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Heliyon Mar 2024Chondrocyte death is the hallmark of cartilage degeneration during osteoarthritis (OA). However, the specific pathogenesis of cell death in OA chondrocytes has not been...
OBJECTIVE
Chondrocyte death is the hallmark of cartilage degeneration during osteoarthritis (OA). However, the specific pathogenesis of cell death in OA chondrocytes has not been elucidated. This study aims to validate the role of CDKN1A, a key programmed cell death (PCD)-related gene, in chondrogenic differentiation using a combination of single-cell and bulk sequencing approaches.
DESIGN
OA-related RNA-seq data (GSE114007, GSE55235, GSE152805) were downloaded from Gene Expression Omnibus database. PCD-related genes were obtained from GeneCards database. RNA-seq was performed to annotate the cell types in OA and control samples. Differentially expressed genes (DEGs) among those cell types (scRNA-DEGs) were screened. A nomogram of OA was constructed based on the featured genes, and potential drugs targeting the featured genes were predicted. The presence of key genes was confirmed using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR), Western blot (WB), and immunohistochemistry (IHC). Micromass culture and Alcian blue staining were used to determine the effect of CDKN1A on chondrogenesis.
RESULTS
Six cell types, namely HomC, HTC, RepC, preFC, FC, and RegC, were annotated in scRNA-seq data. Five featured genes ( and were screened by multiple biological information analysis methods. TAXOTERE had the highest ability to dock with DDIT3. Functional analysis indicated that CDKN1A was enriched in processes related to collagen catabolism and acts as a positive regulator of autophagy. Additionally, CDKN1A was found to be associated with several KEGG pathways, including those involved in acute myeloid leukemia and autoimmune thyroid disease. CDKN1A was confirmed down-regulated in the joint tissues of OA mouse model and OA model cell. Inhibiting the expression of CDKN1A can significantly suppress the differentiation of OA chondrocytes.
CONCLUSION
Our findings highlight the critical role of CDKN1A in promoting cartilage formation in both and and suggest its potential as a therapeutic target for OA treatment.
PubMed: 38463824
DOI: 10.1016/j.heliyon.2024.e27466 -
Osteoarthritis and Cartilage Open Jun 2024Although type II collagen could have marked potential for developing cartilage tissue engineering (CTE) scaffolds, its erratic supply and viscous nature have limited...
OBJECTIVE
Although type II collagen could have marked potential for developing cartilage tissue engineering (CTE) scaffolds, its erratic supply and viscous nature have limited these studies, and there are no studies on the use of marine-derived type II collagen fibrils for CTE scaffold materials. In this study, we aimed to generate a fibril-based, thin-layered scaffold from marine-derived type II collagen and investigate its chondrogenic potential.
METHODS
Time-lapse observations revealed the cell adhesion process. The Cell Counting Kit-8 (CCK-8) assay, light microscopy, and scanning electron microscopy were performed to detect proliferation and filopodium morphology. Alcian blue staining was used to show the deposition of extracellular secretions, and qRT-PCR was performed to reveal the expression levels of chondrogenesis-related genes.
RESULTS
The cell adhesion speed was similar in both fibril-coated and control molecule-coated groups, but the cellular morphology, proliferation, and chondrogenesis activity differed. On fibrils, more elongated finer filopodia showed inter-cell communications, whereas the slower proliferation suggested an altered cell cycle. Extracellular secretions occurred before day 14 and continued until day 28 on fibrils, and on fibrils, the expression of the chondrogenesis-related genes (), (), (), and ( = 0.0049) was significantly upregulated on day 21.
CONCLUSION
Marine-derived type II collagen was, for the first time, fabricated into a fibril state. It showed rapid cellular affinity and induced chondrogenesis with extracellular secretions. We presented a new model for studying chondrogenesis and a potential alternative material for cell-laden CTE research.
PubMed: 38444516
DOI: 10.1016/j.ocarto.2024.100450 -
Diabetes, Metabolic Syndrome and... 2024Obesity is a growing global problem that causes various complications such as diabetes, cognitive dysfunction, cardiovascular diseases, and hepatobiliary disease....
