scholarly journals Culture of equine fibroblast-like synoviocytes on synthetic tissue scaffolds towards meniscal tissue engineering: a preliminary cell-seeding study

2014 ◽  
Author(s):  
Jennifer J Warnock ◽  
Derek B Fox ◽  
Aaron M Stoker ◽  
Mark Beatty ◽  
Mary K Cockrell ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Extracellular Matrix ◽  
Culture Conditions ◽  
Confocal Laser ◽  
Sulfated Glycosaminoglycan ◽  
Dynamic Culture ◽  
Meniscal Tissue ◽  
Static Conditions ◽  
Fibroblast Like Synoviocytes

Introduction: Tissue Engineering is a new methodology for addressing meniscal injury or loss. Synovium may be an ideal source of cells for in vitro meniscal fibrocartilage formation, however, favorable in vitro culture conditions for synovium must be established in order to achieve this goal. The objective of this study was to determine cellularity, cell distribution, and extracellular matrix (ECM) formation of equine fibroblast-like synoviocytes (FLS) cultured on synthetic scaffolds, for potential application in synovium-based meniscal tissue engineering. Scaffolds included open-cell poly-L-lactic acid (OPLA) sponges and polyglycolic acid (PGA) scaffolds cultured in static and dynamic culture conditions, and PGA scaffolds coated in poly-L-lactic (PLLA) in dynamic culture conditions. Materials and Methods: Equine FLS were seeded on OPLA and PGA scaffolds, and cultured in a static environment or in a rotating bioreactor for 12 days. Equine FLS were also seeded on PGA scaffolds coated in 2% or 4% PLLA and cultured in a rotating bioreactor for 14 and 21 days. Three scaffolds from each group were fixed, sectioned and stained with Masson’s Trichrome, Safranin-O, and Hematoxylin and Eosin, and cell numbers and distribution were analyzed using computer image analysis. Three PGA and OPLA scaffolds from each culture condition were also analyzed for extracellular matrix (ECM) production via di-methylmethylene blue (sulfated glycosaminoglycan) assay and hydroxyproline (collagen) assay. PLLA coated PGA scaffolds were analyzed using double stranded DNA quantification as a reflection of cellularity and confocal laser microscopy in a fluorescent cell viability assay. Results: The highest cellularity occurred in PGA constructs cultured in a rotating bioreactor, which also had a mean sulfated glycosaminoglycan content of 22.3μg per scaffold. PGA constructs cultured in static conditions had the lowest cellularity. Cells had difficulty adhering to OPLA and the PLLA coating of PGA scaffolds; cellularity was inversely proportional to the concentration of PLLA used. PLLA coating did not prevent dissolution of the PGA scaffolds. All cell scaffold types and culture conditions produced non-uniform cellular distribution. Discussion/ Conclusion: FLS-seeding of PGA scaffolds cultured in a rotating bioreactor resulted in the most optimal cell and matrix characteristics seen in this study. Cells grew only in the pores of the OPLA sponge, and could not adhere to the PLLA coating of PGA scaffold, due to the hydrophobic property of PLA. While PGA culture in a bioreactor produced measureable GAG, no culture technique produced visible collagen. For this reason, and due to the dissolution of PGA scaffolds, the culture conditions and scaffolds described here are not recommended for inducing fibrochondrogenesis in equine FLS for meniscal tissue engineering.

scholarly journals Culture of equine fibroblast-like synoviocytes on synthetic tissue scaffolds towards meniscal tissue engineering: a preliminary cell-seeding study

2014 ◽  
Author(s):  
Jennifer J Warnock ◽  
Derek B Fox ◽  
Aaron M Stoker ◽  
Mark Beatty ◽  
Mary K Cockrell ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Extracellular Matrix ◽  
Culture Conditions ◽  
Confocal Laser ◽  
Sulfated Glycosaminoglycan ◽  
Dynamic Culture ◽  
Meniscal Tissue ◽  
Static Conditions ◽  
Fibroblast Like Synoviocytes

