New state of nanofibers in regenerative medicine

Abstract

Regenerative medicine has discovered engineered nanofiber scaffolds enhancing regeneration process. These agents have an attractive property to mimic the native environment. They are excellent agents in binding the extracellular matrix of a cell to another cell. They help in the growth and multiplication of the cell and help in the differentiation of the cells which are required before the regeneration process. Regenerative medicine focuses on cellular therapies, origins of stem and progenitor cells, and on explaining how they persevere (or do not) in adult organisms and improvement of biomaterials. The focus of this review is on the application of nanofiber scaffolds.

Keywords:

• Nanofiber
• regenerative medicine
• scaffold
• tissue engineering

Introduction: regenerative medicine

The field of regenerative medicine and tissue engineering has exploded in the last decade (Fisher and Mauck 2013). The approaches of tissue engineering and regenerative medicine are theoretically simple and attractive, yet these have proven to be challenging engineering tasks (Deluzio et al. 2013). In spite of rapid improvements made in this field (Fisher and Mauck 2013, Laflamme and Murry 2011), success is still limited because of major knowledge gaps in our ability to coordinate, control, and direct tissue formation, which are the ultimate goals for both tissue engineering and regenerative medicine (Deluzio et al. 2013).

In regenerative medicine approaches, cellular therapies remain a large focus, and the effectiveness of numerous cell types have been assessed in vivo. Furthermore, huge efforts in regenerative medicine and biology have focused on the origins of stem and progenitor cells, and on explaining how they persevere (or do not) in adult organisms. Another main focus has been on the improvement of biomaterials to either provide a scaffold that can promote appropriate tissue formation and/or release bioactive factors to aid in the healing response (Fisher and Mauck 2013).

Nanofibers and electrospinning

Using electrospinning or electrostatic spinning methods, nanofibrous scaffolds can be fabricated of biodegradable and biocompatible polymers (Table 1), which were reviewed smartly by Ravichandran et al. (2012a).Various biodegradable polymers have been used to produce nanofiber scaffolds such as synthetic, natural, and composite of the both (Ravichandran et al. 2012a).

Table 1. Comparison of various nanofiber scaffold processing methods.

Scaffold processing method	General descriptions	General descriptions	Advantages	Disadvantages
Electrospinning	A process that essentially employs electrostatic forces for the production of polymer nanofibrous scaffolds, typically involves top-down approach.	Relatively easy, lab and industrial scales	Simple and cost-effective, capable to produce long and continuous fibers with control over fiber orientation, mechanical properties, size and shape, versatile to many polymers.	Using apparatus by high voltage
Self-assembly	A process in which atoms, molecules, and supramolecular aggregates organize and arrange themselves into an ordered structure through weak and noncovalent bonds; typically involves a bottom-up approach.	Difficult and lab scale	Mimic the biological process in certain circumstances	Complex process, limited to a few polymers, unable to produce long and continuous fibers with control over fiber orientation.
Phase separation	A process that involves various steps, typically raw material dissolution, gelation, solvent extraction, freezing and drying, leading to the formation of nanofibrous foam-like structure.	Easy and lab scale	Simple process, tailorable mechanical properties	Limited to a few polymers, longer processing time, unable to produce long and continuous fibers with control over fiber orientation.

Natural polymer applied to produce nanofiber scaffolds have completed progressively more research interests, including oxidized cellulose (Son et al. 2004), collagen (Venugopal et al. 2005), hyaluronic acid (Um et al. 2004), elastin (Boland et al. 2004), fibrin (Jukola et al. 2008), silk protein (Jin et al. 2004), elastin-mimetic peptide (Huang et al. 2005), tropoelastin (Li et al. 2005), and fibrinogen (Sindelar et al. 2006).

The most important advantage of natural polymer is its identity and similarity to some molecular biomaterials that exist in the human body. However, the most important disadvantage of natural polymer is its reduced mechanical properties when isolated, thus this natural polymer has a need of further processing for handling. So, synthetic polymer was introduced.

Different synthetic polymers for synthesis of scaffolds have been used such as PCL (Khil et al. 2005, Luong-Van et al. 2006), PLGA (Badami et al. 2006, Chew et al. 2005), PLDLA (Cui et al. 2006), PLLA (Badami et al. 2006), and copolymers for example PCL-PLLA (Nikkola et al. 2005), PCL-PEG, PLGA-PEG (Daraee et al. 2016, Mellatyar et al. 2014), PLLA-PEG (Mellatyar et al. 2014), etc.

