university of minho school of engineering uma escola a reinventar o futuro – semana da escola de...
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University of Minho
School of Engineering
<Nome do Centro>
Uma Escola a Reinventar o Futuro – Semana da Escola de Engenharia - 24 a 27 de Outubro de 2011
Main goal
To develop a new “hydrogel” for application in the cartilage tissue
engineering field through the self-assembly of nanoparticles loaded
with growth factors enabled throught the use of platelets lysates (PL)
in two aways : (i) source of growth factors and (ii) as connecting
hydrogel matrix.
Introduction
The cartilage tissue engineering (TE) strategy described in this work
relies on the combination of natural polymer-based nanoparticles
(NPs), produced from the complexation of chitosan (CH) with
chondroitin sulfate (CS), for the incorporation and sustained release of
bioactive agents, namely platelet lysate (PL). The CH/CS complex
mimics the extracellular matrix (ECM) interactions and PL is an
autologous source of a cocktail of GFs acting on tissue healing and
repair. When used at determined concentrations, the PL-releasing
NPs can assemble in a simple and quick mode to form a three-
dimensional (3D) stable hydrogel while in suspension with human
Adipose derived Stem Cells (hASCs), following a mild centrifugation,
allowing the cells to be entrapped in this enriched 3D environment.
VITOR E. SANTO*, ELENA G. POPA
Supervisors: Rui L. Reis, João F. Mano and Manuela E. Gomes * [email protected]
ASSEMBLY OF PLATELET LYSATE-LOADED NANOPARTICLES AS A NEW HYDROGEL SYSTEM FOR CARTILAGE TISSUE ENGINEERING
PL-adsorbed CH/CS NPs hASCs (isolated from human lipoaspirates)
Spin down
Materials & Methods
Formulations:
(i) hASCs pellets; (ii) assembled empty NPs + hASCs; (iii) assembled
PL-loaded NPs + hASCs.
Characterization:
(i) In vitro GF release; (ii)Histology: Hematoxylin & Eosin (H&E), alcian
blue and safranin-O; (iii) real time Polymerase Chain Reaction (rt-PCR)
for evaluation of collagen type II and type I gene expression.
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CH in acetic acid
CS in distilled water
Centrifugation and resuspension
PL Adsorption
Polyelectrolyte Complexation
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1. Nanoparticles Production and Platelet Lysate Adsorption
2. Assembly of PL-loaded Nanoparticles for hASCs entrapment
3. In vitro culture under chondrogenic stimulus
hASCs+ PL loaded NPs hASCs+ empty NPs hASCs pellets
100µm
H&E
Alcian Blue
Safranin-O
Results and Discussion
2. Histological Characterization
3. Real time PCR – chondrogenic gene expression
Collagen type II Collagen type I
The GF release profile for
PDGF-BB and TGF-β1 is
characterized by a strong
burst release at day 1,
followed by a more controlled
delivery during the remaining
days. After day 7, the GF
release was undetectable.
1. In vitro GF release
The H&E staining reveals that after 28 days, the stability of the PL-loaded NPs
hydrogel is higher in comparison with the empty NPs. Moreover, we can
observe the formation of cartilage ECM comparable with the positive control.
The scaffolds loaded with PL showed a stronger upregulation of col II in
comparison with col I, showing the formation of hyaline articular cartilage ECM.
Materials and Methods Results and Discussion
The presence of PLs in the 3D matrix enhances the in vitro chondrogenic
diferentiation of hASCs. PLs have a multifunctional role as a connective/stability
agent for the matrix and as a GFs supplier for chondrogenic differentiation,
creating an effective hydrogel network for cartilage TE..
Conclusions
Acknowledgments
The authors thank the FCT grants (SFRH/BD/39486/2007 and SFRH/BD/64070/2009), the European
projects (NMP3-CT-2004-500283 and NMP4-SL-2009-229292), IPS and Hospital da Prelada.