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

Organ Number of US patients

Skin >4 millionBone >1 millionHeart 750,000Liver 200,000Neuromuscular 200,000Kidney 600,000

http://www.usatoday.com/educate/health/snaps/organ.htmCheng, J. UIUC, 2010

Tissue Engineering History

• 1980 Yannas: Collagen GAG scaffold for dermal‐regeneration (‘artificial skin’); Integra

• 1985 Wolter/Meyer: 1st use of term, TE; “Sessile Macrophages Forming Clear Endoethelium-like Membrane on Inside of Successful

• 1993 Langer/Vacanti: Science paper on TE; cells inmatrices for tissue formation in vitro

• 1995 Griffith/Vacanti/Langer: Chondrocytes inPGA 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.

Tayalia et al. Adv Mater, 2008.

Multiphoton polymerization

Current 3D Technologies

Stachowiak et al. Adv Funct Mat, 2005, 17, 399 – 403.

Current 3D TechnologiesSolution 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 αvβ3 j• Paxillin• Vinculin• Focal adhesion kinase

α5 and paxillin colocalize in 3D rather than separate to fibrillar and focal sites• “3D Matrix Adhesion”

Fibrillar Adhesions – generate extracellular fibrillars of fibronectin• α5β1 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, α5 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 β1, β4, and α6 integrins

• Distribution patterns radically different• S-1 basally distributed integrins indicative

of polarization• T4-2 integrins randomly distributed

• Western blot revealed overexpression of β1 and β4 in tumorigenc cells

• Malignant behavior a result of integrin changes?

Inhibitory β1 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 β1 integrin expression associated with loss of growth control and pertrubed morphogenesis

• A reduction of β1 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

Experimental Design

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 TemplatingFreeze 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 mm

Lewis and Gratson,,Materials Today (2004).

Direct-Write for Tissue Engineering

Polyacrylamide Scaffolds for 3T3 Murine Fibroblasts pHEMA Scaffolds for Rat Hippocampal Culture

HA-Silk Scaffolds for Osteogenic Growth Bio-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. Parker Wu, W. et al. Adv. Mat. 2011.

Material Requirements

Biocompatible Biodegradable Printable 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

Biocompatible Biodegradable Printable 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

Biocompatible Biodegradable Printable 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 1 Day 3 Day 5

Day 7 Day 9 Day 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.