July 2, 2008

The Development of Biocomposite Nanofibers for Tissue Scaffolding Applications

By Zhang, Y Z Lim, C T

The last decade has seen significant progress in the production of nanoflbers by electrospinning. One of the major drivers to this progress is the potential use ofnanofibrous structures as scaffolds for engineering tissues in regenerative medicine. Electrospun fibers are capable of emulating the nanofibrous architecture of the native extracellular matrix. They can potentially provide in-vivo-like nanomechanical and physicochemical signaling cues to the cells to establish apposite cell-scaffold interactions and promote functional changes between and within cells toward synthesis of a genuine extracellular matrix over time. In this context, this paper presents a brief overview of a scaffold design strategy. It also presents recent research pertaining to developing biomimetic and bioactive nanofibrous tissue scaffolds through electrospinning biocomposite nanofibers of organic-organic and inorganicorganic hybrids, which are potentially applicable to soft and hard tissue engineering. INTRODUCTION

Human tissues are assemblies of one or more types of cells and their associated intercellular materials, biologically termed the extracellular matrix (ECM). The ECM is known to be a complex three- dimensional (3-D) nanofibrous network made of mainly structural proteins (e.g., collagen) and carbohydrates produced and maintained by the cells embedded within this network, just like a spider working with its silk web. The ECM provides mechanical strength and structural support for cell adhesion, migration, proliferation, and differentiation. When tissues are damaged or diseased, especially with a large volume of defects, they can be repaired and/or regenerated by using a tissue-engineering approach1 that usually involves three elements: cells, biomaterials scaffolds, and soluble biomolecules. Throughout the tissue repair or regeneration process, the cells undoubtedly dictate synthesizing and regenerating neo- native functional tissues, but the biomaterials scaffolds, as a provisional ECM analogue, are also deemed to play a pivotal role (at least in the initial developmental stages) because almost all living normal cells are of the anchoring type. This means they will die without a matrix to support and provide a milieu for cellular adhesion, proliferation, spatial organization, and function to form new tissue.2 Thus, tissue engineering to a large extent is reliant on the scaffolding technology, which is closely associated with the materials and fabrication methods of the biomaterials scaffolds.

With respect to the scaffold fabrication methods, numerous techniques as reviewed in the literature3"5 have previously been employed to make a variety of porous scaffolds. Notably today, electrospinning,67 an old fiber-spinning technique, has resurfaced and recently emerged as an outstanding platform technique for tissue scaffold fabrication. Unlike industrial wet/met spinning, electrospinning in principle relies on electrostatic force to drive the formation of extremely fine fibers with typical diameters ranging from a few tens to hundreds of nanometers. The mechanism of forming nanoscale fibers with electrospinning has been identified as a result of the bending instability8 or whipping9,10 of the charged jet. To date, with the electrospinning process, more than 100 different types of materials have been electrospun into ultrafine fibers. Electrospun fibers have found a wide range of applications in such areas as health care, optoelectronics, sensors, and catalysis, just to name a few. In tissue engineering, using electrospun nanofibers enables us to recreate a native ECM- resembling physical environment for the cells, which is geometrically and physicochemically advantageous over those conventional scaffolds. For example, the dimensional smallness and mechanical weakness of nanofibers could potentially provide matched nanomechanical and biodegradation properties for cells to proactively interplay with the provisional nanofibrous scaffold and remodel it via degradation, absorption, and secreting neo-matrix, similar to that of the innate remodeling mechanism. Moreover, the highly porous nanofibrous scaffolds with interconnected pore/ interstice structure and large surface area could facilitate the adsorption of biological molecules that regulates cell activities, exchange of nutrient/waste products, and tissue in-growth for the formation of a 3-D cell-scaffold complex.

Scaffold materials are related to the chemical characteristics that define the surface properties of the scaffold and consequently influence the interactions with cells and those dissociated biomolecules. Since 2001,11-13 synthetic biodegradable polymers like polyOactic acid) (PLA)1 poly(lactic-co-glycolic acid) (PLGA), and polycaprolactone (PCL) have been the major materials being used for nanofibrous scaffolding because of their better electrospinnability. Electrospinning of the naturally occurring biopolymers such as collagen14 and chitosan" is also possible by using specialty organic solvents (e.g., hexafluoroisopropanol [HFP]). Comparatively, the major attractions of synthetic polymers are their availability with a wide range of physical and mechanical properties. However, natural biopolymers are advantageous in terms of biocompatibility, but lack the required mechanical properties. Therefore, hybridization of both the synthetic and the natural polymers to create novel composite materials would be able to combine the merits of both for attaining desired biological and physicochemical characteristics of scaffolds.16-22 As such, to benefit from the advantages of materials hybridization and the ECMmimicking capability of electrospun products, it is necessary to develop biomimetic and bioactive nanofibers from hybrids. Hybridization is particularly important in terms of biomimicking natural bone, which is a hierarchical structure with inorganic hydroxyapatite (HA) nanocrystals incorporated in collagen nanofibers as the basic building blocks.23,24 This article provides an overview of current state-of- the-art biocomposite nanofibers made from hybrids of organic- organic and inorganic-organic materials systems, which can correspondingly be applied to engineer soft and hard tissues, respectively.


