Alginate Culture Research Articles

Conditioned Medium of Intervertebral Disc Cells Inhibits Osteo-Genesis on Autologous Bone-Marrow-Derived Mesenchymal Stromal Cells and Osteoblasts.

Low back pain (LBP) is associated with the degeneration of human intervertebral discs (IVDs). Despite progress in the treatment of LBP through spinal fusion, some cases still end in non-fusion after the removal of the affected IVD tissue. In this study, we investigated the hypothesis that the remaining IVD cells secrete BMP inhibitors that are sufficient to inhibit osteogenesis in autologous osteoblasts (OBs) and bone marrow mesenchymal stem cells (MSCs). A conditioned medium (CM) from primary human IVD cells in 3D alginate culture was co-cultured with seven donor-matched OB and MSCs. After ten days, osteogenesis was quantified at the transcript level using qPCR to measure the expression of bone-related genes and BMP antagonists, and at the protein level by alkaline phosphatase (ALP) activity. Additionally, cells were evaluated histologically using alizarin red (ALZR) staining on Day 21. For judging ALP activity and osteogenesis, the Noggin expression in samples was investigated to uncover the potential causes. The results after culture with the CM showed significantly decreased ALP activity and the inhibition of the calcium deposit formation in alizarin red staining. Interestingly, no significant changes were found among most bone-related genes and BMP antagonists in OBs and MSCs. Noteworthy, Noggin was relatively expressed higher in human IVD cells than in autologous OBs or MSCs (relative to autologous OB, the average fold change was in 6.9, 10.0, and 6.3 in AFC, CEPC, and NPC, respectively; and relative to autologous MSC, the average fold change was 2.3, 3.4, and 3.2, in AFC, CEPC, and NPC, respectively). The upregulation of Noggin in residual human IVDs could potentially inhibit the osteogenesis of autologous OB and MSC, thus inhibiting the postoperative spinal fusion after discectomy surgery.

Enhanced cartilage regeneration by icariin and mesenchymal stem cell-derived extracellular vesicles combined in alginate-hyaluronic acid hydrogel

ObjectiveOsteoarthritis (OA) is characterized by progressive cartilage degeneration and absence of curative therapies. Therefore, more efficient therapies are compellingly needed. Both mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) and Icariin (ICA) are promising for repair of cartilage defect. This study proposes that ICA may be combined to potentiate the cartilage repair capacity of MSC-EVs. Materials and methodsMSC-EVs were isolated from sodium alginate (SA) and hyaluronic acid (HA) composite hydrogel (SA-HA) cell spheroid culture. EVs and ICA were combined in SA-HA hydrogel to test therapeutic efficacy on cartilage defect in vivo. ResultsEVs and ICA were synergistic for promoting both proliferation and migration of MSCs and inflammatory chondrocytes. The combination therapy led to strikingly enhanced repair on cartilage defect in rats, with mechanisms involved in the concomitant modulation of both cartilage degradation and synthesis makers. ConclusionThe MSC-EVs-ICA/SA-HA hydrogel potentially constitutes a novel therapy for cartilage defect in OA.

Human Lung Organoid Culture in Alginate With and Without Matrigel to Model Development and Disease.

Human lung organoids (HLOs) are enabling the study of human lung development and disease by modeling native organ tissue structure, cellular composition, and cellular organization. In this report, we demonstrate that HLOs derived from human pluripotent stem cells cultured in alginate, a fully defined nonanimal product substrate, exhibit enhanced cellular differentiation compared with HLOs cultured in the commercially available Matrigel. More specifically, we observed an earlier onset and increase in the number of multiciliated cells, along with mucus producing MUC5AC+ goblet-like cells that were not observed in HLOs cultured in Matrigel. The epithelium in alginate-grown HLOs was organized in a pseudostratified epithelium with airway basal cells lining the basal lamina, but with the apical surface of cells on the exterior of the organoid. We further observed that HLOs cultured in Matrigel exhibited mesenchymal overgrowth that was not present in alginate cultures. The containment of the mesenchyme within HLOs in alginate enabled modeling of key features of idiopathic pulmonary fibrosis (IPF) by treatment with transforming growth factor β (TGFβ). TGFβ treatment resulted in morphological changes including an increase in mesenchymal growth, increased expression of IPF markers, and decreased numbers of alveolar-like cells. This culture system provides a model to study the interaction of the mesenchyme with the epithelium during lung development and diseased states such as IPF.