BACKGROUND
Obesity is a growing global problem that causes various complications such as diabetes, cognitive dysfunction, cardiovascular diseases, and hepatobiliary disease. Alpha-linolenic acid (ALA) has been reported to exhibit multiple pharmaceutical effects. This study aimed to explore the effects of ALA on obesity-induced adipose tissue accumulation, cognitive impairment, inflammation, and colonic mucosal barrier integrity.
METHODS
Mice were fed with high-fat diet (HFD) and were treated with ALA (60 or 100 mg/kg). Body weight, adipose tissue, serum glucose and lipid levels, glucose resistance, and insulin resistance were measured. Cognitive ability was analyzed using the behavior tests. PTP1B and IRS/p-AKT/p-GSK3β/p-Tau signaling were examined to evaluate inflammation and synaptogenesis. Colon mucosal barrier integrity was examined by Alcian blue staining and expression of the tight junction proteins. The production of pro-inflammatory cytokines and liver damages were evaluated. 3T3-L1 cells were used for in vitro experiments. Cell viability, migration and invasion were detected. The levels of ROS, iron, and ferrous ions were measured to assess ferroptosis. Metabolomic analysis of adipose tissues was performed.
RESULTS
ALA treatment prevented HFD-induced adipose tissue accumulation, improved glucose and lipid homeostasis and metabolism. Administration of ALA repressed the HFD-induced increase in insulin levels and insulin resistance index. Serum and colon levels of pro-inflammatory cytokines were decreased after ALA treatment. ALA elevated mitochondrial content in brown adipose tissues. ALA ameliorated obesity-induced cognitive impairment and hippocampal inflammation, enhanced colon mucosa integrity. ALA treatment ameliorated HFD-induced liver damage and lipid accumulation and inhibited differentiation of preadipocyte 3T3-L1 cells into mature adipocytes and induces ferroptosis. Metabolomic analysis suggested that ALA may target the glycerolipid metabolism pathway to ameliorate obesity. Knockdown of AGPAT2 abolished the protective effects of ALA.
CONCLUSION
ALA treatment suppressed adipose accumulation in adipocytes, improved cognitive ability and colon integrity, and alleviated liver damage by modulating the 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2).
PubMed: 38435630
DOI: 10.2147/DMSO.S434671 -
Cureus Jan 2024Acinic cell carcinoma (ACC) is an exceedingly rare type of triple-negative breast cancer (TNBC). We are reporting a case of a 46-year-old female patient who presented...
Acinic cell carcinoma (ACC) is an exceedingly rare type of triple-negative breast cancer (TNBC). We are reporting a case of a 46-year-old female patient who presented with a palpable lump in her left breast not associated with pain, pruritis, or change of skin color. An open biopsy revealed a mass of about 20 x 25 mm of fleshy, white tan with a lobular configuration and necrosis. The histopathological examination revealed cells with cytoplasmic granularity arranged in a microglandular pattern and a solid pattern, and the case was initially reported as ACC. The most remarkable feature was the presence of small and large, brightly eosinophilic cytoplasmic granules, and some cells are clear or multivacuolated, resembling lipoblasts. Cellular pleomorphism and anaplasia are very mild, and the mitotic activity was very low. The tumor showed a scant and vascularized stroma in the area of hyalinization. Small clusters of lymphoid infiltration in the stroma were seen. Histochemical stains revealed that the acinar cells in ACC contain abundant diastase-resistant, periodic acid Schiff (PAS)-positive cytoplasmic granules. Mucicarmine and Alcian blue were negative. The immunohistochemistry workup revealed that the case was positive for discovered on gastrointestinal stromal tumors-1 (DOG-1) and the positivity pattern ranged from apical membranous, cytoplasmic, and complete membranous. In addition, the tumor cells were positive for low-molecular-weight cytokeratin, carcinoembryonic antigen (CEA), and epithelial membrane antigen (EMA). The FISH workup for the ETV6-NTRK3 fusion was negative, arguing against secretory carcinoma (SC). A diagnosis of acinar cell carcinoma of the breast is very rare, and the presence of cytoplasmic granules is helpful for its diagnosis. In the absence of these granules, the diagnosis is very difficult, and other diagnoses will be put in the differential diagnosis, particularly SC, lactating adenosis, and microglandular adenosis. Immunohistochemical and histochemical stains and genetic workups will support the diagnosis of ACC.
