Li et al. (2009) used electrospun scaffolds in which a gradient of calcium phosphate was formed to mimic tendon-to-bone interfaces. Electrospun PLGA or PCL scaffolds were plasma treated or coated with gelatin respectively and a gradient of calcium phosphate mineral was applied along the length of the scaffold using ten times simulated body fluid. A mineral gradient was formed in these scaffolds as evidenced with SEM and decreasing mineral content was observed when moving from one end of the scaffold to the other. Moreover, the gradient had functional consequences in that it created a mechanical property gradient across the scaffold where the mineralized side had a higher modulus than the nonmineralized side and there was a gradual decrease in modulus from the mineralized end to the non-mineralized end. Finally, MC3T3 cells, a preosteoblastic cell line, were seeded on the scaffolds and showed preferential attachment and spreading on the more mineralized regions of the scaffolds. The cell density of these cells decreased with decreasing mineral content. Thus the authors proposed that these scaffolds may be useful for engineering tendon-bone interfaces (Li et al., 2009).

As tissue engineering moves forward, interfaces between different tissue types will become increasingly important. The first study described above uses electrospinning as a potential an adjuvant to current clinical therapies to improve bone-tendon integration and thus clinical outcomes (Spalazzi et al., 2008). The second study demonstrates the electrospinning can be used as a technique to facilitate the engineering of these transitions. Here, the authors create a mineral gradient along the length of an electrospun scaffold in order to facilitate the formation of the bone-to-tendon transition and promote rotator cuff tendon repair in future studies (Li et al., 2009). Thus, electrospun nanofibers will likely continue to find novel uses in the field of composite tissue engineering.

#### **3.8 Skeletal muscle and muscle-tendon junctions**

When skeletal muscle tissue loses function due to a congenital condition or an acquired condition such as a traumatic injury or tumor excision, there is no effective clinical treatment for replacing that function (Moon et al., 2008). Tissue engineering once again offers the opportunity to one day develop clinical treatments to repair damaged skeletal muscle and restore function. Our laboratory has used electrospinning to develop scaffolds for skeletal muscle tissue engineering (Choi et al., 2008). Electrospun scaffolds were made of a blend of PCL and collagen to investigate the effect that aligned fibers would have on skeletal myotube formation. Fibers were aligned by increasing the rotation speed of a plate mandrel. The aligned-fiber scaffolds enhanced skeletal muscle cell alignment and myofiber length compared to randomly oriented electrospun scaffolds. This suggested, as has already been pointed out in Section 3, that aligned-fiber scaffolds may be advantageous for organizing tissue engineered tissues, in this case skeletal muscle. The scaffolds from this study may one day be useful for forming functional tissue that could be implanted into a patient (Choi et al., 2008).

More recently, there has been increasing interest in composite tissue engineering, which as described in the previous section will become increasingly important as the field of tissue engineering advances. Specifically, we have begun to investigate scaffolds that would be appropriate for muscle-tendon junction tissue engineering (Ladd et al., 2010). Muscle and tendon tissue have different design requirements for scaffolds, but we wanted to create a single scaffold that could accommodate both tissue types. To do this, we decided to design scaffolds that mimic the trends in mechanical properties observed in

Li et al. (2009) used electrospun scaffolds in which a gradient of calcium phosphate was formed to mimic tendon-to-bone interfaces. Electrospun PLGA or PCL scaffolds were plasma treated or coated with gelatin respectively and a gradient of calcium phosphate mineral was applied along the length of the scaffold using ten times simulated body fluid. A mineral gradient was formed in these scaffolds as evidenced with SEM and decreasing mineral content was observed when moving from one end of the scaffold to the other. Moreover, the gradient had functional consequences in that it created a mechanical property gradient across the scaffold where the mineralized side had a higher modulus than the nonmineralized side and there was a gradual decrease in modulus from the mineralized end to the non-mineralized end. Finally, MC3T3 cells, a preosteoblastic cell line, were seeded on the scaffolds and showed preferential attachment and spreading on the more mineralized regions of the scaffolds. The cell density of these cells decreased with decreasing mineral content. Thus the authors proposed that these scaffolds may be useful for engineering

As tissue engineering moves forward, interfaces between different tissue types will become increasingly important. The first study described above uses electrospinning as a potential an adjuvant to current clinical therapies to improve bone-tendon integration and thus clinical outcomes (Spalazzi et al., 2008). The second study demonstrates the electrospinning can be used as a technique to facilitate the engineering of these transitions. Here, the authors create a mineral gradient along the length of an electrospun scaffold in order to facilitate the formation of the bone-to-tendon transition and promote rotator cuff tendon repair in future studies (Li et al., 2009). Thus, electrospun nanofibers will likely continue to find novel uses