Effects of alginate and laminaran on the microbiota and antioxidant properties of human faecal cultures

Alginate and laminaran are water soluble dietary fibres in brown algae. In a previous study, pure cultures of Bacteroides xylanisolvens, Bacteroides uniformis, and Erysipelaclostridium ramosum increased antioxidant capacities in the broth with alginate and/or laminaran fermentation. This study was conducted to evaluate the presence of the alginate- and laminaran-responsive human gut indigenous bacteria (RIB) and their functions. Briefly, human stool samples were cultured in media without (control), or with 1% (w/v) alginate or laminaran at 37 °C for 24 h under anaerobic conditions. Results showed that during incubation, the pH of the culture reduced with the polysaccharides. Phenol production was clearly suppressed via laminaran supplementation in the faecal sample cultures. The 16S rRNA (V4) gene amplicon sequencing confirmed that B. xylanisolvens-, B. uniformis and E. ramosum-like bacteria were alginate- and/or laminaran-RIB in human gut, although their read numbers differed among individuals. Additionally, some butyrate-producers, such as Faecalibacterium and Roseburia, were also detected as the RIB in alginate and laminaran cultures, respectively. Fermentation increased 1,1-diphenyl-2-picrylhydrazyl and superoxide anion radical-scavenging capacities and the Fe-reducing power of the culture medium. Additionally, the fermented cultures increased the nitric oxide secretion in macrophage like RAW 264.7 cells culture. These results suggest that the interaction of alginate and laminaran with their RIB may have desirable effects on the host as next generation prebiotics when brown algae are ingested.

Integration of a miniaturized DMMB assay with high-throughput screening for identifying regulators of proteoglycan metabolism

Defective biosynthesis or function of proteoglycans causes pathological conditions in a variety of tissue systems. Osteoarthritis (OA) is a prevalent degenerative joint disorder characterized by progressive cartilage destruction caused by imbalanced proteoglycan synthesis and degradation. Identifying agents that regulate proteoglycan metabolism may benefit the development of OA-modifying therapeutics. High-throughput screening (HTS) of chemical libraries has paved the way for achieving this goal. However, the implementation and adaptation of HTS assays based on proteoglycan measurement remain underexploited. Using primary porcine chondrocytes as a model, we report a miniaturized dimethyl-methylene blue (DMMB) assay, which is commonly used to quantitatively evaluate sulfated glycosaminoglycan (GAG) content, with an optimized detection range and reproducibility and its integration with HTS. Treatment with TGF-β1 and IL1-α, known as positive and negative proteoglycan regulators, respectively, supported the assay specificity. A pre-test of chemical screening of 960 compounds identified both stimulators (4.48%) and inhibitors (6.04%) of GAG production. Fluorophore-assisted carbohydrate electrophoresis validated the activity of selected hits on chondroitin sulfate expression in an alginate culture system. Our findings support the implementation of this simple colorimetric assay in HTS to discover modifiers of OA or other diseases related to dysregulated proteoglycan metabolism.

Mammary Tumor Organoid Culture in Non-Adhesive Alginate for Luminal Mechanics and High-Throughput Drug Screening.

Mammary tumor organoids have become a promising in vitro model for drug screening and personalized medicine. However, the dependency on the basement membrane extract (BME) as the growth matrices limits their comprehensive application. In this work, mouse mammary tumor organoids are established by encapsulating tumor pieces in non‐adhesive alginate. High‐throughput generation of organoids in alginate microbeads is achieved utilizing microfluidic droplet technology. Tumor pieces within the alginate microbeads developed both luminal‐ and solid‐like structures and displayed a high similarity to the original fresh tumor in cellular phenotypes and lineages. The mechanical forces of the luminal organoids in the alginate capsules are analyzed with the theory of the thick‐wall pressure vessel (TWPV) model. The luminal pressure of the organoids increase with the lumen growth and can reach 2 kPa after two weeks’ culture. Finally, the mammary tumor organoids are treated with doxorubicin and latrunculin A to evaluate their application as a drug screening platform. It is found that the drug response is related to the luminal size and pressures of organoids. This high‐throughput culture for mammary tumor organoids may present a promising tool for preclinical drug target validation and personalized medicine.

