1. Cell and Tissue Engineering, Nanotechnology
Cells
Scaffolds
Biorectors
Signals
BIOE 506
Muqeem Qayyum

2. 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:
farm1.static.flickr.com

3. 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
Structural proteins
Collagen
Elastin
Adhesion proteins
Fibronectin
Laminin

4. Definitions Tissue Engineering: Nanotechnology: Biocompatibility
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
To accomplish this scientist have turned to tissue engineering and nanotechnology.

5. Tissue Engineering (TE)
Scaffolds
Biomaterials, which may be natural or artificially derived, providing a platform for cell function, adhesion and transplantation
Cells
Any class of cell, such as stem or mesenchymal cell
Signals
Proteins and growth factors driving the cellular functions of interest
Bioreactor
System that supports a biologically active environment (ex. Cell culture)
Me-zen-ki-mel
Image sourse: Stke.sciencemag.org, Nature.com

6. Discussion
Barnes, C.P., Nanofiber technology: Designing the next generation of tissue engineering scaffolds1
Me-zen-ki-mel

7. Nanofiber Technology
Techniques of nanofibers for tissue engineering
Characteristics of tissue engineering nanofibrous structures
Electrospinning of synthetic and natural polymers

8. 3 techniques to achieve nanofibers for TE
Self-assembly
Phase separation
Electrospinning

9. 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
peptide-amphiphile
Nanofiber

10. 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

11. 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

12. No consensus of gold standard for creating native ECM
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
Electrospinning
Cost effective
Long continuous fibers
Tailorable mech properties, size & shape
Large scale fibers
No control over 3D pore structure

13. Characteristics of TE nanofibrous structures
ECM
Geometry
Structural role
Physiology
Signaling

14. Scaffolds under Electrospinning
Goal of scaffolds
Properties
Materials
Synthetic
Natural
Limitations

15. Goals/Properties
Goal of TE is to combine cell, scaffold (artificial ECM) and bioreactor to design and fabricate tissues and organs.
Design scaffold with maximum control over:
biocompatibility (chemical)
biodegradability (mechanical)
Premise is regeneration, enable cellular components to heal itself by introducing TE scaffolds that are recognized as ‘self’ and regenreate ‘neo-native’ function.
Scaffolds Properties
Biocompatible
Promote growth
Maintain 3-D structure
Non-immunogenic
Support tissue and cell forces

16. Materials Synthetic Natural
Mimic mechanical properties (strength, elongation and knot retention)
Mass production
Degradation between hosts is minimal
Natural
Cell recognition
Biodegradable
Difficult degradation control between host

17. Synthetic Polyglycolic acid (PGA) Polylactic acid (PLA)
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

18. 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).

19. PGA 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
An elastic modulus, or modulus of elasticity, is the mathematical description of an object or substance's tendency to be deformed elastically (i.e., non-permanently) when a force is applied to it.
The overall results exhibit a correlation between the fiber diameter and orientation and the elastic modulus and strain to failure.
Fig. summarizes mechanical properties in terms of the elastic modulus and strain at failure of PGA in a uniaxial model.

20. 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)

21. 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

22. 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

23. 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
SEM of 180 nm diameter randomly oriented fibrous structures
Results of the fiber diameter analysis versus PDO concentration illustrating the linear relationship between electrospinning solution concentration and fiber
diameter.

24. 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

25. PGA + PCL blend Advantages: 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)

26. PLA + PCL blends Advantages: 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

27. 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
* Further investigation needed
Strain at break values for various PCL concentrations versus PDO:PCL ratio and orientation of the testing specimen

28. Natural Elastin Gelatin collagen Fibrillar collagen Collagen blends
Fibrinogen

29. Elastin Advantages: Linearly elastic biosolid
Insoluble and hydrophobic
Critical role in shape and energy recovery for organs
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
SEM of electrospun elastin scaffold at 250 mg/ml.

30. Collagen Gelatin (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 and gelatin have variable sequences, but are predominately glycine, proline, and hydroxyproline

31. 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.

32. 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.

33. 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 ( MPa)
Fibrin (also called Factor Ia) is a fibrous protein involved in the clotting of blood, and is non globular. Fibrin is made from fibrinogen, a soluble plasma glycoprotein that is synthesised by the liver.
the linear relationship between concentration and fiber diameter composing the structures produced.

34. 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
Fibrin (also called Factor Ia) is a fibrous protein involved in the clotting of blood, and is non globular. Fibrin is made from fibrinogen, a soluble plasma glycoprotein that is synthesised by the liver.
SEM of electrospun hemoglobin in 2,2,2-Trifluoroethanol at 150 mg/ml (A), 175 mg/ml (B), and 200 mg/ml

35. Summary
Scaffolds:
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

36. Discussion
Dalby, M.J., The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder2
Me-zen-ki-mel

37. Overview
3D Scaffolds
electron beam lithography (EBL)
Image source: 6

38. 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

39. Experiment
Culture / nanofeatures
Osteoprogenitors & MSC’s
Square array (SQ)
Hexagonal array (HEX)
Disordered SQ w/50 nm displacement (DSQ50)
Disordered SQ w/20 nm displacement (DSQ20)
Pits randomly placed over 150µm2 (RAND)
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.

40. 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

41. Results (2) formation of dense aggregates raised levels of OPN and OCN
DSQ50
more-dense cell growth but
only limited OPN or OCN levels
RAND

42. 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

43. Results (2) typical osteoblastic morphology expressed foci of OPN
lack of OCN expression
DSQ20
discrete areas of intense
cell aggregation
early nodule formation
positive regions of OPN & OCN
28 days allowed positive identification
of mature bone nodules noted.
DSQ50

44. 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.

45. 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.

46. 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.

47. 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.

48. Reference
Barnes, C.P., Nanofiber technology: Designing the next generation of tissue engineering scaffolds.
Dalby, M.J., The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder.
Gonsalves, K.E., Biomedical Nanostructures, Wiley 2008.
Laurencin, C.T., Nanotechnology and Tissue Engineering: The Scaffold, CRC Press. 2008
Hartgerink, J.D., Self-assembly and Mineralization of Peptide-amphiphile Nanofibers, V. 294, No
Novakovic, G., Culture of Cells for Tissue Engineering. Wiley, 2006