PubMed: 38298310
DOI: 10.7759/cureus.51427 -
Journal of Orthopaedic Translation Jan 2024Mutations in Slc26a2 cause a spectrum of autosomal-recessive chondrodysplasia with a significant and negligible influence on the quality of life. It has been reported...
BACKGROUND
Mutations in Slc26a2 cause a spectrum of autosomal-recessive chondrodysplasia with a significant and negligible influence on the quality of life. It has been reported that Slc26a2 deficiency triggers the ATF6 branch of the UPR, which may, in turn, activate the negative regulator of the FGFR3 signaling pathway. However, the correlation between the deletion of Slc26a2 and the augmentation of downstream phosphorylation of FGFR3 has not been investigated .
METHODS
First, we constructed and double knockout mouse lines and observed gross views of the born mice and histological staining of the tibial growth plates. The second approach was to construct tamoxifen-inducible mouse models to replicate SLC26A2-related non-lethal dysplastic conditions. Pharmacological intervention was performed by administering the FGFR3 inhibitor NVP-BGJ398. The effect of NVP-BGJ398 on chondrocytes was assessed by Alcian blue staining, proliferation, apoptosis, and chondrocyte-specific markers and then verified by western blotting for variations in the downstream markers of FGFR3. The growth process was detected using X-rays, micro-CT examination, histomorphometry staining of growth plates, and immunofluorescence.
RESULTS
Genetic ablation of in embryonic -deficient chondrocytes slightly attenuated chondrodysplasia. Subsequently, in the constructed mild dysplasia model, we found that postnatal intervention with gene in -deficient chondrocytes partially alleviated chondrodysplasia. In chondrocyte assays, NVP-BGJ398 suppressed the defective phenotype of -deficient chondrocytes and restored the phosphorylation downstream of FGFR3 in a concentration-dependent manner. In addition, experiments showed significant alleviation of impaired chondrocyte differentiation, and micro-CT analysis showed a clear improvement in trabecular bone microarchitectural parameters.
CONCLUSION
Our results suggested that inhibition of FGFR3 signaling pathway overactivation and NVP-BGJ398 has promising therapeutic implications for the development of SLC26A2-related skeletal diseases in humans.
THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE
Our data provide genetic and pharmacological evidence that targeting FGFR3 signaling via NVP-BGJ398 could be a route for the treatment of SLC26A2-associated skeletal disorders, which promisingly advances translational applications and therapeutic development.
PubMed: 38282752
DOI: 10.1016/j.jot.2023.09.003 -
JOR Spine Mar 2024Intradiscal transplantation of mesenchymal stromal cells (MSCs) has emerged as a promising therapy for intervertebral disc degeneration (IDD). However, the hostile...
BACKGROUND
Intradiscal transplantation of mesenchymal stromal cells (MSCs) has emerged as a promising therapy for intervertebral disc degeneration (IDD). However, the hostile microenvironment of the intervertebral disc (IVD) may compromise the survival of implanted cells. Interestingly, studies reported that paracrine factors, such as extracellular vesicles (EVs) released by MSCs, may regenerate the IVD. The aim of this study was to investigate the therapeutic effects of Wharton's Jelly MSC (WJ-MSC)-derived EVs on human nucleus pulposus cells (hNPCs) using an in vitro 3D alginate-bead culture model.
METHODS
After EV isolation and characterization, hNPCs isolated from surgical specimens were encapsulated in alginate beads and treated with 10, 50, and 100 μg/mL WJ-MSC-EVs. Cell proliferation and viability were assessed by flow cytometry and live/dead staining. Nitrite and glycosaminoglycan (GAG) content was evaluated through Griess and 1,9-dimethylmethylene blue assays. hNPCs in alginate beads were paraffin-embedded and stained for histological analysis (hematoxylin-eosin and Alcian blue) to assess extracellular matrix (ECM) composition. Gene expression levels of catabolic (), anabolic (), and hNPC marker () genes were analyzed through qPCR. Collagen type I and type II content was assessed with Western blot analysis.
