The potential of nanofibers in tissue engineering and stem cell therapy

Abstract

Electrospinning is a technique in which materials in solution are shaped into continuous nano- and micro-sized fibers. Combining stem cells with biomaterial scaffolds and nanofibers affords a favorable approach for bone tissue engineering, stem cell growth and transfer, ocular surface reconstruction, and treatment of congenital corneal diseases. This review seeks to describe the current examples of the use of scaffolds in stem cell therapy. Stem cells are classified as adult or embryonic stem (ES) cells, and the advantages and drawbacks of each group are detailed. The nanofibers and scaffolds are further classified in Tables I and II, which describe specific examples from the literature. Finally, the current applications of biomaterial scaffolds containing stem cells for tissue engineering applications are presented. Overall, this review seeks to give an overview of the biomaterials available for use in combination with stem cells, and the application of nanofibers in stem cell therapy.

Keywords::

• biomaterial scaffolds
• electrospinning
• nanofiber
• stem cells

Introduction

Electrospinning, which is a technology that enables the manufacture of an ultrafine fiber, was initiated in the 1990 s. From the time the term electrostatic spinning was first used, it has gained popular attention in both the industrial and academic communities (Reneker and Chun 1996, Anton 1934, Huang et al. 2003, Li and Xia 2004). Electrospinning is a technique that enables the fabrication of fibers with diameters in the nanometer range, which produces very large specific surface area, greater than that of the current microfibers produced from conventional dry/wet and melt spinning techniques. Consequently, electrospun nanofibers are very suitable for improving a diversity of structures or products whose purposes are reliant on surface area. Among the promising applications, one of the most potential uses is in increasing nanofibrous cellular scaffolds and nanofibers for tissue engineering and stem cell applications. Up to the present time, electrospun fibers have been used in a wide range of regenerative medicine and stem cell applications.

Stem cells

Adult stem cells, including bone marrow mesenchymal stem cells (MSCs), have been proved for their multipotency in leading to the formation of bone, cartilage, connective tissue, fat or muscle. Defined inductive media can be commonly used for the accurate control of the differentiation lineage in vitro. Scientists have used autologous MSCs in conventional tissue engineering ex vivo, in combination with biomaterials, to generate a cell-based scaffold with the aim of replacing or improving tissue regeneration in vivo. Furthermore, through an incompletely understood mechanism, endogenous MSCs can be located in cell-free implanted scaffolds, which is known as homing. Homing of MSCs contributes to the renewal of the wounded tissue and demonstrates their critical role in the reformative process.

On other hand, embryonic stem (ES) cells are pluripotent cells and they have the potential for constant self-renewal (Han et al. 2012).

Nanofibers and scaffolds

Nanofibers have diameters ranging from tens of nanometers to microns when prepared by electrostatic spinning or electrospinning technologies which enable the production of nonwoven and ultrafine nanoscale nanofibers. This technology was established for the first time in the 1990s by Reneker and coworkers.

There are several methods to produce nanofibers, such as self-assembly (Williamson and Coombes 2004, Naik et al. 2003) template synthesis (Reneker and Chun 1996, Di et al. 2008, Doshi and Reneker 1993, Liang et al. 2007), phase separation (Ma et al. 2005a, Widmer et al. 1998), drawing (Zhong et al. 2011, Guarino et al. 2014), and electrospinning (Badami et al. 2006, Bhattarai et al. 2005).

Phase separation is one of best methods for the production of nanoporous foams and is preferred for use in many areas; however, as this method needs a long time for the completion of the process, it is not the best (Eatemadi et al. 2014a, Eatemadi et al. 2014b, Daraee et al. 2014a, Daraee et al. 2014b). By using this process and by modifying thermodynamic and kinetic factors, it is easy to achieve the production and fabrication of various nanoporous foams and foam scaffolds. Using the phase separation process, this can be achieved in five basic steps—suspension of polymer, phase separation and gelation, extraction of solvent from the gel by means of water, freezing, and then freeze-drying under vacuum (Ma et al. 2005b).

In the self-assembly method, atoms and molecules reform and assemble themselves due to noncovalent and fragile forces, for example electrostatic interactions, hydrophobic forces, and hydrogen bonding, and finally produce a stable construction (Hartgerink et al. 2001, Zhang 2003). Different structures can be produced by the self-assembly method, such as bilayer, unilamellar and multilamellar vesicles, membranes, nanoparticles, films, fibers, tubes, capsules, and micelles (Venugopal and Ramakrishna 2005). In comparison with the electrospinning method, fibers synthesized by self-assembly are much thinner, but the complexity of the procedure as well as the low productivity are the main limitations of this method (Ma et al. 2005a).