When skeletal muscle tissue loses function due to a congenital condition or an acquired condition such as a traumatic injury or tumor excision, there is no effective clinical treatment for replacing that function (Moon et al., 2008). Tissue engineering once again offers the opportunity to one day develop clinical treatments to repair damaged skeletal muscle and restore function. Our laboratory has used electrospinning to develop scaffolds for skeletal muscle tissue engineering (Choi et al., 2008). Electrospun scaffolds were made of a blend of PCL and collagen to investigate the effect that aligned fibers would have on skeletal myotube formation. Fibers were aligned by increasing the rotation speed of a plate mandrel. The aligned-fiber scaffolds enhanced skeletal muscle cell alignment and myofiber length compared to randomly oriented electrospun scaffolds. This suggested, as has already been pointed out in Section 3, that aligned-fiber scaffolds may be advantageous for organizing tissue engineered tissues, in this case skeletal muscle. The scaffolds from this study may one day be useful for forming functional tissue that could be implanted into a patient (Choi et

More recently, there has been increasing interest in composite tissue engineering, which as described in the previous section will become increasingly important as the field of tissue engineering advances. Specifically, we have begun to investigate scaffolds that would be appropriate for muscle-tendon junction tissue engineering (Ladd et al., 2010). Muscle and tendon tissue have different design requirements for scaffolds, but we wanted to create a single scaffold that could accommodate both tissue types. To do this, we decided to design scaffolds that mimic the trends in mechanical properties observed in

tendon-bone interfaces (Li et al., 2009).

in the field of composite tissue engineering.

al., 2008).

**3.8 Skeletal muscle and muscle-tendon junctions**

native MTJ. Our working hypothesis is that a scaffold that mimics these trends would promote the formation of MTJ tissue. We employed a co-electrospinning fabrication technique to begin developing such scaffolds. We showed that by using PCL and PLLA blended with type I collagen, that we could develop scaffolds with regional differences in mechanical properties and that these differences mimicked the trends observed in native MTJ (Ladd et al., 2010). Figure 2 showing the result of co-electrospinning to create a scaffold with regional variations in mechanical properties that mimic the trends observed in native muscle-tendon junction. While our working hypothesis remains to be tested, this is another example of where electrospinning is being used to create novel scaffolds for tissue engineering.

Fig. 2. (A) Image showing the three regions of the scaffold: PCL side, Center, and PLLA side with methylene blue dye added. (B-D) SEM images from the different regions of scaffold showing fiber morphology and diameter (all images ×4k). (B) PCL side (550±98 nm), (C) center (504±93 nm), (D) PLLA side (452±40 nm). (E) Color maps of the true strain profiles of the scaffolds when undergoing uniaxial tensile testing. Maximum principal strain is displayed and the scale is from 0 to 0.25 mm/mm strain. (E-G) The quantification (E) by showing the differences in max strain from each region. Maximum principal true strain for the regions of the scaffold at each overall strain interval for the whole scaffold. (H-J) Analagous data for native MTJ tissue tested in the same manner. Note that (H) has a scale from 0 to 1 mm/mm strain. From these figures, it is clear that the scaffolds mimic the trends in mechanical properties observed in native tissue. Matching symbols indicate statistical difference from one another. Significance was defined as \*P<0.05. Reproduced with permission from all authors, *Biomaterials*, 2010, **32**, 6, 1549. © Elsevier 2010 (Ladd et al., 2010).

In summary, electrospinning provides a unique fabrication technique that could prove to be useful for both skeletal muscle tissue engineering and composite tissue engineering. The ability to form and control the orientation of nanofibers allows scaffolds to incorporate topographical cues to help align skeletal myofibers. The ability to incorporate multiple material types into a single scaffold allows one to create scaffolds with regional variations in mechanical properties that may be useful for MTJ tissue engineering and also may prove useful for other types of composite tissue engineering. Undoubtedly, more work is needed to create clinically useful applications despite its successful use in the laboratory setting. Nevertheless, electrospinning remains as a useful tool in the tissue engineer's armamentarium.