Using high hydrostatic pressure as an abiotic elicitor strategy for improving capsaicin production in free and immobilized cell suspension cultures of Capsicum annuum L.

ABSTRACT In this study, the effects of immobilization and high hydrostatic pressure (HHP) on capsaicin production in cell suspension culture of pepper seeds at different application times were investigated. Callus cultures were obtained from in vitro germinated hypocotyl explants of pepper seedlings and cell suspensions were prepared from calluses. Immobilized cell suspension cultures with calcium alginate and free cell suspension cultures were obtained by using these cell suspensions. 50, 100, 200, 300 and 400 MPa were applied to both free and immobilized cell suspensions as an elicitor. When all the results were compared, the highest amount of capsaicin was achieved in immobilized cell suspensions on the tenth day as 293.187 µg/g f.w at 400 MPa pressure. As a result, it can be concluded that the combined application of immobilization and elicitor (HHP) caused significant increases (p < .05) in the amount of capsaicin.

RPMI-1640 laden alginate hydrogel microcapsule produced by a coaxial electrohydrodynamic method

The microencapsulation of biomaterials has a broad application prospects in the field of biomedicine. This paper introduces design and setup of a coaxial microcapsule generator based on electrohydrodynamic (EHD) method. It includes a liquid supply system, a coaxial nozzle, a collector electrode, and a high voltage power supply. The ejection process is studied via LED illumination and a high speed CMOS camera. For the shell and core materials, alginate and RPMI-1640 culture medium are applied respectively. The effects of electric voltage and flow rate on the ejection frequency and volume of micro-droplet are investigated. The generated micro-droplets react in the collector electrode with CaCl2 solution. Alginate hydrogel microcapsules are produced with RPMI-1640 included. Good encapsulation is confirmed, by fluorescence microscopy, and with RPMI-1640 stained by sodium fluorescein. The produced microcapsule may be used in biological cell encapsulation, in the future.

Physicochemical and swelling properties of composite gel microparticles based on alginate and callus cultures pectins with low and high degrees of methylesterification

Composite gel microparticles based on alginate and callus culture pectins with low and high degrees of methylesterification or apple pectin were produced. By varying the chemical composition of the pectic samples and the ratio of alginate to pectin, the gel strength, morphology, and swelling properties of composite microparticles can be altered. The inclusion of increasing concentrations of alginate in gel formulations promoted an increase in the microparticle gel strength and the formation of a smoother surface microrelief independently of the pectin chemical composition. Microparticles based on the pectin with a low degree of methylesterification (DM) and a higher concentration of alginate exhibited an increased swelling degree in the simulated digestive fluids. Microparticles based on the pectin with high DM and low alginate concentration were destroyed in the simulated intestinal fluid within 1 h due to the low Ca2+ content, gel strength, and grooved and rough surface of these microparticles. An increase in alginate concentration of gel formulations based on pectin with high DM led to increased stability of the microparticles in the simulated intestinal and colonic fluids due to increased Ca2+ content, microparticle gel strength and degree of crosslinking.

