Bioimpresión 3D de Piel: Tecnología, Aplicaciones y

Futuro

José L. Jorcano

Dept. of Bioengineering. University Carlos III (Madrid)

Division of Epithelial Biomedicine. CIEMAT (Madrid)Catedrático Ramón Areces

COSMETORIUMBarcelona, 3-4 de Octubre 2017

The Need for Artificial Skin

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• In the US, the number of burn injuries receiving medical treatment per year are 486000 and the number of hospitalizations per year for acute burn injuries are 40000, per the 2016 report

• The 2016 global wound management market is expected to hit $15 billion and forecasted to be worth over $22 billion in 2024

• The global tissue engineered skin substitutes market was valued at USD 958.8 million in 2014 and is projected to reach USD 3873.5 million by 2023

• There are two compelling reasons as to why the world is in needof artificial skins:(i) wound healing and skin regeneration—especially for

burn victims, and (ii) drug and skin care products (cosmetics) testing.

From: S. Vijayavenkataraman et al., Biofabrication, 2016

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2ª Conferencia Internacional sobre Ensayos Clínicos y Vigilancia Terapéutica de Medicamentos

In Vitro Toxicology Market

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Distribution of the global in vitro toxicology testing market in2016, by region (in billion U.S. dollars)

https://www.statista.com/statistics/679963/global-in-vitro-toxicology-testing-market-share-by-region/

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New technologies are needed that canreliably mimic human skin and can beindustrialized:

3D Bioprinting and Tissues-on-Chips

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Types of 3D printers:Stereolithography(SLA)Digital Light Processing(DLP)Fused deposition modeling (FDM)Selective Laser Sintering (SLS)Selective laser melting (SLM)Electronic Beam Melting (EBM)Laminated object manufacturing (LOM)

Additive Manufacturing (AM) is a term to describe set of technologies that create 3D objects by adding layer-upon-layer of material. Materials can vary from technology to technology

Where are 3D printing technologies

applied in medicine?

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External prosthesis (orthosis)

Lightweight exoskeleton for

Emma

Arthrogryposis multiplex congenita

(Delawere Hospital & Stratasys)

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Dentistry

Prosthesis

But we are talking about

How to Build Tissues and Organs:

3D BIOprinting

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The components of bioinks

Artificial scaffolds

-Natural polymers (collagen, fibrin)-Synthetic polymers

-Materials Science

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Chitosan-based scaffolds for bone tissue engineering

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SCANNING ELECTRON MICROSCOPE:

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Dermal fibroblasts and Col I matrix

2 m²

2.000 million cells

Renewal rate: 70

million/day

What we would like to mimic…..

……What we can mimic

Dermis

Epidermis

St. corneum

St. basale

St. granulosum

St. spinosum

Basal membrane

SKIN: A COMPLEX STRUCTURE

Technology: Bioprinting Methods

From: S. V. Murphy and A. Atala, Nature Biotech., 2014

Continous extrusion

Technology: Bioprinting Methods

(Continous extrusion)

From: S. V. Murphy and A. Atala, Nature Biotech., 2014

Scheme of a Bioprinter

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Bioinks: the Key to Organ Bioprinting

From: H-W Kang et al., Nature Biotech., 2017

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Table 1. Preparation of the cell-laden composite hydrogels for 3D bioprinted tissue constructs

Composite hydrogel

Gelatin Fibrinogen HA Glycerol Cell type & density

Bone (type I) 35 mg/ml 20 mg/ml 3 mg/ml

10 % v/v Human AFSCs, 5x106 cells/ml

Cartilage (Type I) 45 mg/ml 30 mg/ml 3 mg/ml

10 % v/v Rabbit ear chondrocytes, 40x106 cells/ml

Skeletal muscle (type II) 35 mg/ml 20 mg/ml 3 mg/ml

10 % v/v Mouse C2C12 myoblasts, 3x106

cells/ml

A 3D bioprinting system to produce human-scale tissue constructus with structual integrity. Hyun-Wook Kang, Sang Jin Lee, In Kap Ko, Carlos Kengla, James J Yoo & Anthony Atala. Nature biotechnology , 34 (3) pag. 312. DOI: 10.1038/nbt.3413, Mar2016

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Marlborough, an international fund rising firm, places BioDan together withOrganovo as the sector’s leaders (Inflation driving up costs? Your solution lies in Asia;

May 2017; Sally Macdonald).

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The 3D printing Hype Cycle by Gartner: What does the 2017 edition say?

What were we doing?

Manual production of bioengineered skin

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De aquí obtuvimos nuestras biotintas, la clave de nuestro éxito.