Electrospun Nanofibers in Tissue Engineering 367

tissue engineering. After fabricating multi-layered electrospun scaffolds, micro-scaled pores were created by a laser ablation technique. Thus, they were able to maintain the unique nanofibrous structure that supports cellular interactions and to fabricate micro-scaled

An additional limitation of electrospinning is the inability to precisely control where the fibers are deposited (Teo & Ramakrishna, 2005). As fibers whip towards a collecting plate or mandrel, they deposit randomly unless there is some other force to cause them to orient differently. To try to overcome this limitation, researchers have used large cylindrical mandrels with high rotation speeds to cause fiber alignment (Choi et al., 2008; Moffat et al., 2009) or used parallel electrodes to apply an electric field that would force the fiber to align across the plates or in other orientations (Li et al., 2003; Teo & S Ramakrishna, 2005; Li et al., 2005; Yang et al., 2009). Still, electrospinning would be an even more powerful technique if one could precisely control where the polymer jet would land such that computer-assisted design of electrospun scaffolds would be possible. This would allow researchers to easily create nanofibers and create a much broader range of scaffold geometries and thicknesses. Unfortunately, no one has discovered a way to do this to date. The closest anyone has come to approaching this ability was in a study by Kameoka et al. (2003) where they created a scanning tip electrospinning source that used an arrow-shaped tip, polymer droplet, and high voltage to create a Taylor cone and spin fibers. They were able to control deposition to a region of about 5 mm in size. While some researchers have found creative ways to make electrospun scaffolds of differing geometries (besides tubes and sheets), the inability to precisely control the deposition location of the polymer jet limits the variety of possible

Most researchers tend to have electrospinning systems which still require a manual setup, i.e. manual positioning of the needle tip from the collector. Moreover, often humidity and temperature are not well-controlled and these ambient parameters can have an effect on the fibers. Because these parameters are often not precisely controlled, there is an increase in the variation of properties and fiber morphologies from scaffold to scaffold. In order to improve the repeatability and homogeneity between spinning batches, modern technology should be incorporated into electrospinning systems to more precisely control these parameters. For instance, without much difficulty, a computer-controlled spinning apparatus that is selfcontained to control temperature and humidity and that precisely controls air gap distances, mandrel rotation speed, voltage, and spinneret flow rate could be developed. Such a system would allow for more consistent scaffold fabrication and serve as an improved system to

continue to investigate how these spinning parameters affect final fiber morphology.

**5. The future of electrospinning in tissue engineering and regenerative**

Electrospinning has evolved as a powerful tool in various tissue engineering applications. This fabrication technology provides the ability to control biomaterial composition, fiber diameter, fiber alignment, geometry, and drug/protein incorporation into a scaffold. Nanoscaled fibers generated by electrospinning are able to improve the cellular interactions of a wide variety of cell types; moreover, the cells are able to maintain their phenotypic and functional characteristics on nano-scaled fibrous scaffolds. Additionally, a growing body of evidence demonstrates that nano-to micro-scaled topography plays an important role in controlling the adhesion, proliferation, differentiation, and survival of cells in culture.

features within the scaffold.

geometries.

**medicine**

#### **4. Current challenges with using electrospinning in tissue engineering**

As with any fabrication method, there are advantages and disadvantages to using electrospinning as a means of scaffold fabrication for tissue engineering. Many of the advantages have already been highlighted in previous sections such as the mimicking of native ECM and the ability to adapt the technique for versatile applications. Here, we discuss several limitations of the electrospinning and strategies to work towards overcoming them.

An inherent limitation to using electrospun nanofibrous scaffolds in tissue engineering applications is the small pore size. Nanofiber scaffolds typically result in pore sizes on the sub-micron to few micron scale. Because all mammalian cells range in size from several micrometers to several hundred micrometers in size, these cells typically cannot penetrate the scaffold. This poses a problem for tissue engineers because we often aim to form fully cellularized three dimensional tissue constructs. There have been attempts to improve the pore size of electrospun nanofiber scaffolds. The two primary methods are to co-electrospin a set of sacrificial fibers or adding porogens (Baker et al., 2008; Nam et al., 2007). Sacrificial fibers are fibers that are co-electrospun with the main scaffold component, but that are easily dissolved after fabrication leaving behind larger pores. For instance, Baker et al. (2008) used PEO, a water soluble polymer, as the sacrificial component of electrospun scaffolds. After dissolving the PEO, the pore size of the electrospun scaffolds increased. Porogens, primarily salt, have also been added to electrospun scaffolds as they are being electrospun (Nam et al., 2007). Typically the porogens are dropped onto a mandrel or collecting plate while the fibers are being deposited. Then, after fabrication, the porogens can be removed by dissolution. These approaches have improved pore size and allowed for improved cellular infiltration. However, these approaches may compromise the mechanical properties and dimensional stability of the scaffold when the fibers dissolve and collapse. Microintegration is another approach developed by Stankus et al. (2006) for producing a highly cellularized construct. However, this process may limit homogeneity, sterility, and cell survival, due to the long-term processing times required.

