Copyright: the Author(s), 2019 Biomedical Science and ...
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On the transferability of tissue engineering technologies to the design of tissue models Gianluca Ciardelli Department of Mechanical and Aerospace Engineering (DIMEAS), Politecnico di Torino, Turin, Italy Abstract 3D tissue-engineered models are promising tools in the screening and evalu- ation of drugs and therapies as well as in the investigation of the molecular mechanisms involved in disease onset and progression. In this context, we describe our efforts in soft tissue replication, to design in vitro models that have the potential to provide better insight into the development of age- ing process and related pathologies, with particular reference to the cardiovascular field. Introduction 3D tissue-engineered models are promising tools in the screening and evalu- ation of drugs and therapies as well as in the investigation of the molecular mechanisms involved in disease onset and progression. In a 3D bioengineered model, the scaf- fold should finely reproduce the architec- ture and composition of the native tissue, in order to properly regulate cell behaviour, such as spreading, proliferation, differentia- tion, and extracellular matrix (ECM) pro- duction, alike to what they do in vivo. In order to successfully mimic a tissue, several aspects must be considered when designing a tissue model. Among others, cell sources and 3D scaffolds architecture and composi- tion (both in bulk and on the surface) have been investigated in depth (Figure 1). The biomaterial choice is a crucial ele- ment, since it strongly affects cellular func- tions. It should be based on the material mechanical properties, as the one of the scaffolds should match those of the tissue to be modelled, as thoroughly highlighted by mechanobiology studies. 1 In the replication of hard tissues, due to their high stiffness and load- bearing properties, ceramics and their composites are generally used, while natural and synthetic polymers are appro- priate choices in the engineering of soft tis- sues. 2 In soft tissue replication, in vitro models of heart tissue have the potential to provide better insight into the mechanisms of heart diseases, which are the leading cause of mortality in the world. Moreover, myocar- dial models could be a useful tool in the evaluation of drugs cardiotoxicity. In car- diac tissue engineering, the most investigat- ed natural polymers are collagen, gelatin, fibrin, and alginate, and among synthetic polymers, degradable polyesters and their copolymers have been mainly explored. For instance, since the myocardial tissue has to bear the contractile and expansive forces produced at each cardiac cycle, elastomeric polymers, such as poly(glycerol sebacate) and polyurethanes have been mainly stud- ied. Results Our group extensively worked in the synthesis of biodegradable polyurethanes for contractile tissues applications. In a pre- vious article, we identified a promising polyester-urethane (PUR), showing low Young’s modulus and elastomeric behaviour. 3 This PUR was synthesized from poly(ε-caprolactone) diol, 1,4-butanediiso- cyanate and L-lysine ethyl ester. In a suc- ceeding article, this PUR was used to obtain porous scaffolds by thermally induced phase separation (TIPS). The scaffolds were functionalized with fibronectin by plasma treatment, and seeded with primary rat neonatal cardiomyocytes. 4 Morphological and mechanical analysis exhibited a pore- aligned scaffold formation, with mechanical properties and architecture resembling the one of the myocardium. Cardiomyocyte viability was stable for 14 days of experi- mentation, comprising beating activity and cell adhesion. The improved levels of phos- phorylation of AKT and ERK1/2 elucidated the prolonged cell survival. The variation of cardiac muscle and glucose genes indicated a transition phase in cardiomyocyte matura- tion, with an early evolution to an adult phe- notype and a permanence of some fetal characteristics. These results represented a first step towards the development of a new generation of myocardial models. A further step involved the fabrication of scaffolds with improved controlled geometry, which can be obtained via rapid prototyping (RP) procedures.In detail, the PUR was micro- fabricated into 0°/90° square-grid multi- layered scaffolds through a custom-made melt-extrusion additive manufacturing instrument and surface functionalized with laminin-1 (cardiac niche ECM component, sample acronym PUR-LN1) or gelatin (low-cost adhesion protein, sample acronym PUR-G) through plasma treatment followed by carbodiimide chemistry. 