scholarly journals Hyaluronic Acid as a Carrier Supports the Effects of Glucocorticoids and Diminishes the Cytotoxic Effects of Local Anesthetics in Human Articular Chondrocytes In Vitro

2021 ◽  
Vol 22 (21) ◽  
pp. 11503
Author(s):  
Lukas B. Moser ◽  
Christoph Bauer ◽  
Vivek Jeyakumar ◽  
Eugenia-Paulina Niculescu-Morzsa ◽  
Stefan Nehrer

The current study aimed to investigate the cytotoxicity of co-administrating local anesthetics (LA) with glucocorticoids (GC) and hyaluronic acid (HA) in vitro. Human articular cartilage was obtained from five patients undergoing total knee arthroplasty. Chondrocytes were isolated, expanded, and seeded in 24-well plates for experimental testing. LA (lidocaine, bupivacaine, ropivacaine) were administered separately and co-administered with the following substances: GC, HA, and GC/HA. Viability was confirmed by microscopic images, flow cytometry, metabolic activity, and live/dead assay. The addition of HA and GC/HA resulted in enhanced attachment and branched appearance of the chondrocytes compared to LA and LA/GC. Metabolic activity was better in all LA co-administered with HA and GC/HA than with GC and only LA. Flow cytometry revealed the lowest cell viability in lidocaine and the highest cell viability in ropivacaine. This finding was also confirmed by live/dead assay. In conclusion, HA supports the effect of GC and reduces chondrotoxic effects of LA in vitro. Thereby, the co-administration of HA to LA and GC offers an alternative less chondrotoxic approach for treating patients with symptomatic osteoarthritis of the knee.

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Jianghao Gong ◽  
Shangjun Fu ◽  
Zhenghao Zhou

Objective. To explore the effects of silicone gel nanoparticles modified with octacalcium phosphate on the surface (silica/OCP) polymer drugs on the proliferation of osteoblasts and autophagy. Method. Silica/OCP was prepared in vitro, and the quality of the sample preparation was tested through characterization experiments. The osteoblast cell line (hFOB1.19) was treated with silica/OCP, autophagy inhibitor (3-methyladenine (3-MA)), and silica/OCP+3-MA, respectively. The proliferation of hFOB1.19 cells was detected through the methylthiazolyldiphenyl-tetrazolium bromide (MTT) kit. Flow cytometry was used to detect the cell apoptosis. The change in protein beclin1 and P62 expression in hFOB1.19 cells was observed in Western blot. An ROS detection kit was used to detect the content of reactive oxygen species in hFOB1.19 cells. Results. Silica/OCP was a sphere with a particle size of 50 nm to 130 nm and had an OCP phase in electron projection microscopy and X-ray diffraction techniques. The results indicated that OCP successfully modified silica and the material was successfully prepared. An MTT kit and flow cytometry test showed that the cell viability of the cells treated with silica/OCP increased significantly ( P < 0.05 ), and the intracellular apoptosis phenomenon was significantly decreased ( P < 0.05 ) compared to the control group. Moreover, the inhibition of cell viability and promotion of apoptosis caused by the autophagy inhibitor 3-MA can be rescued. Western blotting demonstrated that the protein level of beclin1 in osteoblasts reached the highest after six hours of treatment with silica/OCP, and the protein level of p62, the substrate protein of autophagy, reached the lowest. At the same time, treatment of cells with the autophagy inhibitor 3-MA and silica/OCP+3-MA found that the protein levels of beclin1 and p62 in the silica/OCP+3-MA group were adjusted back compared to the 3-MA group. After adding the autophagy inhibitor, the reactive oxygen content in the cell was significantly increased ( P < 0.05 ) in the silica/OCP group. In the presence of intracellular reactive oxygen inhibitors catalase and silica/OCP, the cell viability of osteoblasts was significantly lower than that of the silica/OCP group but significantly higher than that of the silica/OCP+3-MA group. The apoptosis level of the silica/OCP+catalase group was also significantly lower than that of the silica/OCP+3-MA group ( P < 0.05 ) but was significantly higher than that of the silica/OCP group ( P < 0.05 ). Conclusion. Silica/OCP nanoparticles can upregulate the level of autophagy in osteoblasts and promote the proliferation of osteoblasts.


