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Chondrogenesis-inductive interest. Word count for Abstract: 149 words Word

 

 

Chondrogenesis-inductive nanofibrous
substrate using both biological fluids and mesenchymal stem cells from an
autologous source

 

 

Marta R. Casanova1,2, MSc, Marta Alves da Silva1,2,
PhD, Ana R. Costa-Pinto1,2, PhD, Rui L. Reis1,2, PhD, Albino
Martins1,2, PhD, Nuno M. Neves1,2*, PhD  

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1 3B’s Research Group –
Biomaterials, Biodegradable and Biomimetics, Avepark – Parque de Ciência e
Tecnologia, Zona Industrial da Gandra, 4805-017 Barco – Guimarães, Portugal,

2 ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães
4805-017, Portugal;

*Corresponding
author: [email protected]; Tel. +351-253-510905; Fax: +351-253-

510909

 

 

Funding sources: This work was supported by Portuguese
Foundation for Science and Technology (FCT); The PhD grant of MC (PD/BD/113797/2015)
financed by the FCT Doctoral Program on Advanced Therapies for Health (PATH)
(FSE/POCH/PD/169/2013) The Post-doc fellowships of MAS and ARP (SFRH/BPD/73322/2010
and SFRH/BPD/90332/2012) The IF grant of AM (IF/00376/2014) and the projects SPARTAN
(PTDC/CTM-BIO/4388/2014) and FRONthera (NORTE-01-0145-FEDER-0000232).

The
authors declare no conflicts of interest.

 

 

 

Word
count for Abstract: 149 words

Word
count for manuscript: 4794 words

Number
of references: 51

Number
of tables: 2

Number
of Figures: 7

 

 

 

 

 

 

Abstract. During the last decade, many cartilage tissue engineering
strategies have been developed, being the stem cell-based approach one of the
most promising. Transforming Growth Factor-?3 (TGF-?3) and Insulin-like Growth
Factor-I (IGF-I) are key proteins on the regulation of chondrogenic
differentiation. Therefore, these two growth factors (GFs) were immobilized at
the surface of a single electrospun nanofibrous mesh (NFM) aiming to
differentiate human Bone Marrow-derived Mesenchymal Stem Cells (hBM-MSCs). For
that, the immobilization of defined antibodies (i.e. anti-TGF-?3 and
anti-IGF-I) allows the selective retrieval of the abovementioned GFs from human
platelet lysates (PL). Biochemical assays, involving hBM-MSCs cultured on
biofunctional nanofibrous substrates under basal culture medium during 28 days,
confirms the biological activity of bound TGF-?3 and IGF-I. Specifically, the
typical spherical morphology of chondrocytes and the immunolocalization of
collagen type II confirmed the formation of a cartilaginous ECM. Therefore, the
proposed biofunctional nanofibrous substrate are able to promote chondrogenesis.

 

Keywords: Transforming
Growth Factor-?3 (TGF-?3), Insulin-like Growth Factor-I (IGF-I), Platelet
Lysates, Electrospun Nanofibrous Meshes, Chondrogenic Differentiation

 

 

Background

Articular cartilage is a connective tissue
with low repair potential due to its avascular nature and lack of progenitor
cells.(1) Therefore, articular cartilage injuries present a challenging
problem for the musculoskeletal physicians. Many treatment option have been
developed during the last decades to repair damaged cartilage, such as
microfracture and mosaicplasty.(2) However, an adequate therapy for the long-term repair of cartilage
lesions and which recover totally the function of this tissue is still to be
developed. Tissue engineering and regenerative medicine (TERM) strategies hold
the promise to recover injury in cartilage to its native state by combining
cells, growth factors (GFs) and scaffolds with appropriate environmental
stimulation.(3, 4)

Despite the promising result reported by
chondrocyte implantation techniques, namely autologous chondrocyte implantation
(ACI) and matrix-induced autologous chondrocyte implantation (MACI), in a large
percentage of patients, they present many drawbacks such as the obtainable of
enough autologous chondrocytes during harvesting, loss of cellular
differentiation potential when cultured in vitro, and decreased capacity to
produce extracellular matrix (ECM).(2, 5)
Mesenchymal stem cells (MSCs) present advantages over chondrocytes, since they
can be obtained from an autologous source, in a less invasive procedure, and
present an higher proliferation capability together with their chondrogenic
differentiation potential.(6, 7)

Bone marrow-derived MSCs (hBM-MSC) were
extensively studied in cartilage engineering and regeneration.(4, 8-13)
Conventionally, these MSCs are induced to differentiate into a certain lineage
by the supplementation of culture medium with defined exogenous bioactive
factors. The culture medium for the in vitro chondrogenesis of hBM-MSCs was
firstly described by Johnstone et al. in 1998.(14) The chondrogenic differentiation medium may contains different
combinations of the following bioactive factors: dexamethasone, ascorbic acid,
Transforming Growth Factor-? (TGF-?), Bone Morphogenetic Proteins (BMP),
Fibroblast Growth Factors (FGF) and Insulin-like Growth Factor-1 (IGF-I).(5, 6, 9, 15-21) Among
them, TGF-?3 and IGF-I have been the most effective and commonly used GFs, able
to induce the chondrogenic differentiation of hBM-MSCs, although the IGF-I has
been replaced by insulin-transferrin-selenious acid (ITS) or insulin.(9, 15, 16, 20, 22-24)
Therefore, we herein hypothesize that the availability of TGF- ?3 and IGF-I at
a biomaterial substrate would lead to a stable chondrogenic differentiation of
hBM-MSCs.

Autologous regeneration of tissues, where
both cells and bioactive factors are from the same patient, is an attractive
approach because it avoids the immune response.(25-27) Most
of the works in cartilage tissue engineering are based on mesenchymal stem
cells differentiated into the chondrogenic lineage by using recombinant GFs in
combination with biomaterials.(9, 17-20, 28) More
recently, platelet lysate (PL), consisting in a cocktail of different GFs
(e.g., bFGF, VEGF, TGF- ?, BMPs, PDGF-??, EGF, and IGF-I), provides an
autologous complex mixture of bioactive factors to the cells at the injury site.(27, 29)

The leading goal of this study is to
develop a biofunctionalized electrospun nanofiber mesh (NFM) with chondrogenic
induction capacity, through the immobilization of autologous TGF- ?3 and IGF-I
retrieved from platelet lysates. For that, we will take advantage of the
specific and efficient interactions between specific antibody and its antigen.

Based on this biological strategy, it will be possible to selectively bound the
GFs of interest (TGF-?3 and IGF-I) from a pool of highly concentrated GFs
present in PL. The chondrogenesis potential of electrospun NFMs with
immobilized TGF-?3 and IGF-I will be further assessed by culturing hBM-MSCs.

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