RESULTS
Treatment with WJ-MSC-EVs resulted in an increase in cell content and a decrease in cell death in degenerated hNPCs. Nitrite production was drastically reduced by EV treatment compared to the control. Furthermore, proteoglycan content was enhanced and confirmed by Alcian blue histological staining. EV stimulation attenuated ECM degradation and inflammation by suppressing catabolic and inflammatory gene expression levels. Additionally, NPC phenotypic marker genes were also maintained by the EV treatment.
CONCLUSIONS
WJ-MSC-derived EVs ameliorated hNPC growth and viability, and attenuated ECM degradation and oxidative stress, offering new opportunities for IVD regeneration as an attractive alternative strategy to cell therapy, which may be jeopardized by the harsh microenvironment of the IVD.
PubMed: 38222813
DOI: 10.1002/jsp2.1274 -
Cureus Jan 2024Background The human vertebral column generates movements under versatile, dynamic loads. Understanding how the spine reacts to these movements and loads is crucial for...
Background The human vertebral column generates movements under versatile, dynamic loads. Understanding how the spine reacts to these movements and loads is crucial for developing new spine implants and surgical treatments for intervertebral disc injuries. Mechanically uni-axial compression models have been extensively studied. However, the spine's daily loading is not limited to compression, so it is crucial to measure its behavior in all movements (flexion-extension, rotation, and axial compression). Methods This study utilized L1-L5 segments from 19 healthy adult sheep spines. The L2-L3 disc of the first spine underwent only histological evaluation without biomechanical testing to define basic histological parameters. The remaining 18 were divided into three groups of six and subjected to biomechanical tests. Different mechanisms for three groups of spinal segments were prepared, and tests were performed on Shimadzu AG-IS 10 KN (Universal Drawing Press, Kyoto, Japan). An axial load (800 N) was applied to the first group, an axial load with 15 degrees of flexion to the second group, and an axial load with 10 degrees of rotation plus 15 degrees of flexion to the third group. A biomechanical evaluation of the maximum elongation amounts (MEAs) was performed and compared between the groups. Then, the L2-L3 discs were removed from the sheep spines, and a histological examination of the discs was conducted using Hematoxylin-Eosin (HE), Alcian Blue (AB), and Masson's Trichrome (MT) staining. Results The mean MEA ± Standard Deviation (Range) was 1.39 ± 0.38 (0.91-1.94) for Group 1, 2.02 ± 0.75 (0.91-3.01) for Group 2, and 2.47 ± 1.09 (0.64-3.9) for Group 3. Biomechanically, although MEAs increased from Group 1 to Group 3 (meaning that the mean MEAs increased as the number of types of applied force increased), there was no statistically significant difference between the groups regarding the MEAs (P = 0.092). Histologically, no significant differences were observed between all groups after HE staining. In all groups, hypercellularity, edema in the connective tissue, separation between tissue layers, delamination, and signs of swelling and necrosis in the cells were observed similarly. For the AB staining, there was a decrease in the glycosaminoglycan (GAG) structure in the tissue samples compared to the control tissue, but no significant differences were observed between the groups. However, it was observed that the stratification in Group 3 was slightly more deteriorated than in the other groups. For the MT staining, collagen structure deterioration was observed in all groups. It was observed that the amount of collagen was significantly reduced compared to the control tissue. Conclusion As a result, when the axial load is applied biomechanically, there is more displacement of the vertebral discs in Group 3 with multidimensional movements. Furthermore, histological studies revealed deterioration between tissue layers when exposed to complex movements, and the degradation of stratification in group 3 compared to other loading combinations in groups 2 and 3 may indicate the role of complex loads in the formation of disc herniation.
PubMed: 38196992
DOI: 10.7759/cureus.51941 -
Orthopaedic Journal of Sports Medicine Jan 2024Bone-tendon injury is characterized by poor self-healing. It is established that exosomes are favorable for tissue repair and regeneration. However, their effect on...