Introduction: Tissue Engineering is a new methodology for addressing meniscal injury or loss. Synovium may be an ideal source of cells for in vitro meniscal fibrocartilage formation, however, favorable in vitro culture conditions for synovium must be established in order to achieve this goal. The objective of this study was to determine cellularity, cell distribution, and extracellular matrix (ECM) formation of equine fibroblast-like synoviocytes (FLS) cultured on synthetic scaffolds, for potential application in synovium-based meniscal tissue engineering. Scaffolds included open-cell poly-L-lactic acid (OPLA) sponges and polyglycolic acid (PGA) scaffolds cultured in static and dynamic culture conditions, and PGA scaffolds coated in poly-L-lactic (PLLA) in dynamic culture conditions. Materials and Methods: Equine FLS were seeded on OPLA and PGA scaffolds, and cultured in a static environment or in a rotating bioreactor for 12 days. Equine FLS were also seeded on PGA scaffolds coated in 2% or 4% PLLA and cultured in a rotating bioreactor for 14 and 21 days. Three scaffolds from each group were fixed, sectioned and stained with Masson’s Trichrome, Safranin-O, and Hematoxylin and Eosin, and cell numbers and distribution were analyzed using computer image analysis. Three PGA and OPLA scaffolds from each culture condition were also analyzed for extracellular matrix (ECM) production via di-methylmethylene blue (sulfated glycosaminoglycan) assay and hydroxyproline (collagen) assay. PLLA coated PGA scaffolds were analyzed using double stranded DNA quantification as a reflection of cellularity and confocal laser microscopy in a fluorescent cell viability assay. Results: The highest cellularity occurred in PGA constructs cultured in a rotating bioreactor, which also had a mean sulfated glycosaminoglycan content of 22.3μg per scaffold. PGA constructs cultured in static conditions had the lowest cellularity. Cells had difficulty adhering to OPLA and the PLLA coating of PGA scaffolds; cellularity was inversely proportional to the concentration of PLLA used. PLLA coating did not prevent dissolution of the PGA scaffolds. All cell scaffold types and culture conditions produced non-uniform cellular distribution. Discussion/ Conclusion: FLS-seeding of PGA scaffolds cultured in a rotating bioreactor resulted in the most optimal cell and matrix characteristics seen in this study. Cells grew only in the pores of the OPLA sponge, and could not adhere to the PLLA coating of PGA scaffold, due to the hydrophobic property of PLA. While PGA culture in a bioreactor produced measureable GAG, no culture technique produced visible collagen. For this reason, and due to the dissolution of PGA scaffolds, the culture conditions and scaffolds described here are not recommended for inducing fibrochondrogenesis in equine FLS for meniscal tissue engineering.

scholarly journals 3D Printed Poly(ε-Caprolactone)/Meniscus Extracellular Matrix Composite Scaffold Functionalized With Kartogenin-Releasing PLGA Microspheres for Meniscus Tissue Engineering

2021 ◽  
Vol 9 ◽  
Author(s):  
Hao Li ◽  
Zhiyao Liao ◽  
Zhen Yang ◽  
Cangjian Gao ◽  
Liwei Fu ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Extracellular Matrix ◽  
Composite Scaffold ◽  
Prolonged Release ◽  
Plga Microspheres ◽  
Cell Vitality ◽  
Meniscal Tissue ◽  
Meniscus Tissue ◽  
Meniscus Regeneration

Meniscus tissue engineering (MTE) aims to fabricate ideal scaffolds to stimulate the microenvironment for recreating the damaged meniscal tissue. Indeed, favorable mechanical properties, suitable biocompatibility, and inherent chondrogenic capability are crucial in MTE. In this study, we present a composite scaffold by 3D printing a poly(ε-caprolactone) (PCL) scaffold as backbone, followed by injection with the meniscus extracellular matrix (MECM), and modification with kartogenin (KGN)-loaded poly(lactic-co-glycolic) acid (PLGA) microsphere (μS), which serves as a drug delivery system. Therefore, we propose a plan to improve meniscus regeneration via KGN released from the 3D porous PCL/MECM scaffold. The final results showed that the hydrophilicity and bioactivity of the resulting PCL/MECM scaffold were remarkably enhanced. In vitro synovium-derived mesenchymal stem cells (SMSCs) experiments suggested that introducing MECM components helped cell adhesion and proliferation and maintained promising ability to induce cell migration. Moreover, KGN-incorporating PLGA microspheres, which were loaded on scaffolds, showed a prolonged release profile and improved the chondrogenic differentiation of SMSCs during the 14-day culture. Particularly, the PCL/MECM-KGN μS seeded by SMSCs showed the highest secretion of total collagen and aggrecan. More importantly, the synergistic effect of the MECM and sustained release of KGN can endow the PCL/MECM-KGN μS scaffolds with not only excellent cell affinity and cell vitality preservation but also chondrogenic activity. Thus, the PCL/MECM-KGN μS scaffolds are expected to have good application prospects in the field of MTE.