Another inherent limitation of electrospinning is scaffold thickness, especially when spinning onto a cylindrical mandrel. Electrospun scaffolds tend to be thin, typically less than one millimeter in many cases (Inoguchi et al., 2006; Lee et al., 2008). Scaffolds with thicknesses greater than one millimeter are possible to fabricate, but are less commonly seen in the literature (Pham et al., 2006b). Moreover, in our experience, achieving millimeter or greater thicknesses requires the use of a large volume of polymer solution. The inability to obtain thicker scaffolds may be dictated by the strength of the electric field. When spinning is initiated, there is an electrostatic attraction from the charged needle tip to the grounded mandrel. As spinning continues and fibers are deposited on the mandrel in layers. We presume that the layers act as an insulator, thus preventing sufficient current flow from the newly deposited fibers to the grounded mandrel and resulting in a buildup of charge on the outer fibers. This charge buildup would decrease the electrostatic force the charged polymer feels from the grounded mandrel, resulting in the fibers depositing on the mandrel with less efficiency, an increased attraction of the fibers for other objects, and a decrease in mat density due to loose fiber packing. In other words, the fiber deposition efficiency decreases as fibers are deposited on the mandrel and thus in order to obtain thicker scaffolds, one must use a large volume of polymer solution. McCullen et al. (2010) attempted to increase scaffold thickness using multi-layering electrospun scaffolds with a collagen gel for bone

As with any fabrication method, there are advantages and disadvantages to using electrospinning as a means of scaffold fabrication for tissue engineering. Many of the advantages have already been highlighted in previous sections such as the mimicking of native ECM and the ability to adapt the technique for versatile applications. Here, we discuss several limitations of the electrospinning and strategies to work towards

An inherent limitation to using electrospun nanofibrous scaffolds in tissue engineering applications is the small pore size. Nanofiber scaffolds typically result in pore sizes on the sub-micron to few micron scale. Because all mammalian cells range in size from several micrometers to several hundred micrometers in size, these cells typically cannot penetrate the scaffold. This poses a problem for tissue engineers because we often aim to form fully cellularized three dimensional tissue constructs. There have been attempts to improve the pore size of electrospun nanofiber scaffolds. The two primary methods are to co-electrospin a set of sacrificial fibers or adding porogens (Baker et al., 2008; Nam et al., 2007). Sacrificial fibers are fibers that are co-electrospun with the main scaffold component, but that are easily dissolved after fabrication leaving behind larger pores. For instance, Baker et al. (2008) used PEO, a water soluble polymer, as the sacrificial component of electrospun scaffolds. After dissolving the PEO, the pore size of the electrospun scaffolds increased. Porogens, primarily salt, have also been added to electrospun scaffolds as they are being electrospun (Nam et al., 2007). Typically the porogens are dropped onto a mandrel or collecting plate while the fibers are being deposited. Then, after fabrication, the porogens can be removed by dissolution. These approaches have improved pore size and allowed for improved cellular infiltration. However, these approaches may compromise the mechanical properties and dimensional stability of the scaffold when the fibers dissolve and collapse. Microintegration is another approach developed by Stankus et al. (2006) for producing a highly cellularized construct. However, this process may limit homogeneity, sterility, and

Another inherent limitation of electrospinning is scaffold thickness, especially when spinning onto a cylindrical mandrel. Electrospun scaffolds tend to be thin, typically less than one millimeter in many cases (Inoguchi et al., 2006; Lee et al., 2008). Scaffolds with thicknesses greater than one millimeter are possible to fabricate, but are less commonly seen in the literature (Pham et al., 2006b). Moreover, in our experience, achieving millimeter or greater thicknesses requires the use of a large volume of polymer solution. The inability to obtain thicker scaffolds may be dictated by the strength of the electric field. When spinning is initiated, there is an electrostatic attraction from the charged needle tip to the grounded mandrel. As spinning continues and fibers are deposited on the mandrel in layers. We presume that the layers act as an insulator, thus preventing sufficient current flow from the newly deposited fibers to the grounded mandrel and resulting in a buildup of charge on the outer fibers. This charge buildup would decrease the electrostatic force the charged polymer feels from the grounded mandrel, resulting in the fibers depositing on the mandrel with less efficiency, an increased attraction of the fibers for other objects, and a decrease in mat density due to loose fiber packing. In other words, the fiber deposition efficiency decreases as fibers are deposited on the mandrel and thus in order to obtain thicker scaffolds, one must use a large volume of polymer solution. McCullen et al. (2010) attempted to increase scaffold thickness using multi-layering electrospun scaffolds with a collagen gel for bone

cell survival, due to the long-term processing times required.

