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Bioengineering

Head of Unit: Prof. L. Meseguer-Olmo, MD, PhD. Associated: Francesca Cragnolini Enrique Bautista Francisco J. Gómez

Profile URL: http://www.researcherid.com/rid/A-3141-2011 PProfile: http://es.linkedin.com/pub/luis-meseguer-olmo-md-phd/34/b1/a15 Further info on Facebook (Células madre or Stem cells and Tissue engineering).

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Bioengineering

Nuestros retos clínicos (Our Clinical challenges in orthopaedic)

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Bioengineering

BIOMATERIALES (Biomaterials)

• Naturales (fibroina-seda, hueso deshidratado) (Naturals)

• Sintéticos (cerámicas FC, CaSO4) (Synthetic)

Sistemas liberación controlada moléculas (controlled delivery of molecules)

Ingeniería de Tejidos (Tissue Engineering)

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Bioengineering

Sistemas liberación controlada moléculas (Controlled delivery of molecules) c

b

a

• Local

Efecto (Effect)

(Local)

*

*

*

• Sistémico (Systemic)

• Antibióticos (Antibiotics)

Acción antimicrobiana Efecto citotóxico

• Factores crecimiento (Growth factors)

• MSCs • OBs

- Proliferación celular - Diferenciación - Migración

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Bioengineering

Construcción basada en PRP-Yeso vs PRP-TCP-Yeso

El plasma rico en plaquetas (PRP) aporta los factores de crecimiento (VEGF, TGF, IGF, etc.)

(PRP-Plaster vs PRP-TCP-Plaster constructs)

VEGF release in PBS 250

pg/ml

200 150 TCP+Yeso-PBS

100

Yeso-PBS

50 0 1d

3d

6d

9d

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Bioengineering

Construcción basada en Yeso-Vancomicina-Gentamicina y Yeso-PRP. Aplicación clínica. (Plaster-vancomycin-gentamicin and PRP-plaster constructions. Clinical applications)

X-R plains

2 mth post-OP

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Bioengineering

Bone tissue with / without bone marrow

Ingeniería de Tejidos (Tissue Engineering)

Design of synthetic scaffold

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Porous biomaterials similar to cancellous bone Human cancellous bone

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Bioengineering

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Bioengineering

Células madre adheridas al biomaterial (Stem cells adhered to the biomaterial)

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Osseointegration of Biomaterials

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Bioengineering

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Bioengineering

Queratinocitos adheridos y creciendo en una malla de fibroina (Keratinocytes attached and growing on a fibroin mesh)

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Bioengineering

Multilayer growth

Mesh remains uncovered

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Bioengineering

• General and specific cell cultures • Scanning electron microscopy • Transmission electron microscopy • Energy dispersive spectroscopy

• Electrospinning • Animals laboratory

• Histology & Histopathology techniques • Immuno-histochemical assays • rt-PCR • … and other ralated techniques.

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Bioengineering

•

Instituto Bioingeniería. Universidad Miguel Hernández (Elche, Alicante)

•

Instituto Murciano de Investigación y Desarrollo Agrario Alimentario. Murcia

•

Dpt. Química Orgánica y Bioinorgánica. Universidad Complutense. Madrid

• Instituto Tecnológico de Canarias. Las Palmas de Gran Canarias •

Dpt. de Materiales. E. S. Ingenieros de Caminos. U. Politécnica. Madrid

•

Instituto de Cerámica y Vidrio. CSIC. Madrid

Artificial Skin Challenging the creation of a human skin prototype in vitro.

Support Fibroin matrix

The support chose for this project is a silk fibroin scaffold made using as starting material the fibroin protein extracted from silk cocoons.

Cultures Two cell types from skin are now testing for the capacity to adhere and proliferate normally (as in plastic cultures) in the same culture media and temperature/CO2 conditions.

Culture conditions: 37ºC, 5% CO2, HAM’s F12 medium supplemented with 10% SBF, 1% Pen/Strep, 1% Glutamine, 5% MelanoMax™. The medium is changed 2 time a week. • Keratinocytes: Human Epidermal Keratinocytes (HEK001, ATCC). • Melanocytes: Human Epidermal Melanocytes (HEM, (Gentaur).

Results • Melanocytes and Keratinocytes adhere and proliferate on the silk fibroin matrix.

Fig. 1: Keratinocytes on the left; Melanocytes on the right.

Results • SEM images demonstrate that cells adhere, proliferate and after 28 days in culture cells are confluent on a 1cm2 flat matrix (Fig. 2c).

• For Keratinocytes it is also possible to observe an ulterior organization stage; they organize in areas giving the surface a puzzle like aspect (Fig. 2d).

72 h after seeding

10 days after seeding

Detail

Complete covering of the surface at 28 days

Fig. 2: Keratinocytes after seeding: a) 72h, b) 10 days, c) 28 days, d) puzzled aspect of keratinocytes covered surface after confluence.

Results • SEM imagines confirm that the two types of cells adhere and proliferate each one on a side of the matrix, as seeded, without mix. • For melanocytes is important to underline that they are smaller and thin enough to threaded they way trough the matrix framework (Fig. 3).

Fig.3: Melanocyte into the silk fibroin matrix framework.

Results • Keratinocytes and Melanocytes grow up on multiple layers after reacing confluence.

Some fibers not yet covered

Multiple layers

Conclusions • It is possible to produce prototypes of human skin in vitro, using keratinocytes and melanocytes, two human skin types. In this prototype they organize in layers like in normal human skin. An advantage is offered by the substrate, that is a protein matrix totally compatible with human body and reabsorbable.

Future perspectives • It is possible to produce other skin prototypes, combining other skin cells or more than two types of cells, resembling more the skin composition.

• This model of human skin is directed at everyone researcher interested in test their products on human skin before commercialize this products.