1982 ◽  
Vol 206 (2) ◽  
pp. 329-341 ◽  
Author(s):  
Charles J. Malemud ◽  
Victor M. Goldberg ◽  
Roland W. Moskowitz ◽  
Lee L. Getzy ◽  
Robert S. Papay ◽  
...  

Proteoglycan biosynthesis by human osteochondrophytic spurs (osteophytes) obtained from osteoarthritic femoral heads at the time of surgical joint replacement was studied under defined culture conditions in vitro. Osteophytes were primarily present in two anatomic locations, marginal and epi-articular. Minced tissue slices were incubated in the presence of [35S]sulphate or [14C]glucosamine. Osteophytes incorporated both labelled precursors into proteoglycan, which was subsequently characterized by CsCl-isopycnic-density-gradient ultracentrifugation and chromatography on Sepharose CL-2B. The material extracted with 0.5m-guanidinium chloride showed 78.1% of [35S]sulphate in the A1 fraction after centrifugation. Only 23.0% of the [35S]sulphate in this A1 fraction was eluted in the void volume of Sepharose CL-2B under associative conditions. About 60–80% of the [35S]sulphate in the tissue 4m-guanidinium chloride extract was associated with monomeric proteoglycan (fraction D1). The average partition coefficient (Kav.) of the proteoglycan monomer on Sepharose CL-2B was 0.28–0.33. Approx. 12.4% of this monomer formed stable aggregates with high-molecular-weight hyaluronic acid in vitro. Sepharose CL-2B chromatography of fractions with lower buoyant densities (fractions D2–D4) demonstrated elution profiles on Sepharose CL-2B substantially different than that of fraction D1, indicative of the polydisperse nature of the newly synthesized proteoglycan. Analysis of the composition and chain size of the glycosaminoglycans showed the following: (1) preferential elution of both [35S]sulphate and [14C]glucosamine in the 0.5m-LiCl fraction on DEAE-cellulose; (2) the predominant sulphated glycosaminoglycan was chondroitin 6-sulphate (60–70%), with 9–11% keratan sulphate in the monomer proteoglycan; (3) Kav. values of 0.38 on Sephadex G-200 and 0.48 on Sepharose CL-6B were obtained with papain-digested and NaBH4-treated D1 monomer respectively. A comparison of the synthetic with endogenous glycosaminoglycans indicated similar types. These studies indicated that human osteophytes synthesized in vitro sulphated proteoglycans with some characteristics similar to those of mature human articular cartilage, notably in the size of their proteoglycan monomer and predominance of chondroitin 6-sulphate. They differed from articular cartilage primarily in the lack of substantial quantities of keratan sulphate and aggregation properties associated with monomer interaction with hyaluronic acid.


Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3825-3825
Author(s):  
Hava Glickstein ◽  
Hanspeter Nick ◽  
Zvi I. Cabantchik

Abstract Systemic iron overload (primary or secondary) affects hepatic and extrahepatic functions by damaging endocrine and cardiac tissue. In vitro studies with pancreatic Min6 and pituitary Att20 cells and with cardiac H9c2 cells (all highly active in endocytotic activity) indicated that their exposure to labile iron, acutely or chronically, lead to major intracellular iron accumulation in organelles (endosomes, mitochondria, cytosol) and increased reactive oxygen species (ROS) formation when redox challenged. Among the functions affected by metal-evoked ROS are permselectivity (calcein leakage), mitochondrial ΔΨ (JC1 test), electron transport activity (Alamar Blue) and cell viability (calcein-propidium iodide). The administration at therapeutically achievable doses of deferasirox (30–100 μM) or deferoxamine (DFO) [10 μM] largely (&gt;70%) prevented labile iron from rising in cells if present in the iron-loading medium; however, only deferasirox reduced iron-evoked cell damage and increased cell viability if incubated with cells prior to or post iron-loading (acute or chronic). Thus, deferasirox has both preventive and corrective potential against iron-evoked damage in iron-loaded endocrine and cardiac cells. A gradual reduction of serum concentration from 10% (normally used in culture conditions) to 1% or less (used for experimental testing of drugs) revealed a commensurate increased susceptibility of endocrine and cardiac cells to deferasirox in the higher concentration range of 50–100 μM (48–72 cell viability test). This indicates that the plasma-binding property of deferasirox has a cytoprotective effect.