**4. Current challenges with using electrospinning in tissue engineering**

overcoming them.

tissue engineering. After fabricating multi-layered electrospun scaffolds, micro-scaled pores were created by a laser ablation technique. Thus, they were able to maintain the unique nanofibrous structure that supports cellular interactions and to fabricate micro-scaled features within the scaffold.

An additional limitation of electrospinning is the inability to precisely control where the fibers are deposited (Teo & Ramakrishna, 2005). As fibers whip towards a collecting plate or mandrel, they deposit randomly unless there is some other force to cause them to orient differently. To try to overcome this limitation, researchers have used large cylindrical mandrels with high rotation speeds to cause fiber alignment (Choi et al., 2008; Moffat et al., 2009) or used parallel electrodes to apply an electric field that would force the fiber to align across the plates or in other orientations (Li et al., 2003; Teo & S Ramakrishna, 2005; Li et al., 2005; Yang et al., 2009). Still, electrospinning would be an even more powerful technique if one could precisely control where the polymer jet would land such that computer-assisted design of electrospun scaffolds would be possible. This would allow researchers to easily create nanofibers and create a much broader range of scaffold geometries and thicknesses. Unfortunately, no one has discovered a way to do this to date. The closest anyone has come to approaching this ability was in a study by Kameoka et al. (2003) where they created a scanning tip electrospinning source that used an arrow-shaped tip, polymer droplet, and high voltage to create a Taylor cone and spin fibers. They were able to control deposition to a region of about 5 mm in size. While some researchers have found creative ways to make electrospun scaffolds of differing geometries (besides tubes and sheets), the inability to precisely control the deposition location of the polymer jet limits the variety of possible geometries.

Most researchers tend to have electrospinning systems which still require a manual setup, i.e. manual positioning of the needle tip from the collector. Moreover, often humidity and temperature are not well-controlled and these ambient parameters can have an effect on the fibers. Because these parameters are often not precisely controlled, there is an increase in the variation of properties and fiber morphologies from scaffold to scaffold. In order to improve the repeatability and homogeneity between spinning batches, modern technology should be incorporated into electrospinning systems to more precisely control these parameters. For instance, without much difficulty, a computer-controlled spinning apparatus that is selfcontained to control temperature and humidity and that precisely controls air gap distances, mandrel rotation speed, voltage, and spinneret flow rate could be developed. Such a system would allow for more consistent scaffold fabrication and serve as an improved system to continue to investigate how these spinning parameters affect final fiber morphology.

#### **5. The future of electrospinning in tissue engineering and regenerative medicine**

Electrospinning has evolved as a powerful tool in various tissue engineering applications. This fabrication technology provides the ability to control biomaterial composition, fiber diameter, fiber alignment, geometry, and drug/protein incorporation into a scaffold. Nanoscaled fibers generated by electrospinning are able to improve the cellular interactions of a wide variety of cell types; moreover, the cells are able to maintain their phenotypic and functional characteristics on nano-scaled fibrous scaffolds. Additionally, a growing body of evidence demonstrates that nano-to micro-scaled topography plays an important role in controlling the adhesion, proliferation, differentiation, and survival of cells in culture.

Electrospun Nanofibers in Tissue Engineering 369

Chan-Park, M.B. et al., 2009. Biomimetic control of vascular smooth muscle cell morphology

Choi, J.S. et al., 2008. The influence of electrospun aligned poly(ε-caprolactone)/collagen

Deitzel, J.M. et al., 2001. The effect of processing variables on the morphology of electrospun

Doshi, J. & Reneker, D.H., 1995. Journal of Electrostatics. *Journal of Electrostatics*, 35(2-3),

Ghasemi-Mobarakeh, L. et al., 2008. Electrospun poly(ε-caprolactone)/gelatin nanofibrous scaffolds for nerve tissue engineering. *Biomaterials*, 29(34), pp.4532-4539. Greiner, A. & Wendorff, J.H., 2007. Electrospinning: a fascinating method for the preparation of ultrathin fibers. *Angewandte Chemie,* 46(30), pp.5670-5703. Gupta, P. et al., 2005. Electrospinning of linear homopolymers of poly(methyl methacrylate):

Gupta, D. et al., 2009. Nanostructured biocomposite substrates by electrospinning and

Hunziker, E.B., 2002. Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. *Osteoarthritis and Cartilage,* 10(6), pp.432-463. Inoguchi, H. et al., 2006. Mechanical responses of a compliant electrospun poly(L-lactide-*co*ε-caprolactone) small-diameter vascular graft. Biomaterials, 27(8), 1470-8. Jang, J.-H., Castano, O. & Kim, H.-W., 2009. Electrospun materials as potential platforms for bone tissue engineering. *Advanced Drug Delivery Reviews*, 61(12), pp.1065-1083. Ju, Y.M. et al., 2010. Bilayered scaffold for engineering cellularized blood vessels.