Neutrophil migration through in vitro interstitial matrix

1992 ◽  
Vol 50 (1) ◽  
pp. 894-895
Author(s):  
J. Roemer ◽  
S.R. Simon
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Cell Migration ◽  
Smooth Muscle Cells ◽  
Inflammatory Cell ◽  
Neutrophil Migration ◽  
Culture Conditions ◽  
Aortic Smooth Muscle ◽  
Specific Culture

We are developing an in vitro interstitial extracellular matrix (ECM) system for study of inflammatory cell migration. Falcon brand Cyclopore membrane inserts of various pore sizes are used as a support substrate for production of ECM by R22 rat aortic smooth muscle cells. Under specific culture conditions these cells produce a highly insoluble matrix consisting of typical interstitial ECM components, i.e.: types I and III collagen, elastin, proteoglycans and fibronectin.

scholarly journals Cell-derived Extracellular Matrix as maintaining Biomaterial for adipogenic differentiation

2020 ◽  
Vol 6 (3) ◽  
pp. 410-413
Author(s):  
Petra J. Kluger ◽  
Svenja Nellinger ◽  
Simon Heine ◽  
Ann-Cathrin Volz
Keyword(s):  
Tissue Engineering ◽  
Extracellular Matrix ◽  
Adipogenic Differentiation ◽  
Cellular Activity ◽  
Adipose Tissue Engineering ◽  
Physical Support ◽  
Supporting Effect ◽  
Brdu Assay ◽  
The Impact

AbstractThe extracellular matrix (ECM) naturally surrounds cells in humans, and therefore represents the ideal biomaterial for tissue engineering. ECM from different tissues exhibit different composition and physical characteristics. Thus, ECM provides not only physical support but also contains crucial biochemical signals that influence cell adhesion, morphology, proliferation and differentiation. Next to native ECM from mature tissue, ECM can also be obtained from the in vitro culture of cells. In this study, we aimed to highlight the supporting effect of cell-derived- ECM (cdECM) on adipogenic differentiation. ASCs were seeded on top of cdECM from ASCs (scdECM) or pre-adipocytes (acdECM). The impact of ECM on cellular activity was determined by LDH assay, WST I assay and BrdU assay. A supporting effect of cdECM substrates on adipogenic differentiation was determined by oil red O staining and subsequent quantification. Results revealed no effect of cdECM substrates on cellular activity. Regarding adipogenic differentiation a supporting effect of cdECM substrates was obtained compared to control. With these results, we confirm cdECM as a promising biomaterial for adipose tissue engineering.

scholarly journals Recapitulating Cardiac Structure and Function In Vitro from Simple to Complex Engineering

Micromachines ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 386
Author(s):  
Ana Santos ◽  
Yongjun Jang ◽  
Inwoo Son ◽  
Jongseong Kim ◽  
Yongdoo Park
Keyword(s):  
Tissue Engineering ◽  
Extracellular Matrix ◽  
Computational Modeling ◽  
Cardiac Tissue ◽  
Cardiac Development ◽  
Heart Tissue ◽  
Cardiac Tissue Engineering ◽  
Cardiac Cells

Cardiac tissue engineering aims to generate in vivo-like functional tissue for the study of cardiac development, homeostasis, and regeneration. Since the heart is composed of various types of cells and extracellular matrix with a specific microenvironment, the fabrication of cardiac tissue in vitro requires integrating technologies of cardiac cells, biomaterials, fabrication, and computational modeling to model the complexity of heart tissue. Here, we review the recent progress of engineering techniques from simple to complex for fabricating matured cardiac tissue in vitro. Advancements in cardiomyocytes, extracellular matrix, geometry, and computational modeling will be discussed based on a technology perspective and their use for preparation of functional cardiac tissue. Since the heart is a very complex system at multiscale levels, an understanding of each technique and their interactions would be highly beneficial to the development of a fully functional heart in cardiac tissue engineering.

scholarly journals Transcriptome comparisons of in vitro intestinal epithelia grown under static and microfluidic gut-on-chip conditions with in vivo human epithelia

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kornphimol Kulthong ◽  
Guido J. E. J. Hooiveld ◽  
Loes Duivenvoorde ◽  
Ignacio Miro Estruch ◽  
Victor Marin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Culture Conditions ◽  
Gene Set Enrichment Analysis ◽  
Shear Stresses ◽  
Static Culture ◽  
Dynamic Culture ◽  
Intestinal Segments ◽  
On Chip