Aproximación desde la biomedicina, no desde la ingeniería

3-D plasma matrix

Fibroblasts

Keratinocytes

Bio-Engineered Skin Equivalent

SKIN BIOPSYBLOOD

A. Meana et al., Burns 1998

S. Llames, et al Transplantation, 2004

PATIENT

1cm² → 2m² in 3 weeksExpansion Factor: 105

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IMPLANTATION

EPIDERMOLYSISFOREARM

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• Extensive burns• Chronic vascular and diabetic ulcers• Surgical and traumatic wounds• Skin hereditary diseases

(Epidermolysis Bullosa)• Necrotizing Fasciitis• GVHD• Giant Nevus• Oral mucosa restitution• Urogenital epithelium sustitution

BIOENGINEERED SKIN – THERAPIES:

RESPONSIBLE FOR PRODUCTION AND MARKETING IN SPAINAS A “CONSOLIDATED MEDICAMENT”

UNDER LICENSE

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SkinMed Autologous Bioactive Skin is manufactured in cleanrooms under strict sterile conditions

and analytical controls.

Generation of Autologous Bioactive Skin under GMP conditions

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3-D In Vitro Skin Cultures

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H/E H/E

Col7 Invol. Fillag.

Vimentin

K14/k10

Tricromicro

Gel diferenciado in vitro 16 días

Alta reproducibilidad:Células de un único donante

En colaboración con BioDan

Colágeno humano

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Our two skin model is unique in the market: It is bilayerd and fully human

Next step: How to improve, automatize and standardize the production of skin equivalents.

A collaboration with BioDan

Biofabrication, 2016

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BIOMATERIALS 2014/2015 – S – 17/03/2014BIOMATERIALS EXPERIMENTAL DESIGN 2014/15 –S2 – 17/03/2014 – MOD 7 - 1

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Our bioprinting system

FIRST-GENERATION BIOPRINTER

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THIRD-GENERATION BIOPRINTER

Juan CañizoLucía GullónFátima PérezCristina Quílez

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IN VITRO 3D CULTURE

Hematoxylin-

Eosin Staining

VimentinK10

Suprabasal

strata

Fibroblast

cytoskeleton

E

D E

D

A

D

C

E F

B D

EBM

IN VIVO ASSAY (I)

h Vimentin

Collagen VII h K10 h Filaggrin

H/E h K5

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Conclusiones

- I. Gracias a los conocimientos científicos y tecnológicos

desarrollados, nos encaminamos a una nueva era en la

cosmética y en el testeo de sus productos (COSMETICA

AVANZADA).

- II. La bioimpresión 3D aplicada al testeo facilitará:

1. Incrementar la producción y reducir los costes

significativamente.

2. Estandarizar los métodos de producción. Para ello, necesita

madurar como tecnología de producción.

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3. Incrementar muy significativamente la reproducibilidad

de los datos frente a los obtenidos con biopsias humanas.

4. Generar in vitro piel más parecida a la real: pelo,

glándulas, hipodermis, mayor complejidad celular,

gradientes de moléculas, etc.

III. El trasplante de órganos producidos industrialmente

será una realidad. Es necesario comenzar a diseñar la

FÁBRICA DE ÓRGANOS DEL FUTURO.

ACKNOWLEDGEMENTS

Tissue Engineering: Alvaro Meana (Centro Comunitario de Sangre y Tejidos de Asturias), Marcela Del Rio (UC3M/CIBERER/Fundación Jiménez Díaz), Fernando Larcher (CIEMAT)

BIOPRINTING: Nieves Cubo Mateo (UC3M), Lucía Gullón (BioDan),

Marta García (UC3M), Diego Velasco (UC3M), Juan Cañizo (HUGM), Fátima Pérez (BioDan), Cristina Quílez (UC3M).

….and many dermatologists and plastic surgeons from Spanish and European hospitals

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THANK YOU !!

Which are the main technologies

involved in 3D printing?

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3D PRINTERS: Layer by layer

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3D Bioprinter SolutionsOrganovo U.S. Army

3DS - 3Dynamical Systems

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Andamiaje sintético biodegradable

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Ceramic (e.g. hydroxyapatite) scaffolds for bone tissue engineering

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Technology: Bioprinting Methods

(Continous extrusion)

From: S. V. Murphy and A. Atala, Nature Biotech., 2014

Also: Mixed systems

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THE PRODUCT

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Therapy: Extensive and Severe Burns

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IN VIVO ASSAY (II)

Vascularization:

A relevant issue in bioprinting.

In this case:

Mouse blood vessels invade our engineered tissue.

E

D

sc

BM

BV

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