Kameoka, J. et al., 2003. A scanning tip electrospinning source for deposition of oriented

Kumbar, S.G. et al., 2008. Electrospun poly(lactic acid-*co*-glycolic acid) scaffolds for skin

Ladd, M.R. et al., 2010. Co-electrospun dual scaffolding system with potential for muscletendon junction tissue engineering. *Biomaterials*, 32(6), pp.1549-1559. Lee, C.H. et al., 2005. Nanofiber alignment and direction of mechanical strain affect the ECM production of human ACL fibroblast. *Biomaterials*, 26(11), pp.1261-1270. Lee, S.J. et al., 2007. In vitro evaluation of electrospun nanofiber scaffolds for vascular graft application. *Journal of Biomedical Materials Research, Part A*, 83(4), p.999–1008. Lee, S.J. et al., 2008. Development of a composite vascular scaffolding system that withstands physiological vascular conditions. *Biomaterials*, 29(19), pp.2891-2898. Li, C. et al., 2006. Electrospun silk-BMP-2 scaffolds for bone tissue engineering. *Biomaterials*,

Li, D., Wang, Y. & Xia, Y., 2003. Electrospinning of Polymeric and Ceramic Nanofibers as

Uniaxially Aligned Arrays. *Nano Letters*, 3(8), pp.1167-1171.

*Journal of Biomedical Materials Research. Part A*, 88(4), pp.1104-1121.

*Biomaterials*, 29(19), pp.2899-2906.

*Biomaterials*, 31(15), pp.4313-4321.

27(16), pp.3115-3124.

nanofibres. *Nanotechnology*, 14(10), pp.1124-1129.

tissue engineering. *Biomaterials*, 29(30), pp.4100-4107.

pp.151-160.

2094.

nanofibers and textiles. *Polymer*, 42, pp.261-272.

concentration in a good solvent. *Polymer*, 46, pp.4799-4810.

and phenotype for functional tissue-engineered small-diameter blood vessels.

nanofiber meshes on the formation of self-aligned skeletal muscle myotubes.

exploring relationships between fiber formation, viscosity, molecular weight and

electrospraying for the mineralization of osteoblasts. *Biomaterials*, 30(11), pp.2085-

Therefore, electrospun nano-scaled fibrous scaffolds can serve as a tool for studying the topographical aspects of cellular interactions that would lead to improved tissue formation. Furthermore, these electrospun scaffolds can be functionalized by adding biochemical and mechanical cues to enhance cellular interactions for tissue engineering applications. However, specific interactions between cells and electrospun scaffolds functionalized through surface modifications and bioactive factor incorporation are still poorly understood. Understanding of the specific cues that enhance cell adhesion, proliferation, and guidance of cells seeded on a scaffold, as well as cues that could affect host cell infiltration, differentiation, and vascularization in *in vitro* and *in vivo*, is crucial for the advancement of tissue engineering applications.

The most significant challenges that prohibit the expansion of electrospinning applications are increasing scaffold thickness and pore size. If these two problems were to be solved, electrospun nanofibers would prove to be even more useful than they are currently. Electrospun scaffolds can also be used as a component of a composite scaffold, similar to those developed by Sahoo et al. (Sahoo et al., 2006; Sahoo et al., 2007; Sahoo et al., 2010a; Sahoo et al., 2010b) or incorporated with hydrogel scaffolds to create layered structures. More detailed understanding of how the process parameters interact with one another, especially the electric field, may provide ways to better control fiber deposition. Thus, basic research into the physics of electrospinning will be essential in overcoming the challenges outlined above. For example, in a recent study by Ziabari et al. the simultaneous effects of solution concentration, spinning distance, applied voltage, and flow rate on mean fiber diameter and standard deviation of fiber diameter were investigated (Ziabari et al., 2010). Perhaps with more basic research, we will be able to apply electrospun nanofibers as a controlled fiber jet such that the fibers can be precisely deposited with micron-scale or submicron accuracy. Such a capability would be immensely useful for tissue engineering scaffold design.