Blends of natural polymer and synthetic polymer have been used to encompass characteristics of both. Studies merging polymer involved gelatin-loaded PCL (Ma et al. 2005), composites of PLLA-PCL and collagen (He et al. 2005), collagen-loaded PLLA-PCL (Huang et al. 2003), composites of hyaluronic acid and PCL (Yang et al. 2006), blends of PEO and silk (Li et al. 2002), composites of PLGA, elastin and collagen (Stitzel et al. 2000), composites of PCL and starch (Yang et al. 2006), and composites of PLGA with PHBV (Zhu et al. 2009).

Nanofibers in regenerative medicine

Because of the significant potential of applying biomaterials in wide spectrum applications, field of nanofibers has achieved extensive interest in biotechnology, tissue engineering, and medicine.

Nanofibers have been extensively used in regenerative medicine and nano-biotechnology. Table 2 summarizes several examples of nanofiber application in regenerative medicine.

Table 2. Example of nanofiber application in regenerative medicine.

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/hyaluronn	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, 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 toxic residuals	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 of cell proliferation and cell function	Porcine bone marrow stromal cells	(Sahoo et al. 2006)
Blood vessels	Collagen-blended P(LLA-CL)	Enhance of cell viability, attachment, spreading 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, 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 in 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-coglycolide)	Improve physical properties	ECs and SMCs	(Stitzel et al. 2006)

Skin regeneration

Skin regeneration and repair are based on two important approaches: skin drug delivery to interested sites and ability of the cells surrounding the damaged tissue for regeneration and repair. Cells of these two classes have the potential to regenerate into molecular structures that are similar to the original tissues. The importance of nanofiber scaffolds for skin regeneration and repair and tissue-engineering applications has been greatly studied. Nanofiber scaffolds which have a larger surface to area and nano-scale molecular architectures adsorb efficiently proteins and skin drugs, presenting many binding sites to cell membrane receptors which would be more biomimetic to better support cell–matrix interactions (Fujihara et al. 2005, Venugopal et al. 2005, Venugopal et al. 2008, Yang et al. 2004). Electrospun fibers perform as engineered skin substitutes that are extensively studied as bioactive dressings (Guadalupe et al. 2015).

Wnek et al. engineered a nanofiber scaffold using fibrinogen for skin repair, hemostatic products, and wound dressing (Wnek et al. 2003). Blends of gelatin and chitosan nanofiber have enhanced the biological and cellular activity and skin regeneration, and this composition was assessed in regeneration of various tissues including skin and bone (Bhattarai et al. 2005). Hydrogel scaffolds are prepared for skin regeneration and repair and biological applications, because of their high water content and biocompatibility (Hoffman 2012).

Eross Guadalupe et al. report the fabrication and characterization of bioactive poly-e-caprolactone (PCL) nanofiber matrices incorporated with a model angiogenic factor, PNF1, and a model antibiotic drug, gentamicin (Guadalupe et al. 2015). This study shows that all the fiber matrices were able to support fibroblast growth and maintain normal cell morphology. Such bioactive bandages may serve as versatile and less expensive alternatives for the treatment of complex wounds.

In a study by Venugopal and coworkers, PCL and collagen were used to fabricate nanofiber matrices to observe cell attachment, morphology, proliferation, and cell–matrix interactions (Guadalupe et al. 2015). The results of this work show that this novel biodegradable PCL and collagen nanofiber matrices support the attachment and proliferation of human dermal fibroblasts and might have potential in tissue engineering as a dermal substitute for skin regeneration (Chandrasekaran et al. 2011).

Ravichandran et al. fabricated a mixture of nanofibrous scaffold comprising of PLLA/PAA/Col I&III with stem cell therapy along with the addition of bFGF to promote skin regeneration (Ravichandran et al. 2012b). This system induced the necessary paracrine signaling effect by faster regeneration of the damaged skin tissues.

Kalaipriya Madhaiyan et al. investigated the water soluble vitamin delivery with hydrophobic polymer nanofiber sustaining the release of the vitamin for the transdermal patch applications (Madhaiyan et al. 2013). They showed that the cyanocobalamin loaded nanofibers are suitable for transdermal patch according to the drug release profile in PBS buffer in vitro environment as the release of the energy supplement matches the requirement of the human need.