Reduced graphene oxide facilitates biocompatibility of alginate for cardiac repair

The benefits of combined cell/material therapy appear promising for myocardial infarction treatment. The safety of alginate, along with its excellent biocompatibility and biodegradability, has been extensively investigated for cardiac tissue engineering. Among graphene-based nanomaterials, reduced graphene oxide has been considered as a promising candidate for cardiac treatment due to its unique physicochemical properties. In this study, the reduced graphene oxide incorporation effect within alginate hydrogels was investigated for cardiac repair application. Reduced graphene oxide reinforced alginate properties, resulting in an increase in gel stiffness. The cytocompatibility of the hydrogels prepared with human bone marrow–derived mesenchymal stem cells was assessed by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay. Following reduced graphene oxide addition, alginate-reduced graphene oxide retained significantly higher cell viability compared to that of alginate and cells cultured on tissue culture plates. Acridine orange/propidium iodide staining was also used to identify both viable and necrotic human bone marrow–derived mesenchymal stem cells within the prepared hydrogels. After a 72-h culture, the percentage of viable cells was twice as much as those cultured on either alginate or tissue culture plate, reaching approximately 80%. Quantitative reverse transcription polymerase chain reaction analysis was performed to assess gene expression of neonatal rat cardiac cells encapsulated on hydrogels for TrpT-2, Conx43, and Actn4 after 7 days. The expression of all genes in alginate-reduced graphene oxide increased significantly compared to that in alginate or tissue culture plate. The results obtained confirmed that the presence of reduced graphene oxide, as an electro-active moiety within alginate, could tune the physicochemical properties of this material, providing a desirable electroactive hydrogel for stem cell therapy in patients with ischemic heart disease.

Human Primary Osteoarthritic Chondrocytes Revert to a Healthier Extracellular Matrix Composition Following Long‐term, Serum‐free, Three‐dimensional Alginate Culture

Osteoarthritis (OA) is a common condition involving the loss of articular cartilage which is primarily made up of type II collagen, proteoglycans, and water. In OA, there is an increase in collagenase activity that degrades collagens and proteoglycans necessary for healthy cartilage and leads to the abnormal production of type I collagen. Previous studies in our lab have shown that primary cultures of human osteoarthritic chondrocytes (HOACs) can be reared in serum‐free, three‐dimensional alginate culture and components of the extracellular matrix (ECM) can be measured. However, we had noted that the ECM phenotype of these cells seemed to be changing when cultured past 5 days. In this study, we obtained HOACs from the femoral condyles and the tibial plateau of patients undergoing total knee arthroplasty. HOACs of greater or least pathology, determined by gross observation, were isolated and plated in 12‐well cultures at a density of 1.8 × 106 cells/0.5 mL alginate. Collagens I and II degradation and proteoglycan synthesis were measured in conditioned media and alginate‐associated matrix of the cultures at days 2, 5, 8 and 11. During long term HOAC culture, collagen degradation was reduced (p&lt;0.001 for coll I) while proteoglycans were retained in the ECM. This trend suggests that long term, three‐dimensional, serum‐free culture of HOACs may revert to a healthier phenotype. The largest changes in extracellular matrix production were demonstrated between days 2 and 5 in HOAC culture giving insight of a treatment window to test possible therapeutics.Support or Funding InformationSupport was provided by New Jersey Health Foundation Research Award, Cooper Foundation Research Grant and the Division of Research, PCOM.

Human Primary Osteoarthritic Chondrocytes Reared in Long‐term, Serum‐free, Three‐dimensional Alginate Culture Remain Metabolically Stable: A Nucleic Acid Study

The pathogenic process that defines osteoarthritis (OA) is the imbalance between excess degradation of the extracellular matrix (ECM) in articular cartilage and a decreased ability of chondrocytes to remodel the matrix. There are many factors that contribute to this disease including unchecked actions of matrix metalloproteinase 13 (MMP‐13), an enzyme specific for the breakdown of collagen type II which is the main component of cartilage extracellular matrix. We have developed a three‐dimensional, serum‐free culture system to rear primary, human OA chondrocytes (HOACs). However, we had noted that the ECM phenotype of these cells seemed to be changing when cultured past 5 days. Primary human articular chondrocytes were isolated from patients who underwent surgery for total knee arthroplasty (TKA). The medial and lateral aspects of the joint samples were kept separated and labeled as greatest or least pathology. The number of live cells were determined and plated at 1.8 × 106 cells/0.5ml alginate per well of a 12‐well plate. Cells were cultured in complete serum‐free media for 11 days with RNA and DNA collected at days 2, 5, 8 and 11. Total RNA and DNA were isolated via Trizol reagent protocol. Quantitation of nucleic acids was accomplished with the NanoDrop system. Over the eleven days in culture there was no statistically significant difference in the total amount of RNA isolated either in the least pathology samples (day 2=4.3ugRNA/culture versus day 11=2.8ugRNA/culture) or the greatest pathology samples (day 2=5.3ugRNA/culture versus day 11=6.2ugRNA/culture). The same was true for the total DNA isolated from the least pathology samples (day 2=1.9ugDNA/culture versus day 11=1.6ugDNA/culture) or the greatest pathology samples (day 2=8ugDNA/culture versus day 11=1.7ugDNA/culture). Future studies include quantitative, real‐time PCR to measure expression of metabolic agents including MMPs and collagens present in OA chondrocytes. This study is the first of its kind to isolate RNA and DNA from completely serum‐free, three‐dimensional cultures of primary human OA chondrocytes.Support or Funding InformationThis study has been supported in part by the New Jersey Health Foundation and PCOM Division of Research

