scholarly journals OP0199 EX VIVO BIOMARKER PROFILING IDENTIFIES ONCOSTATIN-M AS SPINE OSTEOARTHRITIS-SPECIFIC OSTEOIMMUNOLOGICAL TARGET

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 120.2-120
Author(s):  
O. Roessinger ◽  
M. Blanchard ◽  
M. Briki ◽  
D. Jurić ◽  
C. Netzer ◽  
...  
Keyword(s):  
Facet Joint ◽  
Ex Vivo ◽  
Oncostatin M ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Type I ◽  
Tgf Beta ◽  
Disease Heterogeneity ◽  
Knee Oa ◽  
Cartilage Proteoglycans

Background:Disease heterogeneity, both clinically and molecularly, has been a major hurdle in the development of efficacious disease-modifying osteoarthritis drugs (DMOADs). Biomechanical, inflammatory, osteoporotic and metabolic OA have been proposed as clinically relevant subtypes for stratification of knee OA patients, yet this remains to be included in clinical trial design. Disease heterogeneity does not only occur within, but also between joint types. However, robust data on joint-specific pathomechanisms of OA are still lacking.Objectives:In this study, we performed ex vivo biomarker profiling of human osteochondral tissue of knee and spine OA to identify joint-specific pathomechanisms and DMOAD treatment responses.Methods:Facet joint and tibial plateaus were obtained from patients undergoing lumbar spinal fusion (n=11, mean age 72.8) and total joint arthroplasty (n=8, mean age 73.0) respectively. Osteochondral specimens were cut in equal-sized samples (100-300 mg wet weight) and randomly assigned to treatment groups: control (DMSO), inflammation (1 μg/mL LPS) or inflammation + DMOAD (TGF-betatype I receptor inhibitor,10 μM SB-505124). Explant culture was conducted for one week and biomarkers of bone metabolism (Pro-Col-Ia, SOST, OPG, SPP1), inflammation (MCP-1, IL-6, MMP3, OSM, TIMP1, VEGFA) and cartilage metabolism (ACAN, COMP) were determined by ELISA. Normalized biomarker secretion was analysed using clusteranalyses and ANOVA. Cartilage proteoglycans were assessed by whole mount Alcian blue staining. Expression of Oncostatin-M (OSM) and its receptors OSMR and LIFR in joint tissues was assessed by RT-PCR and immunohistochemistry.Results:Clusteranalyses revealed that LPS stimulation increased IL-6 and MCP-1 secretion by both facet joint (FJ) and knee joint (KJ) tissues. Interestingly, Oncostatin-M (OSM) and its downstream mediators MMP3 and TIMP1 were increased in the majority of FJ, but not KJ specimens. Statistical analyses corroborated increased OSM, MMP3 and TIMP1 levels in a spine-specific fashion (Figure).Whole mount Alcian blue staining revealed heterogeneous effects of LPS treatment on cartilage proteoglycans, which was negatively correlated with OSM (r=-0.54) and TIMP1 levels (r=-0.45) – yet poorly associated with ACAN (r=0.19). Inhibition of TGF-beta type I receptor signalling in osteochondral tissues led to a drastic reduction of Pro-Collagen-Ia and IL-6 secretion in both spine and knee OA specimens. Interestingly, DMOAD treatment significantly reduced OSM, TIMP1 and MMP3 levels in FJ specimens only. Vice versa, KJ tissues revealed a specific upregulation of monocyte chemoattractant protein-1 (MCP-1) and osteopontin (SPP1) upon inhibition of TGF-beta signalling. OSM was exclusively expressed in subchondral bone marrow macrophages. Isolated chondrocytes and osteoblasts expressed both LIFR and OSMR, yet intact cartilage only showed OSMR expression, while OSMR and LIFR was expressed in marrow tissueConclusion:Oncostatin-M expression and signalling was uncovered as specific pathomechanism of spine OA. DMOAD treatment effects suggested interplay of OSM and TGF-beta signalling pathways in facet joint osteoarthritis. Known to be predominantly expressed by macrophages and immune cells, OSM may be an important osteoimmunological mediator of tissue damage and remodelling in spine, but not knee OA. This study also highlights the value of ex vivo human tissue models for OA phenotyping and preclinical evaluation of DMOADs.Disclosure of Interests:None declared

scholarly journals Suramin attenuates intervertebral disc degeneration by inhibiting NF-κB signalling pathway

