Presentation on theme: "Cell and Tissue Engineering: 3D Effects Lucas Osterbur April 18, 2011 BIOE 506."— Presentation transcript:
Cell and Tissue Engineering: 3D Effects Lucas Osterbur April 18, 2011 BIOE 506
Overview Tissue Engineering Background Tissue Engineering in the Third Dimension Motivation Current Fabrication Technologies Effects of 3D – Literature Review Cell-ECM adhesions Cancer Phenotypes Stem Cell Differentiation Personal Work in 3D Tissue Engineering
An interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function Tissue Engineering Theoretical solution to wide variety of medical diseases and defects Organ failure critical organ shortages expense massive immunorepressive responses Tissue trauma tissues without regenerative ability large defect regenerative tissue Models for human genetic disorders http://biomed.brown.edu/Courses/BI108/BI108_2007_Groups/group12/Homepage.html
Tissue Engineering Demand Surgeries per year related to organ deficiencies OrganNumber of US patients Skin>4 million Bone>1 million Heart750,000 Liver200,000 Neuromuscular200,000 Kidney600,000 http://www.usatoday.com/educate/health/snaps/organ.htm Cheng, J. UIUC, 2010
Tissue Engineering History 1980 Yannas: Collagen‐GAG scaffold for dermal regeneration (‘artificial skin’); Integra 1985 Wolter/Meyer: 1 st use of term, TE; “Sessile Macrophages Forming Clear Endoethelium- like Membrane on Inside of Successful 1993 Langer/Vacanti: Science paper on TE; cells in matrices for tissue formation in vitro 1995 Griffith/Vacanti/Langer: Chondrocytes in PGA scaffold: the earmouse Zhong et al. Nanomed and Nanobio, 2010, 2, 510-525. Vacanti, C., Langer, R. et al. Plast and Reconstr Surg. 1995, 96, 753.
Tissue Engineering Triad Scaffold Porous absorbable material Regulation of cell functions Cells hESCs, MSCs, fibroblasts, chondrocytes, etc. Autologous, allogenic, xenogenic Regulators Chemical – growth factors, small molecules Genetic – viruses, nucleic acids Mechanical – mechanical loading, flow conditions Harley, B., UIUC, 2010
Why 3D? Tissues and organs where cell functions occur are 3D Our ability to understand formation, function, and pathology often depend on 2D culture or animal models 2D Cultures morphology cell-cell interactions cell-matrix interactions differentiation Animal Models discrepancies in gene studies drug therapeutic response autoimmune disease Yamada et al. Cell, 2007, 130, 601-610
Current 3D Technologies Tayalia et al. Adv Mater, 2008. Multiphoton polymerization
Current 3D Technologies Stachowiak et al. Adv Funct Mat, 2005, 17, 399 – 403.
Current 3D Technologies Solution Forming Calieri, S. PhD Thesis, Dr. B. Harley
Literature Taking Cell-Matrix Adhesions to the Third Dimension Cukierman, E.; Pankov, R.; Stevens, D.R.; Yamada, K.M. Science, 2001, 294, 1708-1712.
Motivation Current understanding of cell-matrix adhesions based on in vitro studies of cells and adhesive components cells display altered morphology and polarity compared to in vivo Focal Adhesions – integrin based structures that mediate strong cell-substrate adhesion integrin α v β 3 j Paxillin Vinculin Focal adhesion kinase α 5 and paxillin colocalize in 3D rather than separate to fibrillar and focal sites “3D Matrix Adhesion” Fibrillar Adhesions – generate extracellular fibrillars of fibronectin α 5 β 1 j tensin
Fibroblast Attachment 10 minute attachment assay of fibroblasts to various substrates a.u. = relative number of cells attaching to fibronectin factor of 6 increase in attachment in control population
Morphology Threshholded digital images of 4 cells Only cells distributed on 3D matrix attained elongated in vivo shape within 5 hrs. Although 2D substrate produced elongated cells after 18 hrs, uncommon branched terminals noted Only 3D matrix cells significantly altered by treatment with mAb16
Cell Migration and Proliferation Time lapse video microscopy 16 paths per treatment method Migration was promoted by 3D matrix and prohibited by cell treatment with mAb16 Rates of proliferation more than twice as high in 3D matrix condition
Colocalization in 3D localization of paxillin, α 5 integrin, and fibronectin were examined in 5 substrates cell-derived 3D matrix tissue-derived 3D matrix fibronectin cell-derived 2D matrix 3D fibronectin Triple colocalization only noted in substrates with a 3D matrix and cell-derived components
Conclusion Separate localizing of paxillin and integrins with traditional 2D methods do not adequately describe in vivo fibroblast morphologies A 3D matrix is necessary for colocalization of adhesion proteins aligned with fibronectin within the matrix Other components of the cell-derived matrices are also necessary to demonstrate this behavior Traditional culture methods used to study ECM-Cellular adhesions may not be appropriate for modeling conditions found in vivo
Literature Reversion of Malignant Phenotype of Human Breast Cells in Three-Dimensional Culture and In Vivo by Integrin Blocking Antibodies Weaver, V.M.; Petersen, O.W.; Wang, F.; Larabell, C.A.; Briand, P.; Damsky, C.; Bissel, M.J. The Journal of Cell Biology, 1997, 137, 231-245.