BACKGROUND
Bone-tendon injury is characterized by poor self-healing. It is established that exosomes are favorable for tissue repair and regeneration. However, their effect on bone-tendon healing has not yet been determined.
PURPOSE
To compare the effectiveness of exosomes derived from adipose-derived mesenchymal stromal cells (ADSC-Exos) and bone marrow-derived mesenchymal stromal cells (BMSC-Exos) on bone-tendon interface healing in murine rotator cuff injury model and explore the underlying mechanisms thereof.
STUDY DESIGN
Controlled laboratory study.
METHODS
A total of 63 male C57BL6 mice with rotator cuff injuries underwent surgery and were randomly assigned to a control group treated without exosomes (n = 21), an ADSC-Exos group (n = 21), or a BMSC-Exos group (n = 21). The mice were sacrificed 4 or 8 weeks after surgery, and tissues were collected for histologic examination and radiographic and biomechanical testing. For exosome tracing in vivo, mice were sacrificed 7 days after surgery. A series of functional assays (radiographic evaluation, proliferation assay, Alizarin Red staining, alkaline phosphatase staining and activity, Alcian blue staining, quantitative polymerase chain reaction analyses, and glycosaminoglycans quantification) were conducted to evaluate the effect of exosomes on the cellular behaviors of the BMSCs in vitro. A statistical analysis of multiple-group comparisons was performed by 1-way analysis of variance, followed by the Bonferroni post hoc test to assess the differences between the 2 groups.
RESULTS
The ADSCs and BMSCs were positive for surface markers CD29 and CD90 and negative for surface markers CD34 and CD45 and could differentiate into osteoblasts, chondrocytes, and adipocytes. Exosomes showed a cup- or sphere-shaped morphology and were positive for CD63 and TGS101. Local injection of ADSC-Exos and BMSC-Exos could recruit BMSCs and promote osteogenesis, chondrogenesis, and bone-tendon healing. In vitro, ADSC-Exos and BMSC-Exos could significantly promote the proliferation, migration, osteogenic differentiation, and chondrogenic differentiation ability of BMSCs. In vivo, ADSC-Exos and BMSC-Exos significantly accelerated bone-tendon injury healing, with no significant statistical difference between them.
CONCLUSION
ADSC-Exos and BMSC-Exos exhibited similar therapeutic effects on bone-tendon healing in our murine animal model.
CLINICAL RELEVANCE
ADSC-Exos and BMSC-Exos may be used to develop a new cell-free therapy method for promoting rotator cuff injury repair.
PubMed: 38188618
DOI: 10.1177/23259671231210304 -
Journal of Orthopaedic Translation Jan 2024Hip osteoarthritis (OA) involves structural degeneration of different joint compartments, including femoral head cartilage, periarticular ligaments and the acetabular...
Elevated secretion of pro-collagen I-alpha and vascular endothelial growth factor as biomarkers of acetabular labrum degeneration and calcification in hip osteoarthritis: An explant study.
BACKGROUND
Hip osteoarthritis (OA) involves structural degeneration of different joint compartments, including femoral head cartilage, periarticular ligaments and the acetabular labrum. However, the molecular mechanisms underlying labrum degeneration in hip OA remain poorly understood.
AIM
To assess secretion of putative biomarkers for OA from explanted human labrum tissues under basal and inflammatory conditions and to determine whether these could differentiate between OA and calcification status compared to fracture controls.
METHODS
Intact labrum specimens were collected from patients undergoing joint arthroplasty for primary hip OA ( = 15, mean age 70) or non-OA femoral neck fracture ( = 5, mean age 64). Tissues were dissected in equal-sized samples and explanted for one week. To mimic activation of inflammatory signaling by endogenous damage-associated molecular patterns (DAMP) tissue were stimulated with a toll-like receptor 4 (TLR4) agonist (1 μg/mL LPS). The involvement of transforming growth factor-beta (TGF-beta) signaling was evaluated by treatment with a TGF-beta type 1 receptor inhibitor (10 μM SB-505124). Secretion of aggrecan (ACAN), pro-collagen-I alpha (Pro-Col-Iα), cartilage oligomeric matrix protein (COMP), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) was assessed by enzyme-linked immunosorbent assay (ELISA). Labrum calcification was evaluated by 3D whole mount fluorescent microscopy of ethyl cinnamate-based optically cleared tissues stained with Alcian blue/Alizarin red.