AbstractGut-on-chip devices enable exposure of cells to a continuous flow of culture medium, inducing shear stresses and could thus better recapitulate the in vivo human intestinal environment in an in vitro epithelial model compared to static culture methods. We aimed to study if dynamic culture conditions affect the gene expression of Caco-2 cells cultured statically or dynamically in a gut-on-chip device and how these gene expression patterns compared to that of intestinal segments in vivo. For this we applied whole genome transcriptomics. Dynamic culture conditions led to a total of 5927 differentially expressed genes (3280 upregulated and 2647 downregulated genes) compared to static culture conditions. Gene set enrichment analysis revealed upregulated pathways associated with the immune system, signal transduction and cell growth and death, and downregulated pathways associated with drug metabolism, compound digestion and absorption under dynamic culture conditions. Comparison of the in vitro gene expression data with transcriptome profiles of human in vivo duodenum, jejunum, ileum and colon tissue samples showed similarities in gene expression profiles with intestinal segments. It is concluded that both the static and the dynamic gut-on-chip model are suitable to study human intestinal epithelial responses as an alternative for animal models.

Establishment of a resazurin-based aortic valve tissue viability assay for dynamic culture in a microphysiological system

2021 ◽  
pp. 1-12
Author(s):  
C. Dittfeld ◽  
M. Winkelkotte ◽  
S. Behrens ◽  
F. Schmieder ◽  
A. Jannasch ◽  
...  
Keyword(s):  
Aortic Valve ◽  
Dynamic Condition ◽  
Thermoplastic Polyurethane ◽  
Membrane Integrity ◽  
Tissue Viability ◽  
Preliminary Results ◽  
Dynamic Culture ◽  
Resazurin Assay ◽  
Static Conditions

BACKGROUND/AIM: Tissue pathogenesis of aortic valve (AV) stenosis is research focus in cardiac surgery. Model limitations of conventional 2D culture of human or porcine valvular interstitial/endothelial cells (VIC/VECs) isolated from aortic valve tissues but also limited ability of (small) animal models to reflect human (patho)physiological situation in AV position raise the need to establish an in vitro setup using AV tissues. Resulting aim is to approximate (patho)physiological conditions in a dynamic pulsatile Microphysiological System (MPS) to culture human and porcine AV tissue with preservation of tissue viability but also defined ECM composition. MATERIALS/METHODS: A tissue incubation chamber (TIC) was designed to implement human or porcine tissues (3×5 mm) in a dynamic pulsatile culture in conventional cell culture ambience in a MPS. Cell viability assays based on lactate dehydrogenase (LDH)-release or resazurin-conversion were tested for applicability in the system and applied for a culture period of 14 days with interval evaluation of tissue viability on every other day. Resazurin-assay setup was compared in static vs. dynamic culture using varying substance saturation settings (50–300μM), incubation times and tissue masses and was consequently adapted. RESULTS: Sterile dynamic culture of human and porcine AV tissue segments was established at a pulsatile flow rate range of 0.9–13.4μl/s. Implementation of tissues was realized by stitching the material in a thermoplastic polyurethane (TPU) –ring and insertion in the TIC-MPS-system. Culture volume of 2 ml caused LDH dilution not detectable in standard membrane integrity assay setup. Therefore, detection of resazurin-conversion of viable tissue was investigated. Optimal incubation time for viability conversion was determined at two hours at a saturated concentration of 300μM resazurin. Measurement in static conditions was shown to offer comparable results as dynamic condition but allowing optimal handling and TIC sterilization protocols for long term culture. Preliminary results revealed favourable porcine AV tissue viability over a 14 day period confirmed via resazurin-assay comparing statically cultured tissue counterparts. CONCLUSIONS: Human and porcine AV tissue can be dynamically cultured in a TIC-MPS with monitoring of tissue viability using an adapted resazurin-assay setup. Preliminary results reveal advantageous viability of porcine AV tissues after dynamic TIC-MPS culture compared to static control.

Scaffolds in tissue engineering of blood vessels

2010 ◽  
Vol 88 (9) ◽  
pp. 855-873 ◽  
Author(s):  
Divya Pankajakshan ◽  
Devendra K. Agrawal
Keyword(s):  
Tissue Engineering ◽  
Extracellular Matrix ◽  
Blood Vessels ◽  
Vascular Tissue ◽  
Small Diameter ◽  
Biological Scaffolds ◽  
Hemodynamic Forces ◽  
Biodegradable Scaffolds

