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Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineerin g Cells Scaffol ds Biorect ors Signals

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Page 1: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Cell and Tissue Engineering,

Nanotechnology

BIOE 506

Muqeem Qayyum

Tissue Engineering

Cells Scaffolds

Biorectors Signals

Page 2: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Why?

• In-vitro • In-vivo

Because we can no longer to view a cell as self contained unit existing in a passive structural network. Thus to properly study the cell interactions it must be in a 3D environment.

Image sourse: www.millipore.com/imagesfarm1.static.flickr.com

Page 3: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

EXTRACELLULAR MATRIX (ECM)

• Consists of a 3D array of protein fibers and filaments embedded in a hydrated gel of glycosaminoglycans

• ECM molecules– Glycosaminoglycans– Proteoglycans– Proteins

Proteins in ECM act as either structural members or adhesion sites

Adhesion proteinsFibronectinLaminin

Structural proteinsCollagenElastin

Page 4: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Definitions• Tissue Engineering:

– Interdisciplinary field addressing the improvement, repair, or replacement of tissue/organ function.

• Nanotechnology: – Is study of gaining control of structures at the atomic, molecular levels.

• Biocompatibility– Material should not elicit a significant or prolonged inflammatory

response.4

• Biodegradability– The degraded products of the scaffold must have a safe route for

removal from the host.4

• Mechanical Strength– The scaffold must be able to provide support to the forces applied to it

and the surrounding tissues (especially true for the engineering of weight-bearing orthopedic tissues). 4

Page 5: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Tissue Engineering (TE)• Scaffolds

Biomaterials, which may be natural or artificially derived, providing a platform for cell function, adhesion and transplantation

• CellsAny class of cell, such as stem or mesenchymal cell

• SignalsProteins and growth factors driving the cellular functions of interest

• Bioreactor System that supports a biologically active environment (ex. Cell culture)

Image sourse: Stke.sciencemag.org, Nature.com

Page 6: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

DISCUSSION

Barnes, C.P., Nanofiber technology: Designing the next generation of tissue engineering scaffolds1

Page 7: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Nanofiber Technology

• Techniques of nanofibers for tissue engineering

• Characteristics of tissue engineering nanofibrous structures

• Electrospinning of synthetic and natural polymers

Page 8: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

3 techniques to achieve nanofibers for TE

Self-assembly

Phase separation

Electrospinning

Page 9: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Self-assembly

• Relies on non-covalent interactions to achieve spontaneously assembled 3D structure.

• Biopolymers such as peptides and nucleic acids are used. Example is peptide-amphiphile (PA)

• (A) Chemical structure of (PA)

• (B) Molecular model of the PA showing the narrow hydrophobic tail to the bulkier peptide region

• (C) Schematic of PA molecules into a cylindrical micelle.5

Nanofiberpeptide-amphiphile

Page 10: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Phase separation• This process involves dissolving of a

polymer in a solvent at a high temperature followed by a liquid–liquid or solid–liquid phase separation induced by lowering the solution temperature

• Capable of wide range of geometry and dimensions include pits, islands, fibers, and irregular pore structures

• Simpler than self-assembly a) powder, b) scaffolds with continuous network, c) foam with closed pores.4

SEM of nanofibrous scaffold with interconnected spherical macropores1

Page 11: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Electrospinning

• This process involves the ejection of a charged polymer fluid onto an oppositely charged surface.

• multiple polymers can be combined

• control over fiber diameter and scaffold architecture

A schematic of the electrospinning process to illustrate the basic phenomena and process components1

Page 12: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Overview Nanofiber

No consensus of gold standard for creating native ECM

Process Ease Advantages Limitations

Self-assembly Difficult Produce fiber on lowest ECM scale (5-8 nm)

• Lack of control• Limitation on polymers

Phase separation Easy • Tailorable mechanical prop.• Batch to batch consistency

•Lab scale production• Limitation on polymers

Electrospinning Easy • Cost effective• Long continuous fibers• Tailorable mech properties, size & shape

• Large scale fibers• No control over 3D pore structure

Page 13: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Characteristics of TE nanofibrous structures

Page 14: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Scaffolds under Electrospinning

• Goal of scaffolds• Properties• Materials

– Synthetic – Natural

• Limitations

Page 15: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Goals/Properties

Goal of TE is to combine cell, scaffold (artificial ECM) and bioreactor to design and fabricate tissues and organs.