The most commonly used approach for fabrication of organic- organic biocomposite nanofibers is simply electrospinning a blend of two different polymers from the synthetic and the natural sources, such as the gelatin/PCL system used by the authors.25 In the microstructure, this leads to the formation of components of randomly blended biocomposite nanofibers with different components co-existing within each individual fiber (Figure 1a).26 The blend biocomposite nanofibrous scaffold of gelatin/PCL was found to have very good wettability and/or hydrophilicity and balanced mechanical properties compared to its constituents. In-vitro cell culture results showed significant cell proliferation and infiltration compared to the biologically inert synthetic PCL-only scaffolds. Cell penetration into the gelatin/PCL composite nanofibrous scaffolds up to approximately 110 [mu]m was observed. Because gelatin in the gelatin/PCL scaffold is soluble, the formation of porous fibers after leaching treatment was proven experimentally.26 This suggested that extra spaces in-situ allow for cell in-depth migration and easy transportation of nutrients/waste during cell culture with the nanofibrous gelatin/PCL. Further study suggests the great potential of gelatin/PCL biocomposite nanofibers for application in dermal wound healing.27 A similar approach has been used by other researchers in the last couple of years by blending natural biopolymers, in particular the collagen, with different synthetic polymers to generate biomimetic and bioactive biocomposite nanofibers. These include collagen/PEUU,28 collagen/PLCL,29 collage n/PCL,30,31 collagen/PHBV,32 collagen/elastin/PLGA,33 and chitin/ PGA.34 These studies have similarly demonstrated that natural biopolymer-containing composite nanofibers had outperformed those biologically inert synthetic counterparts in cell adhesion, spreading, migration, proliferation, and differentiation, and are potentially usable for engineering soft tissues like skin, blood vessels, and nerves.

Biocomposite nanofibers can also be fabricated by rearranging the constituents in the form of core-sheath (Figure Ib) through coaxial electrospinning.35-39 Coaxial electrospinning, which slightly differs from the traditional electrospinning setup, requires a compound spinneret consisting of one (or more) inner capillary housed by an outer one from which two spinning dopes can be independently metered into the respective channels and integrated into a core-sheath structured composite fiber as they are ejected from the compound spinneret. Coaxial electrospinning provides a novel route to design and fabricate a variety of functional nanofiber structures.40 When using electrospinnable bioactive macromolecules such as collagen as the shell (to impart bioactivity) and synthetic polymer as core (to retain mechanical and structural advantage), core-shell structured biocomposite nanofibers as a novel bioactive cellular scaffold can be developed. This concept, with its enhanced efficacy, was demonstrated recently41 with an examination of cell proliferation and morphological changes. The study was done by culturing human dermal fibroblasts (HDFs) on the collagen-r-PCL (representing collagen and PCL being the shell and core, respectively) scaffolds. This study suggests that the collagen-r- PCL coreshell biocomposite nanofibers tend to resemble the natural ECM architectural constituent of collagen, thus enabling cells to have a propensity to interact well with them. The above-mentioned blend electrospinning can also produce biocomposite nanofibers incorporated with drugs for controlled release purposes. However, it usually gives rise to severe burst release problems due to poor electrospinnability of the loaded drugs and/or weak molecular interaction between the drug and polymer carriers. In contrast, fabrication of core-shell nanofibers via coaxial electrospinning does not require the encapsulated component to be electrospinnable.42,43 Furthermore, as a type of reservoir release device, core-shell biocomposite nanofibers are promising in preserving those labile biological agents such as DNA and growth factors from being exposed to organic solvents during spinning dope preparation and fabrication stages, or being deactivated/denatured in an aggressive environment. Its capability of sustainably releasing proteins or drugs had also been recently demonstrated.42,44,45 Coaxial electrospinning and core-shell nanofibers afford the simple and effective solution of having novel biomimetic nanofibrous scaffolds integrated with the specific function of sitetargeting controlled drug releases.