#### **6. Acknowledgments**

This work was supported by the Department of Energy (DE-FG02-09ER64711), Telemedicine and Advanced Technology Research Center (TATRC) at the U.S. Army Medical Research and Material Command (USAMRMC) through award W81XWH-07-1- 0718, and Orthopaedic Trauma Research Program (USAMRAA OTRP07-07128091) of Department of Defense.

#### **7. References**

Therefore, electrospun nano-scaled fibrous scaffolds can serve as a tool for studying the topographical aspects of cellular interactions that would lead to improved tissue formation. Furthermore, these electrospun scaffolds can be functionalized by adding biochemical and mechanical cues to enhance cellular interactions for tissue engineering applications. However, specific interactions between cells and electrospun scaffolds functionalized through surface modifications and bioactive factor incorporation are still poorly understood. Understanding of the specific cues that enhance cell adhesion, proliferation, and guidance of cells seeded on a scaffold, as well as cues that could affect host cell infiltration, differentiation, and vascularization in *in vitro* and *in vivo*, is crucial for the advancement of

The most significant challenges that prohibit the expansion of electrospinning applications are increasing scaffold thickness and pore size. If these two problems were to be solved, electrospun nanofibers would prove to be even more useful than they are currently. Electrospun scaffolds can also be used as a component of a composite scaffold, similar to those developed by Sahoo et al. (Sahoo et al., 2006; Sahoo et al., 2007; Sahoo et al., 2010a; Sahoo et al., 2010b) or incorporated with hydrogel scaffolds to create layered structures. More detailed understanding of how the process parameters interact with one another, especially the electric field, may provide ways to better control fiber deposition. Thus, basic research into the physics of electrospinning will be essential in overcoming the challenges outlined above. For example, in a recent study by Ziabari et al. the simultaneous effects of solution concentration, spinning distance, applied voltage, and flow rate on mean fiber diameter and standard deviation of fiber diameter were investigated (Ziabari et al., 2010). Perhaps with more basic research, we will be able to apply electrospun nanofibers as a controlled fiber jet such that the fibers can be precisely deposited with micron-scale or submicron accuracy. Such a capability would be immensely useful for tissue engineering

This work was supported by the Department of Energy (DE-FG02-09ER64711), Telemedicine and Advanced Technology Research Center (TATRC) at the U.S. Army Medical Research and Material Command (USAMRMC) through award W81XWH-07-1- 0718, and Orthopaedic Trauma Research Program (USAMRAA OTRP07-07128091) of

Badami, A.S. et al., 2006. Effect of fiber diameter on spreading, proliferation, and

Baker, B.M. et al., 2008. The potential to improve cell infiltration in composite fiber-aligned

Behonick, D.J. & Werb, Z., 2003. A bit of give and take: the relationship between the

differentiation of osteoblastic cells on electrospun poly(lactic acid) substrates.

electrospun scaffolds by the selective removal of sacrificial fibers. *Biomaterials*,

extracellular matrix and the developing chondrocyte. *Mechanisms of Development*,

tissue engineering applications.

scaffold design.

**6. Acknowledgments**

Department of Defense.

*Biomaterials*, 27(4), pp.596-606.

29(15), pp.2348-2358.

120(11), pp.1327-1336.

**7. References**

Electrospun Nanofibers in Tissue Engineering 371

Sahoo, S., Cho-Hong, J.G. & Siew-Lok, T., 2007. Development of hybrid polymer scaffolds

Sahoo, S. et al., 2010a. Bioactive nanofibers for fibroblastic differentiation of mesenchymal

Sahoo, S., Toh, Siew Lok & Goh, J.C.H., 2010b. A bFGF-releasing silk/PLGA-based

Shin, H.J. et al., 2006. Electrospun PLGA nanofiber scaffolds for articular cartilage

Sill, T.J. & von Recum, H. a, 2008. Electrospinning: applications in drug delivery and tissue

Spalazzi, J.P. et al., 2008. Mechanoactive scaffold induces tendon remodeling and expression

Stankus, J.J. et al., 2006. Microintegrating smooth muscle cells into a biodegradable,

Stoop, R., 2008. Smart biomaterials for tissue engineering of cartilage. *Injury*, 39 Suppl 1,

Sun, T. et al., 2005. Self-organization of skin cells in three-dimensional electrospun

Teo, W.E. & Ramakrishna, S, 2005. Electrospun fibre bundle made of aligned nanofibres

Theron, A., Zussman, E. & Yarin, A., 2001. Electrostatic field-assisted alignment of

Thorvaldsson, A. et al., 2008. Electrospinning of highly porous scaffolds for cartilage

Tillman, B.W. et al., 2009. The in vivo stability of electrospun polycaprolactone-collagen

Vaquette, C. et al., 2010. Aligned poly(L-lactic-*co*-ε-caprolactone) electrospun microfibers

Yang, S. et al., 2001. The design of scaffolds for use in tissue engineering. Part I. Traditional

Yang, Y. et al., 2009. Electrospun Composite Mats of Poly[(D,L-lactide)-*co*-glycolide] and

and knitted structure: a novel composite scaffold for ligament tissue engineering.