5 PUR- G and PUR-LN1 scaffolds both improved cardiac progenitor cell (CPC) adhesion, but LN1 functionalization turned out to be superior in promoting CPC proliferation, protection from apoptosis and expression of differentiation markers for cardiomyocytes, endothelial and smooth muscle cells. PUR- LN1 subcutaneously implanted in mice evoked a weak inflammation and integrated with the host surrounding tissue, evidencing a significant blood vessel density around the scaffolds. In a different approach, ther- mo- and photo-curable bioinks were designed for the bioprinting of cellularized constructs to be used for reparative/regener- ative purposes or as in vitro bioengineered models of soft tissues. The thermo-sensitive nature of the developed bioinks allowed an easy and homogeneous cell dispersion, through the addition of the cells to the hydrogels at a temperature lower than their gelation temperature (below 25 °C), when the systems were in the sol or semi-gel phase. After loading in the syringe, cellular- ized hydrogels were subjected to a fast sol- to-gel transition with increasing tempera- ture. Syringe equilibrium temperature and, as a consequence, bioink viscosity were finely optimized to maximize the outcomes of the printing process, while minimizing cell suffering due to applied shear stress. To this aim, the possibility to print the devel- oped hydrogels as biphasic sol/gel systems or fully developed gels able to keep their shape upon printing was exploited to finely balance the two concurrent needs of high resolution and poor cell suffering and death. Correspondence: Gianluca Ciardelli, Department of Mechanical and Aerospace Engineering (DIMEAS), Politecnico di Torino, Turin, Italy. E-mail: [email protected] Key words: Polyurethanes; bioengineered models; soft tissues; thermoplastic polymers; hydrogels. Conference presentation: this paper was pre- sented at the Second Centro 3R Annual Meeting - 3Rs in Italian Universities, 2019, June 20-21, University of Genoa, Italy. Received for publication: 28 October 2019. Accepted for publication: 6 November 2019. This work is licensed under a Creative Commons Attribution NonCommercial 4.0 License (CC BY-NC 4.0). ©Copyright: the Author(s), 2019 Licensee PAGEPress, Italy Biomedical Science and Engineering 2019; 3(s2):85 doi:10.4081/bse.2019.85 [page 11] [Biomedical Science and Engineering 2019; 3(s2):85] Biomedical Science and Engineering 2019; volume 3(s2):85 Non-commercial use only
Transcript of Copyright: the Author(s), 2019 Biomedical Science and ...
On the transferability of tissueengineering technologies to the design of tissue modelsGianluca CiardelliDepartment of Mechanical andAerospace Engineering (DIMEAS),Politecnico di Torino, Turin, Italy
Abstract3D tissue-engineered models are
promising tools in the screening and evalu-ation of drugs and therapies as well as in theinvestigation of the molecular mechanismsinvolved in disease onset and progression.In this context, we describe our efforts insoft tissue replication, to design in vitromodels that have the potential to providebetter insight into the development of age-ing process and related pathologies, withparticular reference to the cardiovascularfield.
Introduction3D tissue-engineered models are
promising tools in the screening and evalu-ation of drugs and therapies as well as in theinvestigation of the molecular mechanismsinvolved in disease onset and progression.
In a 3D bioengineered model, the scaf-fold should finely reproduce the architec-ture and composition of the native tissue, inorder to properly regulate cell behaviour,such as spreading, proliferation, differentia-tion, and extracellular matrix (ECM) pro-duction, alike to what they do in vivo. Inorder to successfully mimic a tissue, severalaspects must be considered when designinga tissue model. Among others, cell sourcesand 3D scaffolds architecture and composi-tion (both in bulk and on the surface) havebeen investigated in depth (Figure 1).
The biomaterial choice is a crucial ele-ment, since it strongly affects cellular func-tions. It should be based on the materialmechanical properties, as the one of thescaffolds should match those of the tissue tobe modelled, as thoroughly highlighted bymechanobiology studies.1 In the replicationof hard tissues, due to their high stiffnessand load- bearing properties, ceramics andtheir composites are generally used, whilenatural and synthetic polymers are appro-priate choices in the engineering of soft tis-sues.2
In soft tissue replication, in vitromodelsof heart tissue have the potential to provide
better insight into the mechanisms of heartdiseases, which are the leading cause ofmortality in the world. Moreover, myocar-dial models could be a useful tool in theevaluation of drugs cardiotoxicity. In car-diac tissue engineering, the most investigat-ed natural polymers are collagen, gelatin,fibrin, and alginate, and among syntheticpolymers, degradable polyesters and theircopolymers have been mainly explored. Forinstance, since the myocardial tissue has tobear the contractile and expansive forcesproduced at each cardiac cycle, elastomericpolymers, such as poly(glycerol sebacate)and polyurethanes have been mainly stud-ied.