• Biocompatible

Scaffolds Properties

• Promote growth • Maintain 3-D structure • Non-immunogenic

Design scaffold with maximum control over:biocompatibility (chemical)biodegradability (mechanical)

• Support tissue and cell forces

Page 16: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Materials

• Synthetic– Mimic mechanical

properties (strength, elongation and knot retention)

– Mass production– Degradation between

hosts is minimal

• Natural – Cell recognition – Biodegradable – Difficult degradation

control between host

Page 17: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Synthetic• Polyglycolic acid (PGA)

– Highly crystalline, hydrophilic, byproduct is glycolic acid • Polylactic acid (PLA)

– Hydrophobic, lower melting temperature, byproduct is lactic acid• Polydioxanone (PDO)

– Highly crystalline• Polycaprolactone (PCL)

– Semi-crystalline properties, easily co-polymerized, byproduct caproic acid

• Blends– PGA-PLA– PGA-PCL– PLA-PCL– PDO-PCL

Page 18: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Poly(glycolic acid) (PGA)Advantages:• Biocompatible & biodegradable • bioabsorption (2-4 wks)• electrospinning yields diameters ~

200 nm• Good choice for high strength and

elasticity and fast degrading material

Disadvantages:• fast degradation causes pH change

• Tissue may require buffering capacity

SEM showing the random fiber arrangement (left) and the aligned fiber orientation (right) (1600× magnification).

Page 19: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

PGA

Fig. summarizes mechanical properties in terms of the elastic modulus and strain at failure of PGA in a uniaxial model.

• Spinning orientation affects scaffold elastic modulus

• The overall results exhibit a correlation between the fiber diameter and orientation and the elastic modulus and strain to failure

Page 20: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Poly(lactic acid) (PLA)Advantages:• Biocompatible & biodegradable • bioabsorption (30 wks)• Good choice for drug delivery do to

predictable degradation

Disadvantages:• Larger diameter fibers ~ microscale

SEM showing the random oriented PLA from cholorform (left) and the randomly oriented PLA from HFP (right)

Page 21: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

PGA + PLA blends (PLGA)Group Tested copolymers of following

ratios:• 75%PLA–25%PGA• 50%PLA–50%PGA,• blended PLA and PGA together in

HFP at ratios of 100:0, 75:25, 50:50, 25:75

Group found:

• Hydrophilicity proportional to composition of copolymer

• Degradation rate proportional to composition of copolymer

Page 22: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

PLGA

Applications of PLGA:

• cardiac tissue in mice for tissue regeneration

• individual cardiomyocytes attachment at seeding

• scaffold loaded with antibiotics for wound healing

mechanical properties, such as tangential modulus, peak stress, and strain to failure, of these copolymers and blends appear to be controlled by the fiber/polymer composition

Page 23: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Polydioxanone (PDO)Advantages:• Biocompatible & biodegradable • degradation rate between PGA/PLA• Shape memory• Excellent flexibility • Modulus comparable collagen and elastin• Good source for future vascular grafts

Disadvantages:• Lack on knot retention• Lack of adaptability to developing tissue

Results of the fiber diameter analysis versus PDO concentration illustrating the linear relationship between electrospinning solution concentration and fiberdiameter.

SEM of 180 nm diameter randomly oriented fibrous structures

Page 24: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Polycaprolactone (PCL)Advantages:• Biocompatible & biodegradable • Inexpensive • Highly elastic • Slow degradation (1 – 2 yrs)• Good choice for Human

mesenchymal stem cells (hMSCs) seeding to induce differentiation

Disadvantages:• No shape retention (highly elastic)

Applications:• Bone tissue strengthening

• Cardiac grafts

• Collagen and cellular interaction

• Differentiation with MSC cells

Page 25: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

PGA + PCL blendAdvantages:• PGA – high stress tolerance• PCL – highly elastic • Optimum combination PCL/PGA ~ 1/3• Longer degradation time ~ 3 months

