoliosis in chickens, focusing on the cervical vertebrae that are not affected by scoliosis. Methods. 40 newly hatched broiler chickens were divided randomly into four equal groups: sham-operated chickens serving as control (CNT); pinealectomised chickens (PNX); and sham-operated (CNT+MLT) and pinealectomized chickens (PNX+MLT) that received intraperitoneal administration of MLT (8 mg/kg) at 2200 h daily. Pinealectomies were done at the age of 3 days. Before killing the chickens at 2 months of age, blood samples were collected at midnight and MLT concentrations were measured by radioimmunoassay. Post-mortem radiographs were examined for the presence of scoliosis, and microcomputed tomography (micro-CT) images were taken to assess the microstructure of the cervical vertebrae. Histological specimens of the scanned cervical vertebra were prepared, and a mid-sagittal section was stained with haematoxylin and eosin (HE) and tartrate-resistant acid phosphatase (TRAP) to assess the numbers of osteoblasts and osteoclasts, respectively. Results. Scoliosis developed at the thoracic spine in all chickens in the PNX group and in two of the PNX+MLT group. MLT concentrations in the PNX group were substantially reduced, whereas normal concentrations were restored in the PNX+MLT group and were normal in the CNT and CNT+MLT groups. Micro-CT data showed that chickens in the PNX group had a greater degree of generalised osteoporosis than did those in the other groups. The number of osteoblasts was significantly decreased in the PNX group, whereas we recorded no significant difference between the CNT, CNT+MLT, and PNX+MLT groups. The number of osteoclasts was similar in all groups. Conclusions. Our results suggest that MLT deficiency reduces osteoblast proliferation and leads to the development of scoliosis and osteoporosis. The restoration of MLT prevented the development of scoliosis and osteoporosis, indicating that MLT concentrations might be crucial to the development of scoliotic deformity and osteoporosis in AIS


Bone & Joint Research
Vol. 12, Issue 6 | Pages 387 - 396
26 Jun 2023
Xu J Si H Zeng Y Wu Y Zhang S Shen B

Aims

Lumbar spinal stenosis (LSS) is a common skeletal system disease that has been partly attributed to genetic variation. However, the correlation between genetic variation and pathological changes in LSS is insufficient, and it is difficult to provide a reference for the early diagnosis and treatment of the disease.

Methods

We conducted a transcriptome-wide association study (TWAS) of spinal canal stenosis by integrating genome-wide association study summary statistics (including 661 cases and 178,065 controls) derived from Biobank Japan, and pre-computed gene expression weights of skeletal muscle and whole blood implemented in FUSION software. To verify the TWAS results, the candidate genes were furthered compared with messenger RNA (mRNA) expression profiles of LSS to screen for common genes. Finally, Metascape software was used to perform enrichment analysis of the candidate genes and common genes.


Bone & Joint Research
Vol. 10, Issue 5 | Pages 328 - 339
31 May 2021
Jia X Huang G Wang S Long M Tang X Feng D Zhou Q

Aims

Non-coding microRNA (miRNA) in extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) may promote neuronal repair after spinal cord injury (SCI). In this paper we report on the effects of MSC-EV-microRNA-381 (miR-381) in a rodent model of SCI.

Methods

In the current study, the luciferase assay confirmed a binding site of bromodomain-containing protein 4 (BRD4) and Wnt family member 5A (WNT5A). Then we detected expression of miR-381, BRD4, and WNT5A in dorsal root ganglia (DRG) cells treated with MSC-isolated EVs and measured neuron apoptosis in culture by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. A rat model of SCI was established to detect the in vivo effect of miR-381 and MSC-EVs on SCI.