RESULTS
Whole mount microscopy revealed non-OA fracture controls were non-calcified, whereas six OA labra (40%) were partially calcified or ossified. Basal secretion of Pro-Col-Iα and VEGF was increased four-fold in OA versus non-OA labra. Pro-Col-Iα levels were correlated with those of VEGF ( = 0.65) and COMP ( = 0.54). Stimulation of DAMP signaling through TLR4 affected secretion of IL-6, VEGF, COMP and Pro-Col-Iα, with distinct responses between non-OA and OA tissues. Inhibition of TGF-beta signaling specifically reduced elevated secretion of Pro-Col- Iα and VEGF in calcified OA labrum.
CONCLUSIONS
Secretion of the putative OA biomarkers Pro-Col-Iα and VEGF is elevated in degenerated human acetabular labrum and may serve as indicators of OA and calcification status. Secretion of both factors was partially regulated by TGF-beta signaling in calcified OA labrum tissues.Our findings suggest that a biomarker panel consisting of Pro-Col-Iα/VEGF/COMP may be valuable for assessing subradiographic labrum degeneration and calcification in hip OA. Targeting TGF-beta signaling may offer a means to reduce vascular invasion and fibrosis in acetabular labrum tissue.
PubMed: 38179125
DOI: 10.1016/j.jot.2023.08.007 -
Journal of Orthopaedic Surgery and... Jan 2024Osteoarthritis is a chronic disease mainly involving the damage of articular cartilage and the whole articular tissue, which is the main cause of disability in the...
BACKGROUND
Osteoarthritis is a chronic disease mainly involving the damage of articular cartilage and the whole articular tissue, which is the main cause of disability in the elderly. To explore more effective treatment measures, this study analyzed the regulatory role and molecular mechanism of lncRNA LINC00665 (LINC00665) in the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), providing a valuable theoretical basis for the pathogenesis and patient treatment of osteoarthritis.
METHODS
Osteoarthritis tissues and healthy tissues were obtained from 52 patients with osteoarthritis and 34 amputated patients without osteoarthritis, and the levels of LINC00665 and miR-214-3p were assessed by RT-qPCR. BMSCs were cultured and induced chondrogenic differentiation. The proliferation ability of BMSCs was detected by CCK-8 method, and the apoptosis level of BMSCs was evaluated by flow cytometry. The content of proteoglycan-glycosaminoglycan (GAG) in cartilage matrix was determined by Alcian blue staining. In addition, the binding relationship between LINC00665 and miR-214-3p was verified by luciferase reporter assay, and the molecular mechanism was further analyzed.
RESULTS
In osteoarthritis tissues, LINC00665 was elevated and miR-214-3p was down-regulated. With the chondrogenic differentiation of BMSCs, the level of GAG increased, and LINC00665 expression gradually decreased, while miR-214-3p level was on the contrary. After transfection of pcDNA3.1-LINC00665 in BMSCs, cell proliferation capacity was decreased, apoptosis rate was increased, and GAG content was reduced. Moreover, LINC00665 sponged miR-214-3p and negatively regulate its expression. Transfection of pcDNA3.1-LINC00665-miR-214-3p mimic changed the regulation of pcDNA3.1-LINC00665 on the viability and chondrogenic differentiation of BMSCs.
CONCLUSIONS
Overexpression of lncRNA LINC00665 inhibited the proliferation and chondrogenic differentiation of BMSCs by targeting miR-214-3p. The LINC00665/miR-214-3p axis may improve joint damage and alleviate the progression of osteoarthritis.
Topics: Humans; Aged; MicroRNAs; RNA, Long Noncoding; Chondrocytes; Cell Differentiation; Mesenchymal Stem Cells; Cartilage, Articular; Cells, Cultured; Osteoarthritis; Cell Proliferation; Bone Marrow Cells
PubMed: 38167456
DOI: 10.1186/s13018-023-04475-0