ResultsOur group extensively worked in the
synthesis of biodegradable polyurethanesfor contractile tissues applications. In a pre-vious article, we identified a promisingpolyester-urethane (PUR), showing lowYoung’s modulus and elastomericbehaviour.3 This PUR was synthesized frompoly(ε-caprolactone) diol, 1,4-butanediiso-cyanate and L-lysine ethyl ester. In a suc-ceeding article, this PUR was used to obtainporous scaffolds by thermally inducedphase separation (TIPS). The scaffolds werefunctionalized with fibronectin by plasmatreatment, and seeded with primary ratneonatal cardiomyocytes.4 Morphologicaland mechanical analysis exhibited a pore-aligned scaffold formation, with mechanicalproperties and architecture resembling theone of the myocardium. Cardiomyocyteviability was stable for 14 days of experi-mentation, comprising beating activity andcell adhesion. The improved levels of phos-phorylation of AKT and ERK1/2 elucidatedthe prolonged cell survival. The variation ofcardiac muscle and glucose genes indicateda transition phase in cardiomyocyte matura-tion, with an early evolution to an adult phe-notype and a permanence of some fetalcharacteristics. These results represented afirst step towards the development of a newgeneration of myocardial models. A furtherstep involved the fabrication of scaffoldswith improved controlled geometry, whichcan be obtained via rapid prototyping (RP)procedures.In detail, the PUR was micro-fabricated into 0°/90° square-grid multi-layered scaffolds through a custom-mademelt-extrusion additive manufacturinginstrument and surface functionalized withlaminin-1 (cardiac niche ECM component,sample acronym PUR-LN1) or gelatin(low-cost adhesion protein, sampleacronym PUR-G) through plasma treatmentfollowed by carbodiimide chemistry.5 PUR-
G and PUR-LN1 scaffolds both improvedcardiac progenitor cell (CPC) adhesion, butLN1 functionalization turned out to besuperior in promoting CPC proliferation,protection from apoptosis and expression ofdifferentiation markers for cardiomyocytes,endothelial and smooth muscle cells. PUR-LN1 subcutaneously implanted in miceevoked a weak inflammation and integratedwith the host surrounding tissue, evidencinga significant blood vessel density aroundthe scaffolds. In a different approach, ther-mo- and photo-curable bioinks weredesigned for the bioprinting of cellularizedconstructs to be used for reparative/regener-ative purposes or as in vitro bioengineeredmodels of soft tissues. The thermo-sensitivenature of the developed bioinks allowed aneasy and homogeneous cell dispersion,through the addition of the cells to thehydrogels at a temperature lower than theirgelation temperature (below 25 °C), whenthe systems were in the sol or semi-gelphase. After loading in the syringe, cellular-ized hydrogels were subjected to a fast sol-to-gel transition with increasing tempera-ture. Syringe equilibrium temperature and,as a consequence, bioink viscosity werefinely optimized to maximize the outcomesof the printing process, while minimizingcell suffering due to applied shear stress. Tothis aim, the possibility to print the devel-oped hydrogels as biphasic sol/gel systemsor fully developed gels able to keep theirshape upon printing was exploited to finelybalance the two concurrent needs of highresolution and poor cell suffering and death.
Correspondence: Gianluca Ciardelli,Department of Mechanical and AerospaceEngineering (DIMEAS), Politecnico diTorino, Turin, Italy.E-mail: [email protected]
Key words: Polyurethanes; bioengineeredmodels; soft tissues; thermoplastic polymers;hydrogels.
Conference presentation: this paper was pre-sented at the Second Centro 3R AnnualMeeting - 3Rs in Italian Universities, 2019,June 20-21, University of Genoa, Italy.
Received for publication: 28 October 2019.Accepted for publication: 6 November 2019.
This work is licensed under a CreativeCommons Attribution NonCommercial 4.0License (CC BY-NC 4.0).
©Copyright: the Author(s), 2019Licensee PAGEPress, ItalyBiomedical Science and Engineering 2019; 3(s2):85doi:10.4081/bse.2019.85
[page 11] [Biomedical Science and Engineering 2019; 3(s2):85]
Biomedical Science and Engineering 2019; volume 3(s2):85
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Moreover, the fast sol-to-gel transition ofthe developed systems ensured a homoge-neous cell distribution, avoiding cell sedi-mentation during the printing process.Eventually, a further degree of freedom wasprovided to the developed bioinks by mak-ing them photo-responsive, allowing a finemodulation of the final mechanical proper-ties and stability in aqueous environment ofthe scaffolds. By working on bioink compo-
sition, multilayered scaffolds with residencetime in aqueous environment rangingbetween few weeks up to more than 2months were designed. Additionally, gelstiffness was modulated within the range 1-100 kPa, thus making the developed bioinkssuitable for a wide variety of applications inthe biomedical field, such as in brain, skin,liver, kidney and muscle tissue engineering.
References1. Chan BP, Leong KW. Scaffolding in tis-
sue engineering: General approachesand tissue-specific considerations. EurSpine J 2008;17:467-79.
2. Seidi A, Ramalingam M, Elloumi-Hannachi I, et al. Gradient biomaterialsfor soft-to-hard interface tissue engi-neering. Acta Biomater 2011;7:1441-51.
3. Sartori S, Boffito M, Serafini P, et al.Synthesis and structure-property rela-tionship of polyester-urethanes andtheir evaluation for the regeneration ofcontractile tissues. React Funct Polym2013;73:1366-76.
4. Vozzi F, Logrand F, Cabiati M, et al.Biomimetic engineering of the cardiactissue through processing, functional-ization, and biological characterizationof polyester urethanes. Biomed Mater2018;13:055006.
5. Boffito M, Di Meglio F, Mozetic P, etal. Surface functionalization ofpolyurethane scaffolds mimicking themyocardial microenvironment to sup-port cardiac primitive cells. PLoS One2018;13:e0199896.
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Figure 1. TE approach for the development of in vitro models.
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