Chondrogenesis-inductive nanofibroussubstrate using both biological fluids and mesenchymal stem cells from anautologous source Marta R. Casanova1,2, MSc, Marta Alves da Silva1,2,PhD, Ana R. Costa-Pinto1,2, PhD, Rui L. Reis1,2, PhD, AlbinoMartins1,2, PhD, Nuno M. Neves1,2*, PhD 1 3B’s Research Group –Biomaterials, Biodegradable and Biomimetics, Avepark – Parque de Ciência eTecnologia, Zona Industrial da Gandra, 4805-017 Barco – Guimarães, Portugal, 2 ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães4805-017, Portugal; *Correspondingauthor: [email protected]; Tel.
+351-253-510905; Fax: +351-253-510909 Funding sources: This work was supported by PortugueseFoundation 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/73322/2010and 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).Theauthors declare no conflicts of interest. Wordcount for Abstract: 149 words Wordcount for manuscript: 4794 words Numberof references: 51 Numberof tables: 2 Numberof Figures: 7 Abstract. During the last decade, many cartilage tissue engineeringstrategies have been developed, being the stem cell-based approach one of themost promising. Transforming Growth Factor-?3 (TGF-?3) and Insulin-like GrowthFactor-I (IGF-I) are key proteins on the regulation of chondrogenicdifferentiation. Therefore, these two growth factors (GFs) were immobilized atthe surface of a single electrospun nanofibrous mesh (NFM) aiming todifferentiate human Bone Marrow-derived Mesenchymal Stem Cells (hBM-MSCs). Forthat, the immobilization of defined antibodies (i.
e. anti-TGF-?3 andanti-IGF-I) allows the selective retrieval of the abovementioned GFs from humanplatelet lysates (PL). Biochemical assays, involving hBM-MSCs cultured onbiofunctional nanofibrous substrates under basal culture medium during 28 days,confirms the biological activity of bound TGF-?3 and IGF-I. Specifically, thetypical spherical morphology of chondrocytes and the immunolocalization ofcollagen type II confirmed the formation of a cartilaginous ECM. Therefore, theproposed biofunctional nanofibrous substrate are able to promote chondrogenesis. Keywords: TransformingGrowth Factor-?3 (TGF-?3), Insulin-like Growth Factor-I (IGF-I), PlateletLysates, Electrospun Nanofibrous Meshes, Chondrogenic Differentiation BackgroundArticular cartilage is a connective tissuewith low repair potential due to its avascular nature and lack of progenitorcells.(1) Therefore, articular cartilage injuries present a challengingproblem for the musculoskeletal physicians.
Many treatment option have beendeveloped during the last decades to repair damaged cartilage, such asmicrofracture and mosaicplasty.(2) However, an adequate therapy for the long-term repair of cartilagelesions and which recover totally the function of this tissue is still to bedeveloped. Tissue engineering and regenerative medicine (TERM) strategies holdthe promise to recover injury in cartilage to its native state by combiningcells, growth factors (GFs) and scaffolds with appropriate environmentalstimulation.(3, 4) Despite the promising result reported bychondrocyte implantation techniques, namely autologous chondrocyte implantation(ACI) and matrix-induced autologous chondrocyte implantation (MACI), in a largepercentage of patients, they present many drawbacks such as the obtainable ofenough autologous chondrocytes during harvesting, loss of cellulardifferentiation potential when cultured in vitro, and decreased capacity toproduce extracellular matrix (ECM).(2, 5)Mesenchymal stem cells (MSCs) present advantages over chondrocytes, since theycan be obtained from an autologous source, in a less invasive procedure, andpresent an higher proliferation capability together with their chondrogenicdifferentiation potential.
(6, 7)Bone marrow-derived MSCs (hBM-MSC) wereextensively studied in cartilage engineering and regeneration.(4, 8-13)Conventionally, these MSCs are induced to differentiate into a certain lineageby the supplementation of culture medium with defined exogenous bioactivefactors. The culture medium for the in vitro chondrogenesis of hBM-MSCs wasfirstly described by Johnstone et al. in 1998.(14) The chondrogenic differentiation medium may contains differentcombinations 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) Amongthem, TGF-?3 and IGF-I have been the most effective and commonly used GFs, ableto induce the chondrogenic differentiation of hBM-MSCs, although the IGF-I hasbeen 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 ata biomaterial substrate would lead to a stable chondrogenic differentiation ofhBM-MSCs.Autologous regeneration of tissues, whereboth cells and bioactive factors are from the same patient, is an attractiveapproach because it avoids the immune response.(25-27) Mostof the works in cartilage tissue engineering are based on mesenchymal stemcells differentiated into the chondrogenic lineage by using recombinant GFs incombination with biomaterials.(9, 17-20, 28) Morerecently, platelet lysate (PL), consisting in a cocktail of different GFs(e.g., bFGF, VEGF, TGF- ?, BMPs, PDGF-??, EGF, and IGF-I), provides anautologous complex mixture of bioactive factors to the cells at the injury site.(27, 29)The leading goal of this study is todevelop a biofunctionalized electrospun nanofiber mesh (NFM) with chondrogenicinduction capacity, through the immobilization of autologous TGF- ?3 and IGF-Iretrieved from platelet lysates.
For that, we will take advantage of thespecific and efficient interactions between specific antibody and its antigen.Based on this biological strategy, it will be possible to selectively bound theGFs of interest (TGF-?3 and IGF-I) from a pool of highly concentrated GFspresent in PL. The chondrogenesis potential of electrospun NFMs withimmobilized TGF-?3 and IGF-I will be further assessed by culturing hBM-MSCs.