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Patricia Barros

on 26 November 2014

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Transcript of 3D BIOPRINTING

3D bioprinting approaches
Anatomical and phisiological aspects
Skin is complex organ, constituted by elaborated cells, tissues and other structures.
The main layers in the skin are :
- Epidermis, and in this layer we find specialized keratinocyte cells (melanocytes, Langerhans and Merkels cells)

- Dermis, being the most vascularized layer. It also has collagen and reticullar fibres. The hair folicules can be found here

- Hypodermis is strongly irrigated, mainly composed by blood vessels
Tuesday, November 18, 2014
Vol XCIII, No. 311
What is bioprinting?
Bioprinting: A novel technique
First patent of a bioprinter tissue appeared in 2003.

Fabricated in Organovo and specialized for skin

A Novel approach for the fabrication of biological constructs

Process: Cell culture - insertion/dispension into a biocompatible scaffold with chemical and biological stability ( through Ink jet, micro extrusion or laser)

3D bioprinting is defined by the process of generating spatially-controlled cell patterns using 3D printing technologies

Tissue bioprinting strategies
Ink in the cartridge was replaced with a biological material;
Paper was replaced with an electronically controlled elevator stage to provide control of the z axis;
Inkjet printers use thermal or acoustic forces to eject drops of liquid onto a substrate.
Laser with specific energy is emited and the radiation is focused to an absorbing layer
An interlayer of metal promotes the conduction of biomaterial towards the substrate
laser stimulated cell-containing materials underneath the absorving layer deposits on to a collector surface
Control parameters:

Wave length (UV/IR), Energy (Kj), repetition rate and Beam focus diameter have to be controlled depending on the biological material characteristics, such as volume and viscosity

Interlayer must have a film (tens of nm) of a metal (Au,Ag..) to ensure conduction to the biomaterial surface
Aplication of LABP in the creation of artificial ear
3D bioprinting is based on three central approaches:
Manufacture of identical reproductions of the cellular and extracellular components of a tissue or organ
Use embryonic organ development as a guide. The early cellular components of a developing tissue produce their own ECM components, appropriate cell signaling and autonomous organization and patterning to yield the desired biological micro-architecture and function
These can be defined as the smallest structural and functional component of a tissue, such as a kidney nephron.
Inkjet Bioprinting
Electrically heating the print head to produce pulses of pressure that force droplets from the nozzle.
Localized heating (200 °C to 300 °C) is not a problem:
Short duration (~2 μs) Temperature rise of only 4–10 °C.
Risk of exposing cells and materials to thermal and mechanical stress.
Applying a voltage to a piezoelectric crystal induces a rapid change in shape that creates an acoustic wave inside the print head, which in turn generates the pressure needed to eject droplets from the nozzle.
Generate and control a uniform droplet size and ejection directionality as well as to avoid exposure of cells to heat.
Frequencies used by piezoelectric inkjet bioprinters (15–25 kHz):
Potential to induce damage of the cell membrane and lysis.
Imaging and Digital Design
- Diagnostics
-Interventional Procedures
2D cross-sectional images
3D anatomical representations
Computer Tomography (CT)
Magnetic Ressonance Imaging (RMI)
-CAD-CAM software

-Mathematical Modeling
Printer function by robotically controlled extrusion of a material, which is deposited onto a substrate by a microextrusion head;
Microextrusion yields continuous beads of material rather than liquid droplets;
The most common methods to extrude biological materials are pneumatic or mechanical.
The most common and affordable non-biological 3D printers use microextrusion.
Microextrusion bioprinters usually consist of a temperature-controlled material-handling and dispensing system and stage
Capable of movement along the x, y and z axe
Compatible Materials
Biocompatible copolymers
Cell Spheroids
This involves removing a piece of skin from a secondary surgical site for the patient and reapplying the graft on the wound or burn.
Bioprinting for Skin Wounds
Autologous Skin graft
If the wound is extensive, then the number and size of donor sites are limited.
Development of noncellular dermal substitutes
Costly to produce and result in relatively poor cosmetic outcomes.
Alternatively, cell spraying and bioprinting technologies have recently been developed for wound treatment.
Skin application:

1º Laser pulses promote evaporation of
biomaterial layer, and condensation promotes the cells deposition on to the matrix

2º Fibroblasts and keratinocytes on top of the stabilizing matrix

3º Controlled evaporation provides spatial distribution layer by layer

Cell Source
polymeric scaffold
Human keratinocytes
Mesenchymal stem cells

This type of cell constitutes 90% of cells of the epidermis. It is preferable to have this cell type for the printing procedure.
Therapeutic potential for repair and regeneration of tissues damaged by injury or disease.

In particular, MSC treatment of acute and chronic wounds results in accelerated wound closure, increased epithelialization, formation of granulation tissue, and angiogenesis.
Amniotic fluid-derived stem

High proliferation capacity, multipotency, immunomodulatory activity, and lack of significant immunogenicity.
AFS cells remain stable and show no signs of transformation in culture.
Isolation of AFS cells is a simpler process than that for isolation of MSCs.
secondary surgical sites
potential for rejection
Microextrusion bioprinters have been used to fabricate multiple tissue types:
Aortic valves
Tumor models
Branched vascular trees
Cell Culture
Cell Culture
Multipotent stem cells were isolated from human amniotic fluid;
The cells were expanded in culture from a single clone to achieve a relatively homogenous subpopulation;

AFS cells proliferate rapidly in culture without feeder cells for many passages, while maintaining chromosomal stability.

AFS cells and MSCs were separately suspended in the fibrinogen/collagen solution;
While under anesthesia, a single full-thickness skin wound was surgically created with scissors on the mid-dorsalregion of mice.

Fibroblasts were cultivated in DMEM and supplemented with FBS
Keratinocytes were grown with DMEM/Ham’s F12 medium
Cells were then transfered and prepared for printing

LaBP setup consisted of two co-planar glass slides.

Upper one coated with a laser absorbing layer and bottom one is a wet substrate receiver layer.

Spatially defined keratinocytes and fibroblast in the tissue are now ready for in vivo implantation

Several histological tests were performed to evaluate the morphology of the cells after implant, the blood vessel formation and the regeneration of the damaged skin area, whereas the results where positive in all three aspects
MSC-driven wound closure and re-epithelialization were significantly greater than in wounds treated by fibrincollagen gel only.
Increased microvessel density and capillary diameters in the AFS cell-treated wounds compared with the MSC-treated wounds, whereas the skin treated only with gel showed the lowest amount of microvessels.
However, tracking of fluorescently labeled AFS cells and MSCs revealed that the cells remained transiently and did not permanently integrate in the tissue.
"Tissue Engineered Skin Substitutes Created by Laser-Assisted Bioprinting Form Skin-Like Structures in the Dorsal Skin Fold Chamber in Mice"
'Bioprinted Amniotic Fluid-Derived Stem Cells Accelerate Healing of Large Skin Wounds'

Thank you for your attention
Silva. M.L., Silva. P.B., and Mikulich. R.
Fig. 1 - Deteção de vasos sanguíneos através da expressão de colagénio IV
Fig. 2 - Imagens A, B, C a revelar a presença de queratinócitos ( a vermelho) e de fibroblastos (a verde), e Imagens de D, a I revelam a presença de tecido conetivo contendo collagenio e células
Images revealed evenly distributed cells (AFS) in the gels, as viewed from above (B) or from the side (C).
(A): Histology images illustrating wound closure in gel-only, MSC, and AFS treatments. (B): Percentage of unclosed wound remaining at surgery, 1 week, and 2 weeks.
Re-epithelialization was visualized by the extent of formation of keratinocyte layers in hematoxylin and eosin-stained tissue sections. (C): Poorly defined epithelium in the group treated with gel only. (D, E): Well-defined and structurally robust epithelium in MSC-treated (D) and AFS-treated (E) groups.
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