Bone regeneration

The promising application of electrospun nanofiber scaffolds in bone regeneration was initially investigated with fiber mesh of electrospun poly (ɛ-caprolactone) (PCL) to maintain ectopic bone formation in vivo (Shin et al. 2004) and osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro (Yoshimoto et al. 2003). Electrospun nanofiber scaffolds produced from a multiplicity of biomaterials were then used to maintain osteogenic differentiation of a variety of cell types in vitro, including mesenchymal and osteoblasts stem cells (Ngiam et al. 2009, Phipps et al. 2011, Zhang et al. 2008, Zhang et al. 2010). Interaction between cell-nanofiber scaffolds plays a significant role in osteogenesis process. The nanofiber scaffolds significantly improved the total amount of serum proteins adsorbed on the nanofiber scaffolds, particularly the key extracellular matrix proteins involved in cell adhesion (Woo et al. 2003), and enhanced the osteoblast differentiation, growth, and attachment (Woo et al. 2003, 2007). Embryonic stem (ES) cells (Thomson et al. 1998) are attractive enviable cell source for bone tissue regeneration, which is because of the pluripotency and unlimited expansion capacity (Handschel et al. 2010, Jukes et al. 2008). It has been verified that nanofiber matrix can considerably make possible the osteogenic differentiation of ES cells (Smith et al. 2009).

Bone regeneration, using a biomimetic scaffold to assemble a synthetic osteogenic microenvironment, makes possible the natural ossification procedures, which can improve clinical therapy (Shin et al. 2003).

Collagen is the main organic constituent of the bone extracellular matrix (ECM) (Elsdale and Bard 1972), and several studies informed that collagen nanofibers enhanced osteogenesis (Franceschi 1999, Xiao et al. 2002). As a result, complex of scaffolds-nanofibres were prepared to imitate the proosteogenic properties and structural features of collagenous ECM of the bone (Hartgerink et al. 2001, Li et al. 2002).

Bone regeneration is controlled by a variety of bioactive agents such as nucleic acids (Shea et al. 1999), integrin-binding ligands (Hern and Hubbell 1998), and growth factors (Richardson et al. 2001).

In one study, Wei and coworkers encapsulated nanofiber with bone morphogenetic protein-7 (BMP-7) and then implanted in rats subcutaneously (Wei et al. 2007). Then, BMP-7 was released in a regulated fashion with high biological activity and stimulated ectopic bone formation. But, scaffolds with passively adsorbed BMP-7 or without BMP-7 were incapable to provoke osteogenesis, probably due to the insufficient release duration and loss of bioactivity.

Rajeswari Ravichandran et al. investigated the in vitro responses of ADSCs to the surface mineralized PLLA/PBLG/Col/n-HA nanofibrous substrate, in terms of the initial cell adhesion, proliferation, and further osteogenic differentiation and mineralization (Ravichandran et al. 2012c). The results showed that the PLLA/PBLG/Col/n-HA scaffolds increased greater osteogenic differentiation of ADSC as evident from the enzyme activity and mineralization profiles for bone tissue engineering.

Chinnasamy Gandhimathi et al. investigated the in vitro response of hMSCs to the surface mineralized PCL/PAA/Col/n-HA hybrid nanofibrous scaffold, in terms of cell adhesion, proliferation, and further osteogenic differentiation and mineralization for bone tissue regeneration (Gandhimathi et al. 2013). They showed that the PCL/PAA/collagen/n-HA scaffolds promoted greater osteogenic differentiation of hMSCs, proving to be a potential hybrid scaffold for BTE.

Cartilage regeneration

In several studies of tissue-engineered auricle-shaped cartilage, natural or synthetic biodegradable polymers such as poly-ɛ-caprolactone, polylactic acid (PLA), collagen, polyglycolic acid (PGA), and hydrogel (fibrin gel, pluronics, alginate) have been prepared as scaffolds for cell seeding (Bichara et al. 2011, Isogai et al. 2005, Kamil et al. 2004, Vacanti et al. 1988).

Synthetic polymers, predominantly aliphatic polyesters such as polylactic-co-glycolic acid and PGA, have often been used in auricular cartilage engineering (Haisch et al. 2002, Isogai et al. 2005, Kamil et al. 2003, Liu et al. 2010).

Accumulating studies have focused on the interactions of these FDA-approved polymers with chondrocytes (Temenoff and Mikos 2000). For example, copolymer scaffolds of PGA/PLA have been extensively used in cartilage regeneration 17–20 and have revealed success in fabricating human auricle shaped constructs in vitro (Kamil et al. 2003).