2021 ◽  
Vol 10 (8) ◽  
pp. 498-513
Author(s):  
Zi-Miao Liu ◽  
Cheng-Chang Lu ◽  
Po-Chih Shen ◽  
Shih-Hsiang Chou ◽  
Chia-Lung Shih ◽  
...  
Keyword(s):  
Intervertebral Disc ◽  
Disc Degeneration ◽  
Intervertebral Disc Degeneration ◽  
Inflammatory Responses ◽  
Ex Vivo ◽  
Treatment Options ◽  
Underlying Mechanism ◽  
Mitogen Activated Protein Kinase ◽  
Blue Staining ◽  
Alcian Blue Staining

Aims Interleukin (IL)-1β is one of the major pathogenic regulators during the pathological development of intervertebral disc degeneration (IDD). However, effective treatment options for IDD are limited. Suramin is used to treat African sleeping sickness. This study aimed to investigate the pharmacological effects of suramin on mitigating IDD and to characterize the underlying mechanism. Methods Porcine nucleus pulposus (NP) cells were treated with vehicle, 10 ng/ml IL-1β, 10 μM suramin, or 10 μM suramin plus IL-1β. The expression levels of catabolic and anabolic proteins, proinflammatory cytokines, mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-κB-related signalling molecules were assessed by Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), and immunofluorescence analysis. Flow cytometry was applied to detect apoptotic cells. The ex vivo effects of suramin were examined using IDD organ culture and differentiation was analyzed by Safranin O-Fast green and Alcian blue staining. Results Suramin inhibited IL-1β-induced apoptosis, downregulated matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5, and upregulated collagen 2A (Col2a1) and aggrecan in IL-1β-treated NP cells. IL-1β-induced inflammation, assessed by IL-1β, IL-8, and tumour necrosis factor α (TNF-α) upregulation, was alleviated by suramin treatment. Suramin suppressed IL-1β-mediated proteoglycan depletion and the induction of MMP-3, ADAMTS-4, and pro-inflammatory gene expression in ex vivo experiments. Conclusion Suramin administration represents a novel and effectively therapeutic approach, which could potentially alleviate IDD by reducing extracellular matrix (ECM) deposition and inhibiting apoptosis and inflammatory responses in the NP cells. Cite this article: Bone Joint Res 2021;10(8):498–513.

scholarly journals Use of adipose stem cells and polylactide discs for tissue engineering of the temporomandibular joint disc

2009 ◽  
Vol 7 (42) ◽  
pp. 177-188 ◽  
Author(s):  
Katja Mäenpää ◽  
Ville Ellä ◽  
Jari Mauno ◽  
Minna Kellomäki ◽  
Riitta Suuronen ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Temporomandibular Joint ◽  
Adipose Stem Cells ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Type I ◽  
Mrna Levels ◽  
Chondrogenic Medium ◽  
Tmj Disc

There is currently no suitable replacement for damaged temporomandibular joint (TMJ) discs after discectomy. In the present study, we fabricated bilayer biodegradable polylactide (PLA) discs comprising a non-woven mat of poly(L/D)lactide (P(L/D)LA) 96/4 and a P(L/DL)LA 70/30 membrane plate. The PLA disc was examined in combination with adipose stem cells (ASCs) for tissue engineering of the fibrocartilaginous TMJ disc in vitro . ASCs were cultured in parallel in control and chondrogenic medium for a maximum of six weeks. Relative expression of the genes, aggrecan, type I collagen and type II collagen present in the TMJ disc extracellular matrix increased in the ASC-seeded PLA discs in the chondrogenic medium. The hypertrophic marker, type X collagen, was moderately induced. Alcian blue staining showed accumulation of sulphated glycosaminoglycans. ASC differentiation in the PLA discs was close to that observed in pellet cultures. Comparison of the mRNA levels revealed that the degree of ASC differentiation was lower than that in TMJ disc-derived cells and tissue. The pellet format supported the phenotype of the TMJ disc-derived cells under chondrogenic conditions and also enhanced their hyalinization potential, which is considered part of the TMJ disc degeneration process. Accordingly, the combination of ASCs and PLA discs has potential for the development of a tissue-engineered TMJ disc replacement.

scholarly journals Human osteogenic protein-1 induces both chondroblastic and osteoblastic differentiation of osteoprogenitor cells derived from newborn rat calvaria.