2018 ◽  
Author(s):  
Elizabeth Vinod ◽  
Upasana Kachroo ◽  
Solomon Sathishkumar ◽  
P.R.J.V.C Boopalan

AbstractObjectiveCell based therapy optimization is constantly underway since regeneration of genuine hyaline cartilage is under par. Although single source derivation of chondrocytes and chondroprogenitors is advantageous, lack of a characteristic differentiating marker obscures clear identification of either cell type which is essential to create a biological profile and is also required to assess cell type superiority for cartilage repair. This study was the first attempt where characterization was performed on the two cell populations derived from the same human articular cartilage samples.DesignCells obtained from normal/osteoarthritic knee joints were expanded in culture (up to passage 10). Characterization studies was performed using flow cytometry, gene expression was studied using RT-PCR, growth kinetics and tri-lineage differentiation was also studied to construct a better biological profile of chondroprogenitors as well as chondrocytes.Results and conclusionsOur results suggest that sorting based on CD34(-), CD166(+) and CD146(+), instead of isolation using fibronectin adhesion assay (based on CD49e+/CD29+), would yield a population of cells primarily composed of chondroprogenitors which when derived from normal as opposed to osteoarthritic cartilage, could provide translatable results in terms of enhanced chondrogenesis and reduced hypertrophy; both indispensable for the field of cartilage regeneration.


2020 ◽  
Author(s):  
Yue-zhi Zhang ◽  
Shu-jing Ge ◽  
Qing-zhen Leng ◽  
Jian-jun Ma ◽  
Hanchen Liu

Abstract Background: This study aimed to confirm the cytotoxicity of zymosan in AHH-1 cells and HIECs and to determine the treatment time and dose of zymosan at which it exerts radioprotective effects.Methods: AHH-1 cells and HIECs were administered 0, 20, 40, 80 or 160 μg/mL zymosan. The CCK-8 assay and flow cytometry were used to evaluate cell viability and apoptosis 24 h, 48 h, and 72 h after administration. Furthermore, 12 h before irradiation, the cells were treated with 0, 5, 10, or 20 μg/mL zymosan and then irradiated with 4 Gy X-rays. Cell viability and apoptosis were measured by the CCK-8 assay and flow cytometry at 24 h. In addition, the protective effect of zymosan against radiation in vitro was compared to that of 20 μg/mL LPS as a positive control. In vivo, weight, the spleen index and the thymus index were measured to evaluate the toxicity of 0, 5, 10, 20 and 10 mg/kg zymosan. In addition, rats were treated with 0, 2, 4, 8 or 10 mg/kg zymosan and then irradiated with 7 Gy X-rays. The survival rate, spleen index and thymus index were evaluated. The protective effect of zymosan against radiation in vivo was compared to that of 10 mg/kg LPS a positive control. Results: The viability and apoptosis of cells treated with different doses of zymosan for different treatment times were not different from those of control cells (p<0.05). Furthermore, cell viability and apoptosis were clearly improved after zymosan preadministration (p<0.05). The radioprotective effect of zymosan was dose-dependent. In addition, the viability of cells pretreated with zymosan was higher than that of cells pretreated with LPS, and the apoptosis rate of zymosan-treated cells was lower than that of cells pretreated with LPS (p<0.05). In vivo, weight, the spleen index and the thymus index were significantly decreased by zymosan at a concentration of 20 mg/kg (p<0.05). Further experiments showed that the concentration at which zymosan exerted radioprotective effects was 10 mg/kg. The radioprotective effect of zymosan was better than that of LPS pretreatment (p<0.05). Conclusion: Zymosan is nontoxic to cells and exerts a better radioprotective effect than LPS.


Polymers ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 12
Author(s):  
Gerardo Asensio ◽  
Lorena Benito-Garzón ◽  
Rosa Ana Ramírez-Jiménez ◽  
Yasmina Guadilla ◽  
Julian Gonzalez-Rubio ◽  
...  

