Role of Nanofibrous Poly(Caprolactone) Scaffoldsin Human Mesenchymal Stem Cell Attachment

and Spreading for In Vitro Bone Tissue Engineering—Response to Osteogenic Regulators

N.S. Binulal, M.Sc.,1 M. Deepthy, Ph.D.,1 N. Selvamurugan, Ph.D.,2 K.T. Shalumon, M.Sc.,1

S. Suja, M.Sc.,1 Ullas Mony, Ph.D.,1 R. Jayakumar, Ph.D.,1 and S.V. Nair, Ph.D.1

In this study, we evaluated the role of fiber size scale in the adhesion and spreading potential of humanmesenchymal stem cells (hMSCs) on electrospun poly(caprolactone) (PCL) nanofibrous and microfibrous scaf-folds. The effect of in vivo regulators in inducing osteogenic differentiation of hMSCs on PCL nanofibrousscaffolds was investigated using osteogenic differentiation marker gene expression and matrix mineralization.Here, we report for the first time the influence of in vivo regulators in an in vitro setting with hMSCs for bonetissue engineering on PCL nanofibrous matrices. Our results indicated that hMSCs attached and spread rapidlyon nanofibrous scaffolds in comparison to microfibrous PCL. Further, hMSCs proliferated well on the nanofi-brous scaffolds. The cells on the nanofibrous PCL were found to differentiate into the osteoblast lineage andsubsequently mineralize upon addition of in vivo osteogenic regulators. The attachment and spreading of hMSCswere more effective on the nanofibers compared with the microfibers despite the lower protein surface coverage(total adsorbed protein per unit fiber surface area) on nanofibers.

Introduction

Bone tissue engineering is a field of significant researchinterest owing to the continued difficulty in growing

natural implantable bone tissue in sufficient quantity. At-tempts to generate functional bone tissue have shownincreased promise after the emergence of new nanomaterial-based scaffolds. Many studies have found to favor cell–material interactions on nanostructured surfaces, which canhelp cells to adhere, spread, and proliferate on such surfaces.1–3

Natural extracellular matrix (ECM) consists of protein-basednanofibers and nanostructured mineralized deposits that in-fluence the adhesion, proliferation, and differentiation of thecells.4–6 Hence, efforts to simulate the ECM through nanos-tructuring have received widespread attention as a means toengineer tissues in the presence of cells.7

Electrospinning has emerged as a very viable processingoption for nanostructured scaffolds consisting of nanofibrouspolymeric materials.8–10 The process has the capacity to pro-duce three-dimensional scaffolds as well as more complexhierarchical scaffolds consisting of nano- andmicrostructuredfeatures.11 Electrospun scaffolds have been investigated forbone tissue engineering by several investigators, using a

variety of polymers.12,13 In this study our focus was to utilizethis versatile method to structure both a nanofiber and a mi-crofiber-based scaffold of poly(caprolactone) (PCL) to betterunderstand the role of size scale of the fibers in human mes-enchymal stem cell (hMSC) attachment and spreading. Aspointed out in the review by Ballard et al.,14 the influence ofsubstrate structure is directly related to the type and con-centration of specific proteins adsorbed on the structures,because it is the proteins that mediate the interaction of thecells with the underlying structure. We have accordingly at-tempted to understand the differences in specific protein ad-sorption on nanofibrous versus microfibrous scaffolds toinvestigate possible links between the adsorption of specificproteins and the observed cell material interactions.

In our previous study,15 we found a substantial effect ofnanofibrous PCL scaffolds on the morphology and attach-ment of osteosarcoma cell line (MG-63). Morphologically,fibrous structures appeared to have a much stronger effectthan other nanostructured surfaces on cell behavior.16 Hence,in this study, we investigated in detail the behavior of na-nofiber versus microfiber substrates in hMSC attachmentand spreading. A second factor important for tissue engi-neering is the understanding of how in vivo factors play a

1Amrita Centre for Nanosciences, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham University,Kochi, India.

2Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur, India.

TISSUE ENGINEERING: Part AVolume 16, Number 2, 2010ª Mary Ann Liebert, Inc.DOI: 10.1089=ten.tea.2009.0242

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IOP PUBLISHING BIOMEDICAL MATERIALS

Biomed. Mater. 7 (2012) 065001 (13pp) doi:10.1088/1748-6041/7/6/065001

Gelatin nanoparticles loadedpoly( -caprolactone) nanofibroussemi-synthetic scaffolds for bone tissueengineeringN S Binulal1, Amrita Natarajan1, Deepthy Menon1, V K Bhaskaran2,Ullas Mony1,3 and S V Nair1,3

1 Amrita Centre for Nanosciences & Molecular Medicine, Amrita Vishwa Vidyapeetham University,Kochi, India2 Department of Orthopaedics, Amrita Institute of Medical Sciences and Research Centre, AmritaVishwa Vidyapeetham University, Kochi, India

E-mail: ullasmony@aims.amrita.edu and shantinair@aims.amrita.edu

Received 27 January 2012Accepted for publication 30 August 2012Published 9 October 2012Online at stacks.iop.org/BMM/7/065001

AbstractNanofibrous semi-synthetic polymeric nanocomposite scaffolds were engineered byincorporating a maximum of 15 wt% biopolymeric gelatin nanoparticles (nGs) into thesynthetic polymer poly( -caprolactone) (PCL) prior to electrospinning. The effect of nGs inaltering the physico-chemical properties, cell material interaction and biodegradability of thescaffolds was evaluated. Experimental results showed that the inherent hydrophobicity of PCLscaffolds remained unaltered even after the incorporation of hydrophilic nGs. However,breakdown of the continuous nanofibers into lengths less than 7 μm occurred within four toeight weeks in the presence of nGs in contrast with the greater than two year time frame for thedegradation of PCL fibers alone that is known from the literature. In terms of cell–materialinteraction, human mesenchymal stem cells (hMSCs) were found to attach and spread betterand faster on PCL_nG scaffolds compared to PCL scaffolds. However, there was no differencein hMSC proliferation and differentiation into osteogenic lineage between the scaffolds. Theseresults indicate that PCL_nG nanofibrous nanocomposite scaffolds are an improvement overPCL scaffolds for bone tissue engineering applications in that the PCL_nG scaffolds provideimproved cell interaction and are able to degrade and resorb more efficiently.

(Some figures may appear in colour only in the online journal)

1. Introduction

Tissue engineering scaffolds made up of electrospun fibersin the nanoscale range are useful in replicating the physicaldimensions and morphology of the native extracellularcollagen matrix (ECM) [1–4]. There have been many attemptsto combine synthetic and natural polymers and/or ceramicsin order to simulate the mineralized collagenous bone matrix

3 Authors to whom any correspondence should be addressed.

for tissue engineering purposes [5–8]. Synthetic polymers havethe advantage that their mechanical properties and degradationkinetics are more tunable when compared to natural polymers[9, 10], which makes them attractive for tissue engineeringapplications. Poly( -caprolactone) (PCL) is a biocompatible,bioresorbable, Food andDrugAdministration (FDA) approvedlow-cost synthetic and relatively strong polymer which hasbeen successfully electrospun [11]. However, PCL is veryslow to degrade (can take more than two years depending onthe molecular weight) and lacks cell recognition sites on the

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