1993 ◽  
Vol 123 (4) ◽  
pp. 921-933 ◽  
Author(s):  
I Asahina ◽  
T K Sampath ◽  
I Nishimura ◽  
P V Hauschka
Keyword(s):  
Alkaline Phosphatase ◽  
Alcian Blue ◽  
Osteoblastic Differentiation ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Tgf Beta ◽  
Newborn Rat ◽  
Osteoprogenitor Cells ◽  
Osteogenic Protein

Osteogenetic protein-1 (OP-1), a member of the TGF-beta superfamily, induces endochondrial bone formation at subcutaneous sites in vivo and stimulates osteoblastic phenotypic expression in vitro. Primary cultures of newborn rat calvarial cells contain a spectrum of osteogenic phenotypes ranging from undifferentiated mesenchymal osteoprogenitor cells to parathyroid hormone (PTH)-responsive osteoblasts. We examined whether treatment of this cell population with recombinant human osteogenic protein-1 could induce chondrogenesis in vitro. Markers of chondroblastic versus osteoblastic differentiation included alcian blue staining at pH 1, alkaline phosphatase-specific activity, osteocalcin radioimmunoassay, and expression of collagen mRNAs. 6 d of treatment (culture days 1-7) with 4-100 ng OP-1/ml caused dose-dependent increases in alcian blue staining intensity and alkaline phosphatase activity (4.7- and 3.4-fold, respectively, at 40 ng/ml), while osteocalcin production decreased twofold. Clusters of round, refractile, alcian blue-stained cells appeared by day 3, increased in number until day 7, and then became hypertrophic and gradually became less distinct. Histochemically, the day 7 clusters were associated with high alkaline phosphatase activity and became mineralized. mRNA transcripts for collagen types II and IX were increased by OP-1, peaking at day 4, while type X collagen mRNA was detectable only on day 7 in OP-1-treated cultures. Delay of OP-1 exposure until confluence (day 7) amplifies expression of the normal osteoblastic phenotype and accelerates its developmental maturation. In contrast, early OP-1 treatment commencing on day 1 strongly amplifies chondroblastic differentiation. In the same protocol, TGF-beta 1 alone at 0.01-40 ng/ml fails to induce any hypertrophic chondrocytes, and in combination with OP-1, TGF-beta 1 blocks OP-1-dependent chondroinduction. OP-1 is believed to act on a subpopulation of primitive osteoprogenitor cells to induce endochondrial ossification, but does not appear to reverse committed osteoblasts to the chondrocyte phenotype.

scholarly journals Histochemistry of Equine Damaged Tendons, Ligaments and Articular Cartilage

2018 ◽  
Vol 46 (1) ◽  
pp. 8
Author(s):  
Gabriela De Bastiani ◽  
Flávio Desessards De La Corte ◽  
Karin Erica Brass ◽  
Camila Cantarelli ◽  
Stefano Dau ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Connective Tissue ◽  
Alcian Blue ◽  
Collagen Type ◽  
Repair Process ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Type Iii Collagen ◽  
Type I ◽  
Loose Connective Tissue