The electrospinning method has unique properties such as affordability, very high surface-to-volume ratio, availability of a wide range of materials that can be selected, flexibility of adoption for a broad range of sizes and shapes, simplicity, and tunable porosity (Mellatyar et al. 2014, Seidi et al. 2014).

In this process, several biomaterials can be used to fabricate nanofibers, and it requires very low amounts of initial solutions. Fabrication of fibers using the electrospinning method achieves fibers ranging from 3 nm to several micrometers in diameter, but fibers obtained by other methods have diameters in the range of 500 nm to a few microns (Zhang 2003).

Generally, owing to these tremendous properties that offer considerable advantages, the electrospinning method is the most popular method for the fabrication of nanofibers. As nanofibers are very tiny in diameter (micrometers to submicrons or nanometers), they possess numerous remarkable characteristics such as flexibility in surface functionalities, superior mechanical performance (for example, stiffness and tensile strength), and very large surface area to volume ratio compared with any other known form of the material.

As discussed above, these important properties make polymer nanofibers potential and optimal candidates for several major applications.

As nanofibrous materials have considerable potential for a variety of applications and offer some benefits of nanostructured materials, they are being well studied and developed. Nanofibrous materials can be synthesized from biocompatible and biodegradable polymers that are natural or synthetic, and also from composites of the two. These biodegradable polymers have been used to fabricate nanofibers for several purposes, based on the applications. Some polymers have been produced for short-time usage, such as cell carriers, delivery agents, and scaffolds (until new tissue becomes mature and independent). In these cases, the polymer will be substituted by original tissues. Other polymers have been used for long-term usage, such as for implants in surgery (Hohman et al. 2001). Nanofibers have attracted considerable interest in biotechnology and medicine because of the high potential of using biomaterials in different applications (Table I).

Table I. Example of nanofiber applications in tissue engineering.

Tissue	Nanofiber scaffolds	Brief function	Cell or tissue	Reference(s)
Bone	Starch/PCL	Enhance cell attachment, organization, and alkaline phosphatase (AP) activity	Human osteoblast cell line and rat bone marrow stromal cells	Tuzlakoglu et al. 2005.
	PCL	Enhance ECM formation	Neonatal rat bone marrow-derived MSCs	Yoshimoto et al. 2003.
	PCL/hyaluronan	Enhance ECM formation, better cell attachment	Neonatal rat bone marrow-derived MSCs	Yang et al. 2006.
	Bioactive silk fibroin/bone morphogenetic protein-2 (BMP-2),	Higher calcium deposition, enhance transcription levels of bone-specific markers	Human bone marrow-derived MSCs	Li et al. 2006.
	Bioactive silk fibroin/hydroxyapatite	Improve bone formation	Human bone marrow-derived MSCs	Li et al. 2003.
	PCL/CaCO3	Good cell attachment and proliferation of human osteoblasts	Human osteoblasts	Fujihara et al. 2005.
Cartilage	PLGA	Provide mechanical properties appropriate for cartilage tissue	Mouse fibroblast cells and bone marrow-derived MSCs	Li et al. 2002.
	Collagen type II	Adherence, propagation, and infiltration of the chondrocytes, and the creation of pseudopodia	Human articular chondrocytes cell line	Shields et al. 2004.
	PCL	Support chondrocyte proliferation, increase expression of cartilage-specific ECM genes	Fetal bovine chondrocytes (FBCs), rat bone marrow stromal cells (rBMSC)	Li et al. 2005.
Muscle	Poly (caprolactone-co-glycolide)-diol bonded to a diisocyanate, polyesterurethane linked with poly ((R)-3-hydroxybutyric acid)-diol	Satisfactory mechanical properties and the absence of toxicresidues	Rat myoblast cell line (L6), murine myoblast cell line (C2C12) and primary human satellite cells (HSCs)	Riboldi et al. 2005.
Ligament/Tendon	Polyurethane (PU)	Increase ECM production	Human ligament fibroblast (HLF)	Lee et al. 2005.
	PLGA/knitted scaffold.	Increase cell proliferation and cell function	Porcine bone marrow stromal cells	Sahoo et al. 2006.
Blood vessels	Collagen-blended P (LLA-CL)	Enhance cell viability, Attachment, and proliferation, and preserve human coronary artery endothelial cells (EC) markers	Human coronary artery endothelial cells (EC)	He et al. 2005.
	Collagen-coated PCL fibers	Enhance cell attachment, migration, and proliferation, and high quality morphology of-actin filaments	Human coronary artery endothelial cells (HCAECs)	He et al. 2005.
	Gelatin-grafted PCL nanofibers	Improve cell proliferation, maintain morphology of endothelial cells (EC)	Endothelial cells (ECs)	Ma et al. 2005.
	Poly (L-lactide-co- caprolactone)	Increase cell density, migration and proliferation	SMCs and ECs	Mo et al. 2004.
	PLLA-CL	Enhance attachment and proliferation	Human coronary artery SMCs and ECs	Xu et al. 2004.
	Poly (ester urethane) urea (PEUU)	Higher cellular density in perfusion cultures, no significant change in static cultures	Rat aortic SMCs	Son et al. 2004.
	Composition of collagen type I, Elastin, and poly (D,L-lactide-co-glycolide)	Improve physical properties	ECs and SMCs	Stitzel et al. 2006.