Motivation ECM signaling pathways may contain suppressor checkpoints the direct and impinge upon cell architecture and tissue Adherens and other cell-cell junctions are intimately tied into pathways Final tissue phenotypes may be determined by these pathways and signaling sources How can 3D culturing methods modify these pathways and the resulting morphogenesis? About 200,000 new cases of invasive breast cancer will be diagnosed in women in 2011 In vitro culture may provide a model system to better research the proliferation of cancerous cells and the effects of therapeutic drugs In breast cancer models, ECM is known to modulate both biochemical and biomechanical signaling events in vivo American Cancer Society
Experimental Design HMT-3522 breast cancer series used for all experimental cultures Subline of cells became spontaneously tumorigenic after 238 passages All non-malignant cells (S-1) derived from passage 50 Malignant cells (T4-2) derived from passage 238 Cell line offers unique tool for addressing mechanisms behind malignant conversion in breast cancer cells Postulate that morphology and behavior of cells can be modified by altering cell-ECM and cell-cell interactions Commercially available Matrigel used as substrate for 3D matrix Monolayers grown on thinly coated plastic dishes used for 2D model
Initial Morphology Only slightly noticeable differences found in growth rate and morphology in 2D Profound differences evident after just 4 days in 3D system S-1 formed organized structure similar to those found in benign tumors T4-2 formed large, loosely organized and invasive colonies Immunostaining demonstrate basal layer deposition for S-1 cells Cetenin/Cadherin interaction reduced in T4-2 cells
Integrin Distribution Both cell types expressed β 1, β 4, and α 6 integrins Distribution patterns radically different S-1 basally distributed integrins indicative of polarization T4-2 integrins randomly distributed Western blot revealed overexpression of β 1 and β 4 in tumorigenc cells Malignant behavior a result of integrin changes?
Inhibitory β 1 Antibody S-1 and treated T4-2 exhibit localized nuclei and well organized F-actin E-cadherins and β-cetenins are colocalized in S-1 and treated T4-2 cells Control T4-2 cells highly disorganized Reversible process
Conclusions The use of related human cell lines, one malignant and one benign allowed for the study of the fundamental role of cell junctions in tissue morphogenesis Cells produced drastically different results depending on culture in 2D or 3D substrates T4-2 tumorigenic cells have increased β 1 integrin expression associated with loss of growth control and pertrubed morphogenesis A reduction of β 1 integrin activity is sufficient to revert the tumor phenotype Malignancy of breast tumor cells could potentially be controlled and restored to normal cell function by experimenting with these interactions
Literature Osteogenic Differentiation of Mouse Embryonic Fibroblast Cells in a Three-Dimensional Self-Assembling Peptide Scaffold Garreta, E.; Genove, E.; Borros, S.; Semino, C.E. Tissue Engineering, 2006, 8, 2215-2227.
Embryonic Stem Cells Long-Term Self-Renewal: Can be proliferated for over 100 passages in culture Keep normal karyotype Pluripotency: Human embryonic stem cells have the potential to differentiate into all cell types How can we best use bioengineering to develop methods for in vitro development of functional tissues from this cell source?