Background: The injury repair process in tendons and ligaments includes different phases such as inflammation, neovascularization, fibroblast proliferation and fibrosis. Collagen type and tissue characteristics of tendon and ligament repair are described such as type collagen differentiation and properties of the scars tissue. The degeneration of articular cartilage when, characterized by loss of the articular layers associated of the decreased of proteoglycans. The aim of this study is to describe by histochemistry techniques the characteristics of tissue scar, collagen type in the repair process of tendons and ligaments, as well as articular cartilage degeneration.Materials, Methods & Results: Tissue samples of equine tendons, ligaments and articular cartilage of the metacarpophalangeal joint region were evaluated by ultrasonography, macroscopically and prepared for routine histopathology (H&E staining). The inclusion criterion of the samples in this study was based on the presence of lesions characterized in H&E stain as fibroplasia, neovascularization, collagenolysis, chondroid metaplasia in tendons and ligaments and fibrillation and cartilaginous eburnation lesions in the articular cartilage samples. The Masson’s trichrome, Picrosirius red and Alcian blue staining techniques were also performed in addition to H&E. Pathologic findings in the tendons and ligaments included fibroplasia, collagenolysis, chondroid metaplasia and lymphohistioplasmacytic inflammation. Tendons and ligaments scars were composed of type III collagen but there was also some type I collagen. Fiber alignment of tendons and ligaments in the reorganization tissue was not flawless and the fiber appearance was characterized by a lack of the fiber crimp and parallelism. The fibroplasia was characterized by endotendinous tickening areas associated with the presence of loose connective tissue. In the areas of loose connective tissue substitution, collagen type fibers are intercalated to a lesser extent by type-III collagen fibers. In the Alcian blue stained samples of articular cartilage observed the surface layer and the matrix zone of calcified cartilage were weakly stained in blue.Discussion: Three special stains were utilized in this study along with the H&E evaluation elucidating the behavior tendons, ligaments and articular cartilage injury. The important observation in this study was fibroplasia in tendons and ligaments seems to be composed by abundant of loose connective tissue, chondrocytes and intermingled collagen type I and III fibers associated with lack of crimps alignment of the fibers. The fragile structure suggested by the Masson’s trichrome stain results (presence of the loose connective tissue) in this study perhaps make the tendons and ligaments receptive to other lesions. The characteristic blue discoloration of collagen fibers was only observed in the loose connective tissue may be because the dye penetration becomes easier when compared to the dense connective tissue (stained in red). The Masson’s trichrome made possible the differentiated the dense connective tissue of the loose connective tissue. The combined histochemistry staining technics allowed an improved characterization of fiber alignment, collagen type, inflammatory cell infiltration and neovascularization, which happens during the repair process of tendons and ligaments. The fibrillation and eburnation of the articular cartilage were associated with the decrease Alcian Blue staining characterized by degeneration process of articular cartilage.

scholarly journals Tissue-Engineering the Fibrous Pancreatic Tumour Stroma Capsule in 3D Tumouroids to Demonstrate Paclitaxel Response

2021 ◽  
Vol 22 (8) ◽  
pp. 4289
Author(s):  
Judith Pape ◽  
Katerina Stamati ◽  
Rawiya Al Hosni ◽  
Ijeoma F. Uchegbu ◽  
Andreas G. Schatzlein ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Drug Testing ◽  
Collagen Type I ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Type I ◽  
Tumour Mass ◽  
Stromal Compartment ◽  
Pancreatic Tumour ◽  
Initial Drug

Pancreatic cancer is a unique cancer in that up to 90% of its tumour mass is composed of a hypovascular and fibrotic stroma. This makes it extremely difficult for chemotherapies to be delivered into the core of the cancer mass. We tissue-engineered a biomimetic 3D pancreatic cancer (“tumouroid”) model comprised of a central artificial cancer mass (ACM), containing MIA Paca-2 cells, surrounded by a fibrotic stromal compartment. This stromal compartment had a higher concentration of collagen type I, fibronectin, laminin, and hyaluronic acid (HA) than the ACM. The incorporation of HA was validated with alcian blue staining. Response to paclitaxel was determined in 2D MIA Paca-2 cell cultures, the ACMs alone, and in simple and complex tumouroids, in order to demonstrate drug sensitivity within pancreatic tumouroids of increasing complexity. The results showed that MIA Paca-2 cells grew into the complex stroma and invaded as cell clusters with a maximum distance of 363.7 µm by day 21. In terms of drug response, the IC50 for paclitaxel for MIA Paca-2 cells increased from 0.819 nM in 2D to 3.02 nM in ACMs and to 5.87 nM and 3.803 nM in simple and complex tumouroids respectively, indicating that drug penetration may be significantly reduced in the latter. The results demonstrate the need for biomimetic models during initial drug testing and evaluation.