(PCL-2 yrs, PGA 2-4 wks)

Page 26: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

PLA + PCL blendsAdvantages:• Greater elasticity than PGA+PCL• Similar tensile strength to PLA• ~5% addition of PCL increased

strain by 8 fold• Overall best synthetic ECM for

cardiac applications

Disadvantages:

• Decreasing PLA+PCL ratios decreases strain capacity, optimized at 95:5

Strain to failure of a tissue matrix a function of both composition (varying blend ratios of PLA and PCL) and fiber alignment

Page 27: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

PDO + PCL blends

Advantages:• PCL high elasticity• PDO shape memory

Disadvantages:

• lower tensile capacity than PDO

• lower elasticity than PDO

• larger fiber diameter

Peak stress values for various PCL concentrations versus PDO:PCL ratio and orientation of the testing specimen

Strain at break values for various PCL concentrations versus PDO:PCL ratio and orientation of the testing specimen

* Further investigation needed

Page 28: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Natural

• Elastin

• Gelatin collagen

• Fibrillar collagen

• Collagen blends

• Fibrinogen

Page 29: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

ElastinAdvantages:

• Linearly elastic biosolid

• Insoluble and hydrophobic

• Critical role in shape and energy recovery for organs

SEM of electrospun elastin scaffold at 250 mg/ml.

Disadvantages:

• Less elastic than native elastin

• Needs to be combined with PDO to increase tensile strength

• Fiber ~300 nm (not as small as PDO ~ 180 nm)

• Varying diameter

Page 30: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

CollagenGelatin (denatured collagen)

Advantages:

• Biocamptibale and biodegradable

• Inexpensive

Disadvantages:

• Quick to dissolve

Fibril-forming (Types I, II, III)

• Most abundant natural polymers in body

• Important role in ECM

• Type I: principal structure in ECM

• Type II: pore size and fiber diameter easily controlled

• Type III: still under investigation

Page 31: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Collagen blends (1st attempts)

• Artery – Intima: innermost layer, composed of single layer of endothelial

cells on basement membrane of elastin and Type IV collagen– Media: thickest layer, several layers consisting of collagen type I

& III, elastin and proteoglycans – Adventitia: made of fibroblast and collagen type I

(Left) Photograph of 2 and 4 mm ID electrospun scaffolds. (Middle) SEM of tubular electrospun composite. (Right) SEM of electrospun 40:40:20 blend of collagen type I, collagen type III, and elastin with random orientation.

Page 32: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Collagen blends (1st attempts)

• Collagen Type I & III + PDO– indication that blends of PDO and collagen may

match mechanical and morphological requirements of a blood vessel's microenvironment (similar to PDO section).

Tangential modulus presented as a function of the ratio of PDO to collagen type I & III. collagen I highest tensile capacity, optimal ratio for all collagens was 70:30 collagen-PDO.

Page 33: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Globular proteins

• Fibrinogen (protein in blood plasma – wound healing)– Low concentration produced fibers within range of fibrinogen

fibers in plasma clots (80, 310, 700 nm)– High surface area to volume ratio: increases area available for

clot formation– Stress capacity comparable to collagen (80-100 MPa)

the linear relationship between concentration and fiber diameter composing the structures produced.

Page 34: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Globular proteins• Hemoglobin & myoglobin

– Fiber sizes 2-3 µm & 490 – 990 nm– Spun with fibrinogen for clotting and healing improvements– High porosity means higher oxygenation– Clinical applications:

• Drug delivery• Hemostatic bandages• Blood substitutes

SEM of electrospun hemoglobin in 2,2,2-Trifluoroethanol at 150 mg/ml (A), 175 mg/ml (B), and 200 mg/ml

Page 35: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

SummaryScaffolds:• Electrospinning viable for both synthetic and biological

scaffolds/mats

• synthetic polymers– PGA, PLA and PLGA most commonly used– PDO most similar to Elastin collagen blend (limited by shape memory)– PCL most elastic and mixed frequenlty with other material – Provide nanoscale physical features

• Natural polymers– Collagen Type I & III + PDO: best possible match for blood vessels

Limitations on Scaffolds• Mechanical material failure• Immunogenic reaction to material

Page 36: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

DISCUSSION

Dalby, M.J., The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder2

Page 37: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Overview

Image source: 6

electron beam lithography (EBL)

3D Scaffolds

Page 38: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

TE for Bone

Mesenchymal: Derived from mesoderm tissue consists of undifferentiated cells loosely organized within ECM.6

Osteoprogenitor cells: located in periosteum and bone marrow giving rise to osteoblasts.