Comparison of Bovine Small Antral Follicle Development in Two- and Three-Dimensional Culture Systems.

To compare the effects of two-(2D, microplate) and three-dimensional (3D, alginate) culture systems on the in vitro growth of small antral follicles in cattle, individual follicles were separately cultured in the two culture systems for 8 days. Half of the culture medium was replaced by fresh medium every 2 days; the former medium was used to assess the amount of follicular hormone secretion using ELISA. Individual follicle morphology, diameter, and survival rate were recorded every alternate day. The results showed that in 4 days, there was no significant difference between the two systems, except that the growth rate of follicles in 2D system was relatively faster. After 4 days, estradiol concentration in 3D system was higher than that in 2D system. However, progesterone concentration was lower than that in the 2D system. The survival rate and oocyte quality of follicles in 2D system were significantly lower than those in 3D system on day 8. The follicle diameter slightly increased (30-60 μm) in the entire process. Taken together, for in vitro culture of follicles within 4 days, the 2D culture system is more suitable. However, when the culture duration is >4 days, the 3D culture system is more suitable.

Utilizing Calcium Alginate for the Assessment of Bone Morphogenetic Protein 15 Induction Effect on the Differentiation of Mesenchymal Stem Cell Derived from Human Follicular Fluid to Oocyte-Like Structure.

Background:Follicular fluid (FF)-derived mesenchymal stem cells (MSCs) are possible new source of cells in the study of oogenesis and regenerative medicine. Several biomaterials have been used as scaffolds to mimic ovarian tissue stroma. Using good matrix is essential for increasing the cell survival rate, proliferation, and differentiation. However, no study has been performed to investigate the effects of BMP15 and calcium alginate hydrogel on the differentiation potential of FF-derived MSCs to oocyte-like structures (OLSs).Materials and Methods:In this work, FF MSCs, which were collected from women in routine in vitro fertilization procedure, were capsulated with 0.5% calcium alginate, and then the encapsulated cells were cultured in medium containing BMP15 for 2 weeks. Trypan blue staining was carried out to determine cell viability. Real-time polymerase chain reaction (PCR) and immunofluorescence (ICC) staining method were performed to characterize the expression of OCT4, Nanog, ZP2, and ZP3 genes and protein. The encapsulation process did not change the morphology and viability of the encapsulated cells.Results:Reverse-transcription-PCR and ICC showed that MSCs expressed germ line stem cell markers such as OCT4 and Nanog. After 4 days of culture, OLSs formed and expressed zona pellucida markers. OLSs at least reached 180–230 μm in diameter in the control and BMP15-treated groups. Finally, a reduction in the expression pattern of pluripotency and ZP markers was detected in the encapsulated cells cultured in the BMP15-supplemented medium.Conclusion:The three-dimensional alginate culture system seems to be a promising method of getting in vitro differentiation and development of ovarian cells, which could mimic the native ovarian condition.

Evidence That Calebin A, a Component of Curcuma Longa Suppresses NF-B Mediated Proliferation, Invasion and Metastasis of Human Colorectal Cancer Induced by TNF-β (Lymphotoxin).

