synthetic materials have been evaluated for the production of tra-

cheal scaffolds [3]. Although promising, the results obtained met

limited success because of immunological complications and bacte-

rial infections. In addition, the materials tested lacked many of the

organ-specific biomechanical properties, namely flexibility, strength

to avoid collapse, and the formation of airtight seals [4, 5].

We here describe a two-step protocol for the ex vivo creation of

a bioprosthetic trachea. The first step involves a decellularization

technique that allows for the production of a naturally derived

extracellular matrix (ECM)-based porcine bio-scaffold (Fig. 1). In

the second step, human chondrocytes are seeded onto the decel-

lularized trachea, using a rotating bioreactor to ensure a correct

scaffold repopulation (Fig. 2).

Nonimmunogenic tracheal bio-scaffolds were derived by using

a physical-chemical method to successfully eliminate the cell com-

partment, while preserving the macro- and micro-architecture and

maintaining an intact ECM protein composition [6, 7]. The

bio-scaffold was obtained from the pig which is an ideal source of

organs for xenotransplantation because of its anatomical and phys-

iological similarities to humans [8, 9]. In addition, the adult por-

cine trachea has been recently demonstrated to match the

biomechanical properties of the human organ, including bending

stiffness, radial supporting force, longitudinal elongation, residual

stress, and bursting strength [10]. In the second step, human

chondrocytes were used to repopulate the porcine bio-scaffold to

generate “semi-xenografts,” where the ECM-based scaffold is

animal-derived, and the repopulating cells have human origin,

thus combining the advantages of both xenotransplantation and

tissue bioengineering.

A key aspect of the protocol described is represented by the use

of a rotating bioreactor that ensures a dynamic repopulation system

with several important advantages compared to the static culture

approach. Indeed, the adoption of a bioreactor favors and positively

impacts on ex vivo cell and tissue re-organization, ensuring the

Fig. 1 Decellularization protocol and macroscopic images illustrating changes in trachea color, turning from

red to white, while maintaining original shape

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