Tissue engineering of small diameter (<5 mm) blood vessels is a promising approach for developing viable alternatives to autologous vascular grafts. It involves in vitro seeding of cells onto a scaffold on which the cells attach, proliferate, and differentiate while secreting the components of extracellular matrix that are required for creating the tissue. The scaffold should provide the initial requisite mechanical strength to withstand in vivo hemodynamic forces until vascular smooth muscle cells and fibroblasts reinforce the extracellular matrix of the vessel wall. Hence, the choice of scaffold is crucial for providing guidance cues to the cells to behave in the required manner to produce tissues and organs of the desired shape and size. Several types of scaffolds have been used for the reconstruction of blood vessels. They can be broadly classified as biological scaffolds, decellularized matrices, and polymeric biodegradable scaffolds. This review focuses on the different types of scaffolds that have been designed, developed, and tested for tissue engineering of blood vessels, including use of stem cells in vascular tissue engineering.

scholarly journals Gravity-Based Flow Efficient Perfusion Culture System for Spheroids Mimicking Liver Inflammation

Biomedicines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1369
Author(s):  
Young-Su Kim ◽  
Arun Asif ◽  
Abdul Rahim Chethikkattuveli Salih ◽  
Jae-Wook Lee ◽  
Ki-Nam Hyun ◽  
...  
Keyword(s):  
Culture System ◽  
Disease Modeling ◽  
Perfusion Culture ◽  
Drug Analysis ◽  
Culture Conditions ◽  
Current Approach ◽  
Spheroid Culture ◽  
Organ Specific ◽  
Static Conditions

The spheroid culture system provides an efficient method to emulate organ-specific pathophysiology, overcoming the traditional two-dimensional (2D) cell culture limitations. The intervention of microfluidics in the spheroid culture platform has the potential to enhance the capacity of in vitro microphysiological tissues for disease modeling. Conventionally, spheroid culture is carried out in static conditions, making the media nutrient-deficient around the spheroid periphery. The current approach tries to enhance the capacity of the spheroid culture platform by integrating the perfusion channel for dynamic culture conditions. A pro-inflammatory hepatic model was emulated using a coculture of HepG2 cell line, fibroblasts, and endothelial cells for validating the spheroid culture plate with a perfusable channel across the spheroid well. Enhanced proliferation and metabolic capacity of the microphysiological model were observed and further validated by metabolic assays. A comparative analysis of static and dynamic conditions validated the advantage of spheroid culture with dynamic media flow. Hepatic spheroids were found to have improved proliferation in dynamic flow conditions as compared to the static culture platform. The perfusable culture system for spheroids is more physiologically relevant as compared to the static spheroid culture system for disease and drug analysis.

scholarly journals Engineering large cartilage tissues using dynamic bioreactor culture at defined oxygen conditions

2018 ◽  
Vol 9 ◽  
pp. 204173141775371 ◽  
Author(s):  
Andrew C Daly ◽  
Binulal N Sathy ◽  
Daniel J Kelly
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Culture Conditions ◽  
Cartilage Tissue ◽  
Bioreactor Culture ◽  
Dynamic Culture ◽  
Oxygen Conditions ◽  
Static Conditions

Mesenchymal stem cells maintained in appropriate culture conditions are capable of producing robust cartilage tissue. However, gradients in nutrient availability that arise during three-dimensional culture can result in the development of spatially inhomogeneous cartilage tissues with core regions devoid of matrix. Previous attempts at developing dynamic culture systems to overcome these limitations have reported suppression of mesenchymal stem cell chondrogenesis compared to static conditions. We hypothesize that by modulating oxygen availability during bioreactor culture, it is possible to engineer cartilage tissues of scale. The objective of this study was to determine whether dynamic bioreactor culture, at defined oxygen conditions, could facilitate the development of large, spatially homogeneous cartilage tissues using mesenchymal stem cell laden hydrogels. A dynamic culture regime was directly compared to static conditions for its capacity to support chondrogenesis of mesenchymal stem cells in both small and large alginate hydrogels. The influence of external oxygen tension on the response to the dynamic culture conditions was explored by performing the experiment at 20% O2 and 3% O2. At 20% O2, dynamic culture significantly suppressed chondrogenesis in engineered tissues of all sizes. In contrast, at 3% O2 dynamic culture significantly enhanced the distribution and amount of cartilage matrix components (sulphated glycosaminoglycan and collagen II) in larger constructs compared to static conditions. Taken together, these results demonstrate that dynamic culture regimes that provide adequate nutrient availability and a low oxygen environment can be employed to engineer large homogeneous cartilage tissues. Such culture systems could facilitate the scaling up of cartilage tissue engineering strategies towards clinically relevant dimensions.

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