Electrospun polymeric nanofibers and scaffolds have been fabricated using copolymers, homopolymers, polymeric blends, and organic-inorganic hybrid materials. Among accessible biopolymers, the most extensively studied nanofiber systems for the regeneration of tissues and in stem cell applications which have been studied are poly-L-lactide (PLA), poly-glycolide (PGA), poly (e-caprolactone) (PCL), and their copolymers (Burg et al. 2000). Nanofibers can be used globally in stem cell applications and they have significant potential in the field of stem cell research and therapy.

Bone tissue engineering by stem cells

Numerous kinds of cells are used for nanofiber and scaffold seeding in this field, comprising bone cells and stem cells. The use of stem cells embodies a current approach in the field of bone tissue engineering, and the revolution is embodied by the assessment of proliferation, cell adhesion, and mostly differentiation on the scaffold and nanofiber, as well as motivating cell differentiation in the direction of the desired phenotype. The mesenchymal stem cells (MSCs) are the central cell lines used for bone tissue engineering applications. Adult bone marrow comprises MSCs that contribute to the regeneration of mesenchymal tissues, such as bone, muscle, cartilage, tendon, ligament, and stromal cells from adipose tissue. Actually, under suitable stimulation given by the scaffold, MSCs experience osteogenic differentiation over a definite pathway, as well as acquire osteoblastic indicators and secrete calcium crystals and extracellular matrix (Yoshimoto et al. 2003).

Scaffolds for stem cell growth and transfer

One of the main problems related to stem cell therapy remains the deficiency of an appropriate carrier to transfer stem cells to the exact location in tissue. Up to now, numerous materials and scaffolds have been studied for the transfer of stem cells, such as self-assembling peptide nanofibers studied for cell-based therapy on infarcted myocardium (Dubois et al. 2008), macroporous hydrogels used to transfer SCs for spinal cord injury repair (Syková et al. 2006), polymers and collagen sponges, (Rama et al. 2001) and fibrin glue used for cell transfer onto the ocular surface (Schwab et al. 2006). Also, stem cells for therapeutic application on the ocular surface cannot be directly injected to the damaged eye as a cell suspension. To permanently anchor transferred cells, numerous carriers have been suggested and studied. Up to now, human amniotic membrane symbolizes the gold standard. In the majority of cases studied, the amniotic membrane was applied as a cell carrier for the transfer and growth of stem cells (Nakamura et al. 2004, Jiang et al. 2010, Shimazaki et al. 2002). Nevertheless, amniotic membrane is a biomaterial of human origin, with initial challenges in regulating its preparation, and therefore an exploration for substitute cell carriers and modes of stem cell transplantation on the ocular surface continues. In Table II, some of the electrospun polymeric scaffolds for bone tissue engineering based on stem cells have been summarized.

Table II. Electrospun polymeric scaffolds for bone tissue engineering.

