cell and tissue engineering, nanotechnology bioe 506 muqeem qayyum tissue engineering cellsscaffolds...
TRANSCRIPT
Cell and Tissue Engineering,
Nanotechnology
BIOE 506
Muqeem Qayyum
Tissue Engineering
Cells Scaffolds
Biorectors Signals
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
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
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
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
DISCUSSION
Barnes, C.P., Nanofiber technology: Designing the next generation of tissue engineering scaffolds1
Nanofiber Technology
• Techniques of nanofibers for tissue engineering
• Characteristics of tissue engineering nanofibrous structures
• Electrospinning of synthetic and natural polymers
3 techniques to achieve nanofibers for TE
Self-assembly
Phase separation
Electrospinning
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
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
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
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
Characteristics of TE nanofibrous structures
Scaffolds under Electrospinning
• Goal of scaffolds• Properties• Materials
– Synthetic – Natural
• Limitations
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
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
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
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).
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
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)
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
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
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
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
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)
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
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
Natural
• Elastin
• Gelatin collagen
• Fibrillar collagen
• Collagen blends
• Fibrinogen
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
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
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.
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.
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.
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
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
DISCUSSION
Dalby, M.J., The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder2
Overview
Image source: 6
electron beam lithography (EBL)
3D Scaffolds
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
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.
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
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
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
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.
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.
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.
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.
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.
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