Regenerative therapies based on tissue engineering are becoming the most promising alternative for the treatment of osteoarthritis and rheumatoid arthritis. However, regeneration of full-thickness articular osteochondral defects that reproduces the complexity of native cartilage and osteochondral interface still remains challenging. Hence, in this work, we present the fabrication, physic-chemical characterization, and in vitro and in vivo evaluation of biomimetic hierarchical scaffolds that mimic both the spatial organization and composition of cartilage and the osteochondral interface. The scaffold is composed of a composite porous support obtained by cryopolymerization of poly(ethylene glycol) dimethacrylate (PEGDMA) in the presence of biodegradable poly(D,L-lactide-co-glycolide) (PLGA), bioactive tricalcium phosphate β-TCP and the bone promoting strontium folate (SrFO), with a gradient biomimetic photo-polymerized methacrylated hyaluronic acid (HAMA) based hydrogel containing the bioactive zinc folic acid derivative (ZnFO). Microscopical analysis of hierarchical scaffolds showed an open interconnected porous open microstructure and the in vitro behaviour results indicated high swelling capacity with a sustained degradation rate. In vitro release studies during 3 weeks indicated the sustained leaching of bioactive compounds, i.e., Sr2+, Zn2+ and folic acid, within a biologically active range without negative effects on human osteoblast cells (hOBs) and human articular cartilage cells (hACs) cultures. In vitro co-cultures of hOBs and hACs revealed guided cell colonization and proliferation according to the matrix microstructure and composition. In vivo rabbit-condyle experiments in a critical-sized defect model showed the ability of the biomimetic scaffold to promote the regeneration of cartilage-like tissue over the scaffold and neoformation of osteochondral tissue.


Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3172
Author(s):  
Magdalena Rausch ◽  
Andrea Weiss ◽  
Marloes Zoetemelk ◽  
Sander R. Piersma ◽  
Connie R. Jimenez ◽  
...  

Clear cell renal cell carcinoma (ccRCC) is characterized by high histone deacetylase (HDAC) activity triggering both cell motility and the development of metastasis. Therefore, there is an unmet need to establish innovative strategies to advance the use of HDAC inhibitors (HDACIs). We selected a set of tyrosine kinase inhibitors (TKIs) and HDACIs to test them in combination, using the validated therapeutically guided multidrug optimization (TGMO) technique based on experimental testing and in silico data modeling. We determined a synergistic low-dose three-drug combination decreasing the cell metabolic activity in metastatic ccRCC cells, Caki-1, by over 80%. This drug combination induced apoptosis and showed anti-angiogenic activity, both in original Caki-1 and in sunitinib-resistant Caki-1 cells. Through phosphoproteomic analysis, we revealed additional targets to improve the translation of this combination in 3-D (co-)culture systems. Cell–cell and cell–environment interactions increased, reverting the invasive and metastatic phenotype of Caki-1 cells. Our data suggest that our optimized low-dose drug combination is highly effective in complex in vitro settings and promotes the activity of HDACIs.


Author(s):  
Minwook Kim ◽  
Jason A. Burdick ◽  
Robert L. Mauck

Mesenchymal stem cells (MSCs) are an attractive cell type for cartilage tissue engineering in that they can undergo chondrogenesis in a variety of 3D contexts [1]. Focused efforts in MSC-based cartilage tissue engineering have recently culminated in the formation of biologic materials possessing biochemical and functional mechanical properties that match that of the native tissue [2]. These approaches generally involve the continuous or intermittent application of pro-chondrogenic growth factors during in vitro culture. For example, in one recent study, we showed robust construct maturation in MSC-seeded hyaluronic acid (HA) hydrogels transiently exposed to high levels of TGF-β3 [3]. Despite the promise of this approach, MSCs are a multipotent cell type and retain a predilection towards hypertrophic phenotypic conversion (i.e., bone formation) when removed from a pro-chondrogenic environment (e.g., in vivo implantation). Indeed, even in a chondrogenic environment, many MSC-based cultures express pre-hypertrophic markers, including type X collagen, MMP13, and alkaline phosphatase [4]. To address this issue, recent studies have investigated co-culture of human articular chondrocytes and MSCs in both pellet and hydrogel environments. Chondrocytes appear to enhance the initial efficiency of MSC chondrogenic conversion, as well as limit hypertrophic changes in some instances (potentially via secretion of PTHrP and/or other factors) [5–7]. While these findings are intriguing, articular cartilage has a unique depth-dependent morphology including zonal differences in chondrocyte identity. Ng et al. showed that zonal chondrocytes seeded in a bi-layered agarose hydrogel construct can recreate depth-dependent cellular and mechanical heterogeneity, suggesting that these identities are retained with transfer to 3D culture systems [8]. Further, Cheng et al. showed that differences in matrix accumulation and hypertrophy in zonal chondrocytes was controlled by bone morphogenic protein [9]. To determine whether differences in zonal chondrocyte identity influences MSC fate decisions, we evaluated functional properties and phenotypic stability in photocrosslinked hyaluronic acid (HA) hydrogels using distinct, zonal chondrocyte cell fractions co-cultured with bone marrow derived MSCs.