Objective: Natural polyphenol Calebin A has been recently discovered as a novel derivate from turmeric with anti-cancer potential. Pro-inflammatory cytokine TNF-β (lymphotoxin α) is a stimulant for cancer cell malignity via activation of NF-κB pathway, also in colorectal cancer (CRC). Here, we investigated the potential of Calebin A to suppress TNF-β-induced NF-κB signalling in CRC. Materials and Methods: Three distinct CRC cell lines (HCT116, RKO, SW480) were treated in monolayer or 3-dimensional alginate culture with TNF-β, Calebin A, curcumin, BMS-345541, dithiothreitol (DTT) or antisense oligonucleotides-(ASO) against NF-κB. Results: Calebin A suppressed dose-dependent TNF-β-induced CRC cell vitality and proliferation in monolayer culture. Further, in alginate culture, Calebin A significantly suppressed TNF-β-enhanced colonosphere development, as well as invasion and colony formation of all three CRC cell lines investigated. Calebin A specifically blocked TNF-β-induced activation and nuclear translocation of p65-NF-κB, similar to curcumin (natural NF-κB inhibitor), BMS-345541 (specific IKK inhibitor) and ASO-NF-κB. Moreover, Immunofluorescence and Immunoblotting showed that Calebin A, similar to curcumin or BMS-345541 suppressed TNF-β-induced activation and nuclear translocation of p65-NF-κB and the transcription of NF-κB-promoted biomarkers associated with proliferation, migration and apoptosis, in a dose- and time-dependent manner. Those findings were potentiated by the specific treatment of extracted nuclei with DTT, which abrogated Calebin A-mediated nuclear p65-NF-κB-inhibition and restored p65-NF-κB-activity in the nucleus. Conclusion: Overall, these results demonstrate, for the first time, that multitargeted Calebin A has an anti-cancer capability on TNF-β-induced malignities through inhibitory targeting of NF-κB activation in the cytoplasm, as well as by suppressing the binding of p65-NF-κB to DNA.

Exploring peptide‐functionalized alginate scaffolds for engineering cardiac tissue from human embryonic stem cell‐derived cardiomyocytes in serum‐free medium

Engineering human cardiac tissue is a promising solution for myocardial repair of injured hearts and for drug screening. Herein, we examined the capability of chemically defined alginate scaffolds to promote cardiac tissue regeneration from human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs) in serum‐free, chemically defined medium. The cells were single seeded or coseeded with human dermal fibroblasts (HFs) in macroporous scaffolds made from pristine alginate or alginate modified with arginine‐glycine‐aspartate (RGD) peptide and heparin‐binding peptide (HBP). Our results show that the addition of fibroblasts to the 3‐D culture is indispensable for the formation of functional cardiac tissues and that the presence of RGD/HBP attached to the alginate matrix further improves its functionality. The engineered tissue displayed the typical fiber morphology with massive striation. An increase in contraction amplitude and calcium transients with time, together with a decrease in excitation threshold, indicated advancement toward tissue maturation. Our results thus point to the importance of co‐cultivating fibroblasts with hESCs‐CMs in chemically defined peptide‐functionalized alginate scaffolds and culture medium for regenerating functional cardiac tissue in vitro.

Extracellular Matrix Protection Factor Increases Sulfated Proteoglycan Production in Serum‐free, Primary Cultures of Human Osteoarthritic Chondrocytes