Biomaterial	Cells seeded on the scaffold	Reference(s)
PCL	MSCs derived from the bone marrow of neonatal rats	Yoshimoto et al. 2003, Shin et al. 2004.
PCL	hMSCs	Binulal et al. 2010.
PCL	MSCs isolated from male Wistar rats	Ruckh et al. 2010.
Collagen type I	Bone marrow hMSCs	Shih et al. 2006.
Silk fibroin	Human mesenchymal stem cells (hMSCs)	Meinel et al. 2009.
Silk fibroin	Human bone marrow stromal cells (BMSCs)	Jin et al. 2004.
Chitosan	Human osteosarcoma cell line MG63	Jin et al. 2004.
PCL/collagen	Pig bone marrow mesenchymal cells (pBMMCs)	Ekaputra et al. 2009.
PLA/DBP	Human mandible-derived mesenchymal stem cells (hMSCs)	Ko et al. 2008.
PLGA/MWNTs/HA	Rat bone mesenchymal stem cells (BMSCs)	Zhang and Chen 2010.
Gelatin-siloxane (3-glycidoxypropyl trimethoxysilane)	Bone marrow-derived mesenchymal stem cells (BMSCs)	Ren et al. 2010.
Silk/PEO/BMP-2, Silk/PEO/nHAP, Silk/PEO/nHAP/BMP-2 (control: silk/PEO)	Human bone marrow-derived mesenchymal stem cells (hMSCs)	Li et al. 2006.

The use of MSCs for ocular surface reconstruction

Ocular surface reconstruction, challenged by the low number of available cells and the frequent absence of autologous limbal stem cells (LSC), led to attempts to use MSCs developed in vitro to substitute LSCs for corneal reconstruction and regeneration. These attempts are based on the potential of MSCs to differentiate into other cell types, as well as their immunomodulatory properties which can be valuable for the assessments of the local inflammatory response after transplantation of stem cells.

For the first time, (Ma et al. 2006) showed that MSCs derived from human bone marrow and transplanted by means of an amniotic membrane onto chemically burned rat ocular surface supported corneal healing and were involved in the reconstruction of the damaged cornea. Their healing effects contributed to the inhibition of angiogenesis and inflammation.

The helpful application of MSCs was approved in another study using a model of chemically burned rat ocular surface and the subconjunctival administration of rat MSCs (Yao et al. 2012), the application of MSCs with a special hollow plastic tube (Oh et al. 2008), or the transplantation of rat MSCs onto the ocular surface using amniotic membrane (Jiang et al. 2010). In another study, scientists (Martínez-Conesa et al. 2012) injected a suspension of human MSCs into a rabbit model of corneal stromal defect and observed the restoration of the stromal organization, the maintenance of transparency, and the acquisition of vision. These studies were developed by a model of LSC deficiency in a murine model of a mechanically damaged ocular surface and transplantation of syngeneic MSCs seeded on nanofiber scaffolds (Zajicova et al. 2010), or in rabbits injected with autologous MSCs under an amniotic membrane (Reinshagen et al. 2011). All of these experimental studies have shown that MSCs significantly attenuate inflammation, increase corneal wound healing, and reduce corneal neovascularization.

The use of MSCs to treat congenital corneal diseases

Liu et al. (2010) launched an experimental model to study the potential of overcoming the lack of corneas for keratoplasty to heal congenital corneal diseases of genetic mutation and heal the cloudy and thin corneas of lumican-null mice with human umbilical MSCs. Corneal stromal thickness and corneal transparency were significantly improved by transplanted MSCs. This study suggests that MSCs may also be a competent key for the cure of congenital corneal diseases, including keratocyte dysfunction.

Conclusion

Electrospinning has gained a reputation within the tissue engineering and stem cell research community as a promising means of fabricating scaffolds and nanofibers. This technology of constructing nanofibers enables the assessment of fiber diameter, geometry, fiber alignment, biomaterial composition, and drug/protein combination in a scaffold. Nanoscale fibers manufactured using electrospinning technology are competent to develop the cellular interactions of a wide-ranging diversity of cell types; furthermore, the cells are capable of conserving their functional and phenotypic characteristics on nanofibrous scaffolds. Generally, these electrospun scaffolds and nanofibers can be functionalized by anchoring mechanical and biochemical signals, to improve cellular interactions for stem cell therapy and tissue engineering applications.

Authors’ contributions

SG conceived of the study and participated in its design and coordination. AA participated in the sequence alignment and drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgments

The authors thank the Department of Molecular Medicine, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, for all support provided. This work is funded by a 2015 grant by the Drug Applied Research Center, Tabriz University of Medical Sciences.

Declaration of interest

The authors have no declaration of interest. The authors alone are responsible for the content and writing of the paper.