Electron-beam lithography is a technique that employs a focused beam of electrons in order to pattern a mask or a silicon wafer.

Image source: 6

Page 39: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Experiment

Experiment looking to model osteoblastic differentiation of two cell types, osteoprogenitors and MSCs, and their interactions following culture on nanofeatures of different symmetry and with varying degrees of disorder.

Page 40: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Results (osteoprogenitors)• Immunofluorescence was used to detect expression of the bone-

specific ECM proteins osteopontin (OPN) and osteocalcin (OCN) by osteoprogenitors in response to the materials.

• good cell density• cells remained fibroblastic appearance• lack of OPN & OCN production

• decerase osteoprogenitors cell density• poor cell adhesion

SQ

HEX

Page 41: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Results (2)

• formation of dense aggregates• raised levels of OPN and OCN

• more-dense cell growth but only limited OPN or OCN levels

DSQ50

RAND

Page 42: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Results (MSCs)• Two MSC batches were cultured: one for 21 days before labeling OPN and

OCN and a second population for 28 days before alizarin red staining (stain for calcium present in bone mineral, indication of osteospecific differentiation).

• fibroblastic in appearance• highly elongated and aligned morphology

• more typical osteoblastic morphology• negligible OPN or OCN presence

SQ

RAND

Page 43: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Results (2)

• typical osteoblastic morphology• expressed foci of OPN• lack of OCN expression

• discrete areas of intense cell aggregation• early nodule formation• positive regions of OPN & OCN

DSQ20

DSQ5028 days allowed positive identification of mature bone nodules noted.

Page 44: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Osteospecific macroarrays

• To compare MSC differentiation for cells cultured on (1) untreated cells cultured on a planar control, (2) planar material with dexamethasone (DEX) (steroid to induce bone formation) and (3) DSQ50

Cells cultured with DEX exhibited the highest levels of osteogenic up-regulation (24 gene hits) followed by MSCs cultured on DSQ50 (11 gene hits). Cells cultured on the planar control, however, demonstrated only three gene hits.

Page 45: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

qPCR

• To confirm these results, a quantitative polymerase chain reaction (qPCR) was used with primers for the bone marker genes OCN and alkaline phosphatase

In agreement with the macroarray data, MSCs cultured with DEX or on the DSQ50 material had significantly higher expressions of genes of interest.

Page 46: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Summary

• The qPCR and macroarray results together clearly indicated that the disordered materials have an osteogenic potential close to that of DEX and a higher osteogenic potential than that of highly ordered or totally random topographies.

• It was shown that surface topography caused significant changes in MSC response and that topography may induce a broader response than simply changing osteoblast-specific genes.

Page 47: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Summary (2)

• Results demonstrate that highly ordered nanotopographies produce low to negligible cellular adhesion and osteoblastic differentiation.

• Cells on random nanotopographies exhibited a more osteoblastic morphology by 14 days

• It seems more likely that as long as cell spreading is permitted by the surface, cells will follow a classical differentiation timelines.

Page 48: Cell and Tissue Engineering, Nanotechnology BIOE 506 Muqeem Qayyum Tissue Engineering CellsScaffolds BiorectorsSignals

Reference

1. Barnes, C.P., Nanofiber technology: Designing the next generation of tissue engineering scaffolds.

2. Dalby, M.J., The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder.

3. Gonsalves, K.E., Biomedical Nanostructures, Wiley 2008.

4. Laurencin, C.T., Nanotechnology and Tissue Engineering: The Scaffold, CRC Press. 2008

5. Hartgerink, J.D., Self-assembly and Mineralization of Peptide-amphiphile Nanofibers, V. 294, No. 5547. 2001.

6. Novakovic, G., Culture of Cells for Tissue Engineering. Wiley, 2006