Shieh and coworkers assessed scaffolds made of PGA coated with poly-4-hydroxybutyrate, PCL, and poly-l-lactic acid (PLLA) and compared the growth of engineered cartilage and ear-like shape preservation in vivo in both rabbit and nude mouse models (Shieh et al. 2004). The constructs were fabricated over 40 weeks in vivo in the nude mouse, thus detecting neocartilage creation in all specimens. PCL constructs revealed the best shape retention which is possibly because of the slowest degradation rate of this scaffold material, although they reduced in size over time.

Isogai and coworkers later informed numerous studies, one of which assessed the preservation of neocartilage integrity in an auricle-shaped construct in a nude mouse model, by using PLLA/PCL copolymer (Isogai et al. 2004). The results revealed that neocartilage had not been consistently dispersed all over the scaffold; insufficient cell-seeding methods were accountable, according to the authors. Isogai et al. prepared the similar PLLA/PCL copolymer blended with basic fibroblast growth factor (bFGF) encapsulated into microspheres generated by gelatin (Isogai et al. 2005). The prepared bFGF release enhanced chondrogenesis and improved vascularization of cell-seeded constructs blended in nude mice; shape maintenance was enhanced with addition of FGF because of enhanced chondrogenesis.

Ligament/tendon regeneration

The functions of the ligament are to prepare aid and support in joint movements. Basically ligament/tendon damages were treated by using autografts, allografts, or biological grafts (Bell 1995). In applications of ligament tissue engineering, scaffolds should make available appropriate shape and required mechanical strength during degrade and reconstruction at a rate similar to the regeneration rate of tissue. For ligament reconstruction both synthetic and natural materials have been used in the form of gels, membranes, or 3-D scaffolds (Figueroa et al. 2014, Lu et al. 2005, Rathbone et al. 2010, Wiig et al. 1990).

One of the most common ligament injuries of the knee is the rupture of the anterior cruciate ligament (ACL) (Fetto and Marshall 1980). Natalie Luanne Leong and coworkers examined the regenerative potential of tissue-engineered ACL grafts using immunohistochemistry, histology, and mechanical testing up to 16 weeks postoperatively (Leong et al. 2015). Mechanical testing of the grafts revealed significantly higher mechanical possessions than immediately post-implantation. Histology exhibited infiltration of the grafts with cells and immunohistochemistry revealed aligned collagen deposition with minimal inflammatory reaction. In this in vivo rodent model study for ACL reconstruction, the histological and mechanical evaluation revealed tremendous regeneration and healing possible of electrospun PCL ligament graft. The scaffold of electrospun polymer assisted both cell and matrix alignment in the regenerated ACL. These grafts caused inefficacious bone integration with augmented strength over time; load to failure augmented three-fold as compared with the recreated ACL instantly postoperatively. In an ovine study, it was established that at 52 weeks, the autografts had 67 and 38% the peak stiffness and load, respectively, of control ligaments (Scheffler et al. 2008).

Deepthi and coworkers, with the aim of developing a scaffold that could mimic the native ligament fibrous morphology, along with an ECM mimicking coating that can act as a cell or nutrient reservoir, developed aligned PCL micro/random nano- (PCL aligned multiscale) and random micro/nano (PCL random multiscale) fibers through electrospinning following which a coating with chitosan–hyaluronic acid hydrogel was given (Deepthi et al. 2015). This study implies the use of hydrogel coated systems to provide a reservoir for cells and nutrients and further modifications of these systems would make it promising for ligament regeneration.

Conclusion

Regenerative medicine and tissue engineering are the developing branches providing the requirement for regeneration of tissues damaged due to injury. The emerging field of regenerative medicine and tissue engineering targets to three main approaches: cellular therapies, origins of stem and progenitor cells, and on explaining how they persevere (or do not) in adult organisms and improvement of biomaterials to either provide a scaffold that can promote appropriate tissue formation and/or release bioactive factors to aid in the healing response. This filed regenerates damaged tissues by combining cell from the body with highly porous scaffold biomaterials which guide the growth of new tissues (Noorjahan and Sastry 2005, Pallela et al. 2012) and an effective technique in treating this is the use of nanofiber scaffolds. Finally, the recent progress in these fields needs to be combined to develop nanofiber scaffolds that would respond to the requirement of regenerative medicine.

Acknowledgements

The authors thank the Department of Medical Nanotechnology, Faculty of Advanced Medical Science of Tabriz University of Medical Sciences for all the support provided.

Disclosure statement

The authors declare that they have no competing interests.