Osteoarthritis (OA) is a common chronic condition which involves the loss of articular cartilage, the cushion between joints that provides a smooth surface for the bones to glide. Three compounds that make up most of the articular cartilage are type II collagen, proteoglycans, and water. In OA there is an increase in collagenase activity that degrades collagens and proteoglycans necessary for healthy cartilage. The composition of the extracellular matrix changes in OA as evidenced by production of type I collagen. Previous studies in our lab have shown that primary cultures of human osteoarthritic chondrocytes (HOACs) can be reared in serum‐free alginate culture but measurable extracellular matrix endpoints of the OA profile are dampened when chondrocytes isolated from sides of greater and least pathology in the joint are pooled together in culture. In this study, we obtained HOACs from patients undergoing total knee arthroplasty. Femoral condyles and the tibial plateau were labeled greater or least pathology, determined by gross anatomical observation, the cells were isolated and plated separately in three‐dimensional, twelve‐well, alginate cultures at a density of 1.8 × 106 cells per 0.5 mL. Samples were treated for 48 hours from day of plating with either 250nM Extracellular Matrix Protective Factor‐1 (ECPF‐1) or hyaluronic acid conjugated ECPF‐1 (HA‐ECPF‐1). Collagen I degradation and sulfated proteoglycan synthesis were measured in alginate‐associated matrix of the treated cultures. HOACs isolated from the side of greatest pathology responded to treatment by reducing collagen type I degradation (8.42ng/culture Control, 7.25ng/culture ECPF‐1 and 4.25ng/culture HA‐ECPF‐1) and increasing retention of sulfated proteoglycans in the extracellular matrix (0.36ug/culture Control versus 0.56ug/culture ECPF‐1 treated). These data confirm that 48 hour treatment in serum free HOAC culture is sufficient to measure the therapeutic efficacy of an OA drug intervention and that both formulations of ECPF‐1 can slow the progression of OA by altering production of sulfated proteoglycan and collagen type I.Support or Funding InformationThis work was supported in part by a grant from the New Jersey Health Foundation and Philadelphia College of Osteopathic Medicine's Center for Chronic Disorders of Aging and the Division of Research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Induction of the Epithelial-to-Mesenchymal Transition of Human Colorectal Cancer by Human TNF-β (Lymphotoxin) and its Reversal by Resveratrol.

Objective: Tumor necrosis factor-beta (TNF-β), as an inflammatory mediator that has been shown to promote tumorigenesis, induces NF-κB. Natural multi-targeted agent resveratrol in turn shows anti-inflammatory and anti-cancer properties. Epithelial-to-mesenchymal transition (EMT) allows cancer cells to turn into a motile state with invasive capacities and is associated with metastasis and development of cancer stem cells (CSC). However, TNF-β-induced EMT and the anti-invasion mechanism of resveratrol on CRC are not yet completely understood. Methods: We investigated the underlying molecular mechanisms of resveratrol on TNF-β/TNF-βR-induced EMT and migration of CRC cells (HCT116, RKO, SW480) in monolayer or 3D alginate cultures. Results: TNF-β, similar to TNF-α, induced significant cell proliferation, morphological change, from an epithelial to a spindle-like mesenchymal shape with the formation of filopodia and lamellipodia associated with the expression of EMT parameters (elevated vimentin and slug, reduced E-cadherin), increased migration/invasion, and formation of CSC in all CRC cells. Interestingly, these effects were dramatically decreased in the presence of resveratrol or anti-TNF-βR with TNF-β co-treatment, inducing biochemical changes to the mesenchymal-epithelial transition (MET), with a planar cell surface and suppressed formation of CSC cells. This was associated with a significant increase in apoptosis. Furthermore, we found that resveratrol suppressed TNF-β-induced NF-κB and NF-κB-regulated gene biomarkers associated with growth, proliferation, and invasion. Finally, TNF-βR interacts directly with focal adhesion kinase (FAK) and NF-κB. Conclusion: These results suggest that resveratrol down-regulates TNF-β/TNF-βR-induced EMT, at least in part via specific suppression of NF-κΒ and FAK in CRC cells.

Extracellular Matrix Protection Factor‐1, a Novel Osteoarthritis Therapeutic, Decreases Expression of Interleukin‐1 beta by Primary Cultures of Human Osteoarthritic Chondrocytes