Motivation Mouse embryonic fibroblast (MEFs) typically employed as a feeder layer for ESCs Chondrogenic induction of MEFs in high concentration in the presence of bone morphogenic proteins has been found to lead to ossification, providing a model for further bone formation Cell – matrix adhesions are critical in determining development, differentiation and remodeling of bone 2D cultures have mostly been used as the substrates for osteoblast-specific differentiation 3D environments may improve upon the process by attaining the proper niche to simulate the in vivo environment
Loss of Pluripotency Tagged Oct-4 promoter in mESC cells were monitored throoughout experimental procedure Loss of fluorescence indicated loss of pluripotency Confirmation via immunostaining with antibody anti-Oct 4 (red) Western blot final verification
MEF Osteogenic Differentiation von Kassa staining (black) confirm mineralization in 3D culture systems No mineralization found in 2D system Osteopontin, marker for early stage osteoblast formation, only expressed in 3D immunostaining 3D culture only system to develop small, elongated phenotype Phenotype lost when replated in 2D
mESC Osteogenic Differentiation von Kassa staining confirm mineralization in culture systems Immunostaining shows presence of ostepontin (red) in cultures Control staining demonstrates effect of osteogenic pathway Most notable 2D vs 3D difference in cell proliferation
Conclusion Experimental procedure let to controlled loss of pluripotency in mESCs Culture systems were successfully induced into an osteogenic pathway Dimensionality of culture system is important in determining progress of osteogenic differentiation in mESCs and MEFs Evidence for MEF cultures are much more supportive of postulation
3D Fabrication Tayalia et al. Adv Mater, 2008. Lee et al. J Mater Chem, 2006. Kim et al. Biomaterials, 2005. Tayalia et al. Adv Mater, 2008. Fabrication limitations: Available materials Random architecture Severe processing conditions Multiphoton polymerization Particle Templating Freeze forming Stachowiak et al. Adv Funct Mat, 2005, 17, 399 – 403. Calieri, S. PhD Thesis, Dr. B. Harley
Direct-Write Assembly Robotic x-y-z control Layer-by-layer assembly Pressure driven flow of continuous filament through deposition nozzle Materials flexibility (polymers, ceramics, metals…) Range of architectures and feature sizes from ~ 1 μm to mm’s 200 m Lewis and Gratson,,Materials Today (2004).
Direct-Write for Tissue Engineering Polyacrylamide Scaffolds for 3T3 Murine FibroblastspHEMA Scaffolds for Rat Hippocampal Culture HA-Silk Scaffolds for Osteogenic GrowthBio-Inspired Microvascular Networks Barry, R, Shepherd, R. et al. Adv Mat, 2009, 21, 1-4.Hansen Shepherd, J., Parker, S. et al. Adv Funct Mat. 2011, 21, 47-54 PhD thesis – S. ParkerWu, W. et al. Adv. Mat. 2011.
Material Requirements BiocompatibleBiodegradablePrintable Rheology
Material Requirements Biocompatible Poly(Hyaluronic Acid) Natural biopolymer; primary component of ECM Bioactive material Proliferation and Survival Cell Motility Cell-Cell/Cell-Substrate Adhesion Long term proliferation in bulk hydrogel Biodegradable Gerecht, S. Burdick, J. et al. PNAS, 2005, 104, 11298–11303. Pluripotent hESCs post-20 day culture Printable Rheology
Material Requirements BiocompatibleBiodegradablePrintable Rheology K.P. Vercruysse et al. J Chromatogr B, 1994, 6, 179-190 Hyaluronidase Natural enzyme to degrade pHA Well defined kinetics Degradation of pHA in vivo
Material System Ink Composition high and low MW methacrylated HA PEGDA Irgacure Water/glycerol solvent BiocompatibleBiodegradablePrintable Rheology Shear thinning gel allows for nozzle deposition without clogging G’ > G” for spanning features
Hyaluronic Scaffolds 200 µm Scaffolds successfully printed with hyaluronic acid ink via direct-write assembly Printing and UV curing carried out simultaneously Feature sizes can be varied systematically 100 µm
Cell Growth Day 1Day 3Day 5 Day 7Day 9Day 11 with Yijie Geng, Dr. Fei Wang
Conclusion Cell and tissue engineering are moving into the third dimension as researchers seek to investigate cell behavior in the most biomimetic environment possible. As new technologies and methods make this a reality, new cell activities related to differentiation, morphogenesis, and cell adhesions are being discovered that continue to widen the gap between 2D and 3D milieus. The field has an exciting and practically limitless future, and the push from in vitro to in vivo on the lab bench will be an integral part how the progression unfolds.