Inorganic-organic biocomposite nanofibers can be used to engineer hard tissues such as bone and dental tissues. Compositionally, natural bone consists of 65-70 wt.% inorganic crystals (mainly HA) and 30-35 wt.% of organic matrix (mainly collagen), and structurally is hierarchically organized from the macro- to micro- to nano- scale. At the nano-scale, the basic building blocks are the plate- like HA nanocrystals incorporating collagen nanofibers with the crystallographic c axis of HA being aligned along the long axis of collagen fibers.23,46-48 The unique compositional and structural characteristics of bone have been inspiring researchers to use biomimetic approaches to prepare different bone-like substitutes. In this regard, electrospinning holds a great potential for a "bottom- up" strategy to reconstruct bone tissue through developing compositionally and nanostructurally bone-mimicking biocomposite nanofibers. Currently, four different routes have been employed for preparation of inorganic-organic biocomposite nanofibers:

* One-step method: dispersing inorganic components, primarily HA nanoparticles, in polymer solutions by simple blend mixing for electrospinning49-55

* Two-step method: in-situ synthesis of inorganic-organic composites followed by electrospinning (Figure 2a)56

* Two-step method: electrospinning polymer nanofibers containing entrapped calcium or phosphorus precursors, followed by in-situ growth of calcium phosphate on and/or within the polymer nanofibers (Figure 2b)57

* Three-step method: preparing electrospun polymer nanofibers, having them surface-modified to generate reactive functional groups (for subsequent nucleation and growth of apatite minerals), then mineralizing in simulated body fluids58-60

Here, the first two routes produce components of blended biocomposite nanofibers (Figure 2a), whereas the third and fourth route usually lead to coatings of minerals formed on the electrospun polymer nanofiber templates (Figure 2b).


The strategy of introducing natural bioactive components into biologically inert but mechanically meritorious synthetics and converting such combinations into nanofibrous form offers a facile approach to bioactivate and functionalize nanofibrous scaffolds. Yet, there are problems and challenges to be addressed for better control of microstructures, mechanical properties, and functions of biocomposite nanofibers in future research activities of this niche area. For example, due to the incorporation of poor electrospinnable natural components, formulating a robust electrospinnable composite materials system by using a novel solvent system and fiber-forming additives is critical.61 Another concern is the limited improvement or sometimes decreased mechanical properties in the electrospun biocomposite nanofibers.25,28,29,50,62 For organic-organic hybrids, severe phase separation and weak physical interactions between constituents are probably responsible for the weakening mechanical performance.25,26,34 For the inorganic-organic hybrids, a key aspect is to ensure molecular recognition between the organic matrix and the nucleating/ growing mineral phase while carrying out biomimetic synthesis and process.63 Native cells reside in a 3-D nanofibrous ECM network, which is subjected to a continuous renewal and remodeling process by the cells. However, it seemed difficult to have cells evenly and threedimensionally distributed within the electrospun nanofibrous scaffolds due to the small pores/ interstices, formed from nanofiber interlaces. Encouraged by previously observed cellular ingrowth phenomena,25,28,64-66 future biocomposite nanofibrous structures need to be endowed with more precise biophysical and biochemical motifs to motivate a cell's in- depth migration, and to be physically spatialized with large pore size and porosity.

How would you...

...describe the overall significance of this paper?

Electrospinning has recently provided an enormous impetus to the pursuit of fabricating extracellular matrix-like fibrous scaffolds. While a variety of polymers had been attempted for this purpose, this paper focuses on nanofibers made from a composite of more than one material. These nanofibers have been demonstrated to outperform single material nanofibers as hiomimetic scaffolds for tissue engineering applications.

...describe this work to a materials science and engineering professional with no experience in your technical specialty?

Electrospinning relies on electrostatic forces to drive the formation of ultrafine submicrometer- and/or nanofibrotts structures. Individual nanofibers containing different phases/ structures and signaling molecules can be made from hybrids of organic-organic or inorganicorganic materials. Thus, physical, chemical, and biological properties can be tailored for engineering particular functional tissues effectively.

...describe this work to a layperson?

A biocomposite is made from two or more components with different properties better than either of its constituents alone. For tissue repair and regeneration applications, this paper aims to illustrate different biocomposite nanofibers that were prepared based on an ultrafine fiber-manufacturing technology-electrospinning.


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Y.Z. Zhang and CT. LIm are with the Division of Bioengineering, National University of Singapore (NUS). Lim is also with the Department of Mechanical Engineering and the NUS Nanoscience & Nanotechnology Initiative, 9 Engineering Drive 1, Block EA #050 - 10, Singapore 117576; +65-65167801; fax +65-67791459; e-mail [email protected]

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