Osteoarthritis (OA) is characterized by an imbalance of cartilage extracellular matrix (ECM) production and degradation, due, in part, to increased cytokine production. It has been demonstrated that the cytokine, Interleukin‐1 beta (IL‐1β), is produced by OA articular chondrocytes and plays a prominent role in altering ECM metabolism by targeting the matrix metalloproteases (MMPs) that degrade the ECM. We are developing a novel, OA therapeutic, Extracellular Matrix Protection Factor‐1 (ECPF‐1) that targets MMP interaction with its ECM substrate. We have demonstrated a reduction in ECM degradation when primary, human OA chondrocyte (HOAC) cultures are treated with ECPF‐1. In this study, HOACs isolated from total knee arthroplasty were reared in three‐dimensional, serum‐free, alginate cultures. The sides of least and greatest pathology of the femoral condyles and tibial plateaus were determined by gross inspection and cells isolated were cultured separately (LP and GP). Chondrocytes were plated at 2.5×106 cells per milliliter of alginate and incubated in serum‐free medium for five days. Cells were released from the alginate and the RNA extracted from the pellet, reverse‐transcribed into cDNA and quantitative, real time PCR (qRT‐PCR) was performed with TaqMan Gene Expression assays for IL‐1β, collagen type I, collagen type II and 18s rRNA. In a previous study, IL‐1β production was detected in the conditioned media of HOAC cultures with the highest concentration of this cytokine present in the cultures established from the region of greatest pathology (GP). In this study, IL‐1β expression was detectable in mRNA produced by LP and GP HOAC cultures. The expression was reduced 2‐fold in LP cultures treated 24 hours with 2.5 uM ECPF‐1 and 1.4 fold in GP cultures. These results demonstrate the ability of ECPF‐1 to alter expression of a molecule involved in the feedback loop of ECM destruction. ECPF‐1 provides a tool with which to further define the cellular mechanisms responsible for OA chondrocyte pathology.Support or Funding InformationNew Jersey Health Foundation, Center for Chronic Disorders of Aging Small Grant, PCOM's Division of ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Optimizing a Novel, Serum‐Free, Three‐Dimensional Culture System of Primary Human Osteoarthritic Chondrocytes: A Clinically Relevant Platform to Test and Develop Osteoarthritis Therapeutics

Osteoarthritis (OA), a progressive and degenerative disease, affects millions of aging adults and results in the loss of cartilage. In healthy joints, articular cartilage provides a smooth and cushioned surface that allows bones to glide over each other with little friction. However, when OA begins to develop, degradation of the cartilage extracellular matrix begins to outweigh its synthesis. Production of growth factors, cytokines, and matrix‐degrading enzymes increases. Development of OA therapeutics is the gold standard in orthopedic medicine, but the complexity of the joint makes it a difficult system to manipulate. In this study, we optimize our novel, serum‐free, three‐dimensional culture system for human osteoarthritic articular chondrocytes (HOACs) obtained from patients with end‐stage osteoarthritis who have undergone a total knee arthroplasty. We altered the parameters of culture size, tissue harvest sites and the number of cultures pooled to determine whether clinically‐relevant endpoints are within measurable ranges. In previous studies, sides of greatest and least pathology, as determined from gross examination of the femoral condyles and tibial plateau, were separated and cells were isolated and plated within serum‐free, three‐dimensional alginate cultures on 35 mm plates at a plating density of 2.5×106 cells/milliliter. In this study, we combine both sides of the joint tissue (greatest and least pathology) together to increase the number of cells and cultures obtained from each patient. In addition, we scaled down the size of the cultures to 22 mm plates at a plating density of 1.8×106 cells/milliliter. The scaled‐down, serum‐free, three‐dimensional alginate cultures were maintained for five days, media fractions collected on the second and fifth day of the culture and the alginate‐associated matrix collected at termination of the cultures on day five. Intact and degraded collagens were measured by immunoassay. Both collagens type I and type II, intact and degraded, were detectable within the scaled‐down culture system. Pooling three cultures together provided adequate measurable material of the clinically‐relevant endpoints. Collagen I intact and degraded values were lower than collagen II intact and degraded regardless of the number of cultures pooled (n= 2, 3 or 4). In summary, our optimization techniques have provided more cultures per patient thus enabling measurement of clinically‐relevant endpoints of OA and a screening system to test potential OA therapeutics.Support or Funding InformationNew Jersey Health Foundation Research Award, Center for Chronic Disorders of Aging Small Grant, and PCOM's Division of ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.