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXVII | Pages 21 - 21
1 Jun 2012
Zaidman A Zaidman MN Mikhailovsky M Suzdalov V Zavyalova EL Sadovoy MA
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Introduction. Type 1 neurofibromatosis is a serious hereditary disease in which mainly skin, nervous, muscular, and bone systems are damaged. In bone systems the most common deformities are thoracic kyphosis and scoliosis. Data for morphological changes in the structural components of spine in neurofibromatosis are scarce. Thus our study aimed to investigate morphological changes in structural components of the spine in NF1 neurofibromatosis. Methods. Growth plates, intervertebral discs, and fragments of vertebral bodies from deformed and adjacent segments of the spine were obtained from 15 patients aged 10–14 years with scoliosis (Cobb angle 90–120°) caused by neurofibromatosis. Preoperative examination included MRI study of the spine and brain to exclude intracanal masses, and radiographic study of the spine. Patients did not present any neurological symptoms. All children underwent anterior release and interbody fusion. Structural spinal components from children aged 12–14 years collected at forensic autopsy were used as controls. Tissues were investigated by conventional histochemical and ultrastructural methods. The levels of aggrecan and NF1 gene expression were studied with the PCR method. Results. The study of growth plate and intervertebral disc specimens removed during surgery for scoliosis in neurofibromatosis showed a clear boundary between their convex and concave sides. Both growth plate and intervertebral disc in convex side retain their architectonic and histochemical characteristics. The concave side of the growth plate is presented by small chondroblasts densely spaced without a definite orientation and surrounded by homogeneous matrix, which is made up of chondroitin sulphates. These embryonic-type chondroblasts are poorly differentiated. Chondroblasts proliferate beyond the growth plate. Proliferating cells invade into vertebral body and are bordered by thin bone lamellae, causing the scalloping of vertebral body as a radiological symptom of the pathology. Changes occurring in the intervertebral disc are of considerable interest. Concave-side disc is characterised by isolated proliferation zones containing poorly differentiated chondroblasts and fibroblasts, and neurinoma-like masses. Bone trabeculae inside a concave-side vertebra are passing the stage of osteogenesis imperfecta. Detected morphological changes in spinal structures are consistent with findings of Stevenson, who registered cartilage and bone deficiencies in animal model (mice with NF1 genemutation). Thus, morphological studies testify to structural disorder in concave side of the growth plate, but unchanged regularities and stages of chondroblast differentiation and adequate osteogenesis in the convex side. NF1 gene regulates the growth, differentiation, and proliferation of chondroblasts at the early stage of embryogenesis. Gene inactivation at a somite stage results in altered development of definitive spinal structures. Continued growth with adequate proliferation, differentiation, osteogenesis, and topochemical characteristics occurs in the convex-side growth plate, and growth disorder in the concave-side part with continued load cause growth asymmetry and development of spinal deformity. Scoliosis associated with neurofibromatosis is notable for deformity progression and pseudoarthrosis development after surgery. Deformity progression (modulation) should be considered in connection with disorder in osteogenic potency of osteoblasts. Conclusions. The causal factor of spinal deformity development in NF1 neurofibromatosis is NF1 gene mutation. Inactivation of NF1 gene results in disorder in chondrogenesis and osteogenesis within structurally altered zones. A continued load causes development of scoliotic spinal deformity