TGF-beta type I receptor

2007 ◽  
Author(s):  
Mythreye Karthikeyan ◽  
Gerard Blobe
Keyword(s):  
Type I ◽  
Tgf Beta

Hyperactivation of RAP1 and JAK/STAT Signaling Pathways Contributes to Fibrosis during the Formation of Nasal Capsular Contraction

2021 ◽  
pp. 1-12
Author(s):  
Meng Wu ◽  
Ming Li ◽  
Hong-Ju Xie ◽  
Hong-Wei Liu
Keyword(s):  
Signaling Pathways ◽  
Type I Collagen ◽  
Ex Vivo ◽  
Capsular Contracture ◽  
Silicone Implant ◽  
Type I ◽  
Loss Of Function ◽  
Sequencing Data ◽  
Functional Changes ◽  
Stat Signaling

Silicone implant-based augmentation rhinoplasty or mammoplasty induces capsular contracture, which has been acknowledged as a process that develops an abnormal fibrotic capsule associated with the immune response to allogeneic materials. However, the signaling pathways leading to the nasal fibrosis remain poorly investigated. We aimed to explore the molecular mechanism underlying the pathogenesis of nasal capsular contracture, with a specific research interest in the signaling pathways involved in fibrotic development at the advanced stage of contracture. By examining our recently obtained RNA sequencing data and global gene expression profiling between grade II and grade IV nasal capsular tissues, we found that both the RAP1 and JAK/STAT signaling pathways were hyperactive in the contracted capsules. This was verified on quantitative real-time PCR which demonstrated upregulation of most of the representative component signatures in these pathways. Loss-of-function assays through siRNA-mediated Rap1 silencing and/or small molecule-directed inhibition of JAK/STAT pathway in ex vivo primary nasal fibroblasts caused a series of dramatic behavioral and functional changes, including decreased cell viability, increased apoptosis, reduced secretion of proinflammatory cytokines, and synthesis of type I collagen, compared to control cells, and indicating the essential role of the RAP1 and JAK/STAT signaling pathways in nasal capsular fibrosis. Our results sheds light on targeting downstream signaling pathways for the prevention and therapy of silicone implant-induced nasal capsular contracture.

scholarly journals Numerical and Experimental Investigations on Vocal Fold Approximation in Healthy and Simulated Unilateral Vocal Fold Paralysis

2021 ◽  
Vol 11 (4) ◽  
pp. 1817
Author(s):  
Zheng Li ◽  
Azure Wilson ◽  
Lea Sayce ◽  
Amit Avhad ◽  
Bernard Rousseau ◽  
...  
Keyword(s):  
Computational Model ◽  
Vocal Fold ◽  
High Speed ◽  
Computational Models ◽  
Ex Vivo ◽  
Vocal Fold Paralysis ◽  
Future Application ◽  
Experimental Investigations ◽  
Type I ◽  
Mri Scans

We have developed a novel surgical/computational model for the investigation of unilat-eral vocal fold paralysis (UVFP) which will be used to inform future in silico approaches to improve surgical outcomes in type I thyroplasty. Healthy phonation (HP) was achieved using cricothyroid suture approximation on both sides of the larynx to generate symmetrical vocal fold closure. Following high-speed videoendoscopy (HSV) capture, sutures on the right side of the larynx were removed, partially releasing tension unilaterally and generating asymmetric vocal fold closure characteristic of UVFP (sUVFP condition). HSV revealed symmetric vibration in HP, while in sUVFP the sutured side demonstrated a higher frequency (10–11%). For the computational model, ex vivo magnetic resonance imaging (MRI) scans were captured at three configurations: non-approximated (NA), HP, and sUVFP. A finite-element method (FEM) model was built, in which cartilage displacements from the MRI images were used to prescribe the adduction, and the vocal fold deformation was simulated before the eigenmode calculation. The results showed that the frequency comparison between the two sides was consistent with observations from HSV. This alignment between the surgical and computational models supports the future application of these methods for the investigation of treatment for UVFP.

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