2011 ◽  
Vol 19 (7) ◽  
pp. 1198-1205 ◽  
Author(s):  
Tsuyoshi Miyazaki ◽  
Shigeru Kobayashi ◽  
Kenichi Takeno ◽  
Takafumi Yayama ◽  
Adam Meir ◽  
...  

2018 ◽  
Vol 12 (5) ◽  
pp. E234-42
Author(s):  
Abdulmuttalip Simsek ◽  
Anthony J. Bullock ◽  
Sabi Roman ◽  
Chirstoper R. Chapple ◽  
Sheila MacNeil

Introduction: We aimed to compare alternative synthetic scaffolds suitable for future implantation and to examine the use of an inhibitor of lysyl oxidase (beta-amino-propionitrile [β-APN]) to reduce contraction in these implants.Methods: Three synthetic scaffolds were compared to natural dermis as substrates for the production of tissue-engineered skin. For natural dermis, Euroskin was used to provide a cell-free cadaveric dermis. Synthetic scaffolds consisted of microfibrous poly-L-lactic acid (PLA), nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and a micro-/nanofibrous trilayer of PLA-PHBV-PLA. The latter were all electrospun and then all four scaffolds (three synthetic, one natural) were placed in six well plates. A culture well was formed on the scaffold using a 1 cm diameter stainless steel ring and 1.5x105 oral fibroblasts were seeded one side; after two days of culture, the ring was placed on the other side of the scaffolds and 3x105 oral keratinocytes were seeded on to the scaffolds and cultured with keratinocytes uppermost. After a further two days of culture, scaffolds were cut to 1 cm2 and raised to an air-liquid interface on stainless steel grids; some were treated with 200 μg/ mL β-APN throughout the culture period (28 days). Contraction in vitro was assessed by serial digital photography of cell-seeded scaffolds and cell-free scaffolds three times a week for 28 days. All cell-seeded scaffolds were assessed for cell metabolic activity, mechanical properties, histology, and morphology by scanning electron microscopy (SEM).Results: The mean fibre diameters and pore sizes of PLA and PHBV scaffolds were 2.4±0.77, 0.85±0.21 μm (p<0.001), and 10.8±2.3, 4.3±1.1 μm (p<0.001), respectively. Oral fibroblasts and keratinocytes were tightly adhered and grew well on both surfaces of trilayer. The ultimate tensile strength (UTS) and Young’s modulus (YM) of PLA samples were significantly lower than Euroskin (p<0.001 and p<0.05, respectively); only the UTS of the trilayer samples was slightly significantly lower (p<0.05). Metabolic activity was significantly increased for cells on all scaffolds, without significant differences between them from Day 0 to Day 28. There were no adverse effects of β-APN on cell viability. With respect to contraction, cells on trilayer and PHBV monolayers did not undergo any significant contraction; however, cells on PLA monolayer and Euroskin contracted 25.3% and 56.4%, respectively, over 28 days. The addition of 200 μg/ml β-APN significantly reduced contraction of Euroskin compared with the control (p<0.01); however, β-APN did not affect PLA contraction during this culture period (p>0.05).Conclusions: This study shows that a trilayer micro-nano-3D porous synthetic scaffold is suitable for oral keratinocyte and fibroblast growth with good cell viability and minimal contraction. This material also has good mechanical properties and histological analyses showed its ability to mimic normal human oral mucosal morphology. Furthermore, synthetic trilayer scaffolds have advantages over biological scaffolds — there is no risk of disease transmission or immunological rejection and they appear resistant to contraction. We suggest they present a good alternative to allodermis for future use in urethral reconstruction.


Sign in / Sign up

Export Citation Format

Share Document