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXVII | Pages 48 - 48
1 Jun 2012
Moreau A Yuan Q Akoume M Karam N Taheri M Bouhanik S Rompre P Bagnall K Labelle H Poitras B Rivard C Grimard G Parent S
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Introduction. From the many human studies that attempt to identify genes for adolescent idiopathic scoliosis (AIS), the view emerging is that AIS is a complex genetic disorder with many predisposing genes exhibiting complex phenotypes through environmental interactions. Although advancements in genomic technology are transforming how we undertake genetic and genomic studies, only some success has been reached in deciphering complex diseases such as AIS. Moreover, the present challenge in AIS research is to understand the causative and correlative effects of discovered genetic perturbations. An important limitation to such investigations has been the absence of a method that can easily stratify patients with AIS. To overcome these challenges, we have developed a functional test that allows us to stratify patients with AIS into three functional subgroups, representing specific endophenotypes. Interestingly, in families with multiple cases of AIS, a specific endophenotype is shared among the affected family members, indicating that such a transmission is inherited. Moreover, increased vulnerability to AIS could be attributable to sustained exposure to osteopontin (OPN), a multifunctional cytokine that appears to be at the origin of the Gi-coupled receptor signalling dysfunction discovered in AIS. We examined the molecular expression profiles of patients with AIS and their response to OPN. Methods. Osteoblasts isolated from patients with AIS were selected for each functional subgroup and compared with osteoblasts obtained from healthy matched controls. We used the latest gene chip human genome array Affymetrix (HuU133 Plus 2.0 array) that allows for the analysis of the expression level of 38 000 well characterised human genes. Raw data were normalised with robust multiarray analysis method. Statistical analysis was done by the EB method with FlexArray software. Selection criteria for in-depth analysis include the magnitude of change in expression (at least □} 3-fold) and 5% false discovery rate as stringency selection. Validation of selected candidate genes was done by qPCR and at the protein level by Western blot and ELISA methods. Plasma OPN concentrations were measured by ELISA on a group of 683 consecutive patients with AIS and were compared with 262 healthy controls and 178 asymptomatic offspring, born from at least one scoliotic parent, and thus considered at risk of developing the disorder. The regulation of OPN signalling pathway in normal and AIS cells were validated in vitro by cellular dielectric spectroscopy (CDS). Results. Of 38 000 human genes tested, we have found eight genes specifically associated with the functional subgroup 1, 16 genes with the functional subgroup 2, and 11 genes with the functional subgroup 3. Interestingly, only 19 genes were shared and affected to the same extent in all AIS functional subgroups exhibiting a similar curve pattern (double major), suggesting their role in the formation of this curve pattern. Indeed, most of these genes encode for regulatory proteins such as transcription factors regulating axial skeleton, somite development, and extracellular matrix proteins. Mean plasma OPN concentrations were significantly increased in patients with AIS and correlated with disease severity. Increased plasma OPN concentrations were also detected in the asymptomatic at-risk group, suggesting that these changes precede scoliosis onset. CDS experiments clearly showed that OPN exposure triggers a Gi-coupled receptor signalling dysfunction, which is exacerbated by oestrogens. Conclusions. Our data further support our functional method of stratification of patients with AIS and allow the identification of genes triggering scoliosis onset versus those predisposing to the development of a specific curve pattern. Furthermore, our clinical and experimental data show that OPN is essential for scoliosis onset and curve progression, thus offering a first molecular concept to explain the pathomechanism leading to the asymmetrical growth of the spine in AIS. Acknowledgments. This research project was supported by grants from La Fondation Yves Cotrel de l'Institut de France, Canadian Institutes of Health Research, and Paradigm Spine LLC


Bone & Joint Research
Vol. 7, Issue 2 | Pages 124 - 130
1 Feb 2018
Coric D Bullard DE Patel VV Ryaby JT Atkinson BL He D Guyer RD

Objectives

Pulsed electromagnetic field (PEMF) stimulation was evaluated after anterior cervical discectomy and fusion (ACDF) procedures in a randomized, controlled clinical study performed for United States Food and Drug Administration (FDA) approval. PEMF significantly increased fusion rates at six months, but 12-month fusion outcomes for subjects at elevated risk for pseudoarthrosis were not thoroughly reported. The objective of the current study was to evaluate the effect of PEMF treatment on subjects at increased risk for pseudoarthrosis after ACDF procedures.

Methods

Two evaluations were performed that compared fusion rates between PEMF stimulation and a historical control (160 subjects) from the FDA investigational device exemption (IDE) study: a post hoc (PH) analysis of high-risk subjects from the FDA study (PH PEMF); and a multicentre, open-label (OL) study consisting of 274 subjects treated with PEMF (OL PEMF). Fisher’s exact test and multivariate logistic regression was used to compare fusion rates between PEMF-treated subjects and historical controls.


The Bone & Joint Journal
Vol. 100-B, Issue 5 | Pages 617 - 621
1 May 2018
Uehara M Takahashi J Ikegami S Kuraishi S Fukui D Imamura H Okada K Kato H

Aims

Although we often encounter patients with an aortic aneurysm who also have diffuse idiopathic skeletal hyperostosis (DISH), there are no reports to date of an association between these two conditions and the pathogenesis of DISH remains unknown. This study therefore evaluated the prevalence of DISH in patients with a thoracic aortic aneurysm (AA).

Patients and Methods

The medical records of 298 patients who underwent CT scans for a diagnosis of an AA or following high-energy trauma were retrospectively examined. A total of 204 patients underwent surgery for an AA and 94 had a high-energy injury and formed the non-AA group. The prevalence of DISH was assessed on CT scans of the chest and abdomen and the relationship between DISH and AA by comparison between the AA and non-AA groups.