Collagen with High Porosity as Potential Scaffolds for Skin Tissue Engineering.

scaffolds in vascular reconstruction. *Biomaterials*, 30(4), pp.583-588.

*Journal of Biomedical Materials Research. Part A*, 94(4), pp.1270-1282. Wong, D.J., Chang, H.Y. & Biology, E., 2009. Skin tissue engineering. *StemBook*, pp.1-9. Xu, C., 2004. Aligned biodegradable nanofibrous structure: a potential scaffold for blood

*Macromolecular Materials and Engineering*, 294(9), pp.611-619.

*Materials (Bristol, England)*, 2(3), pp.169-173.

*Research in Biological Diversity*, 79(2), pp.102-110.

progenitor cells. *Biomaterials*, 31(11), pp.2990-2998.

engineering. *Biomaterials*, 29(13), pp.1989-2006.

elastomeric fiber matrix. *Biomaterials*, 27(5), pp.735-744.

polystyrene scaffolds. *Tissue Engineering*, 11(7-8), pp.1023-1033.

over two fixed points. *Nanotechnology*, 16(9), pp.1878-1884.

electrospun nanofibres. *Nanotechnology*, 12, pp.384-390.

regeneration. *Biomacromolecules*, 9(3), pp.1044-1049.

vessel engineering. *Biomaterials*, 25(5), pp.877-886.

factors. *Tissue Engineering*, 7(6), pp.679-689.

2), pp.103-119.

1948.

pp.S77-87.

for potential applications in ligament and tendon tissue engineering. *Biomedical*

precursor cells for ligament/tendon tissue engineering applications. *Differentiation;*

biohybrid scaffold for ligament/tendon tissue engineering using mesenchymal

reconstruction: mechanical stability, degradation and cellular responses under mechanical stimulation in vitro. *Journal of Biomaterials Science. Polymer Edition*, 17(1-

of fibrocartilage markers. *Clinical Orthopaedics and Related Research*, 466(8), pp.1938-

Li, W.-J. et al., 2005. A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells. *Biomaterials*, 26(6), pp.599-609. Li, X. et al., 2009. Nanofiber scaffolds with gradations in mineral content for mimicking the

Liao, S. et al., 2008. Processing nanoengineered scaffolds through electrospinning and

Lodish, H. et al., 2008. *Molecular Cell Biology* 6th ed., New York, New York: W. H. Freeman

McCullen, S.D. et al., 2010. In situ collagen polymerization of layered cell-seeded

Megelski, S. et al., 2002. Micro- and nanostructured surface morphology on electrospun

Moffat, K.L. et al., 2009. Novel nanofiber-based scaffold for rotator cuff repair and

Moon, D.G. et al., 2008. Cyclic mechanical preconditioning improves engineered muscle

Moore, K.L. & Dalley, A.F., 1999. *Clinically Oriented Anatomy* 4th ed., Baltimore, MD:

Nam, J. et al., 2007. Improved cellular infiltration in electrospun fiber via engineered

Nie, H. & Wang, C.-H., 2007. Fabrication and characterization of PLGA/HAp composite

Pham, Q.P., Sharma, U. & Mikos, A.G., 2006. Electrospinning of polymeric nanofibers for tissue engineering applications: a review. *Tissue Engineering*, 12(5), pp.1197-1211. Plotkin, L.I. et al., 1999. Prevention of osteocyte and osteoblast apoptosis by

Powell, H.M. & Boyce, S.T., 2009. Engineered human skin fabricated using electrospun

Prabhakaran, M.P., Venugopal, J.R. & Ramakrishna, Seeram, 2009. Mesenchymal stem cell

Priya, S.G., Jungvid, H. & Kumar, A., 2008. Skin tissue engineering for tissue repair and

Reneker, D. H., & Chun, I., 1996. Nanometre diameter fibres of polymer, produced by

Ross, M.H., Kaye, G.I. & Pawlina, W., 2003. Integumentary System. In *Histology A Text and*

Sahoo, S. et al., 2006. Characterization of a novel polymeric scaffold for potential application in tendon/ligament tissue engineering. *Tissue Engineering*, 12(1), pp.91-99.

regeneration. *Tissue Engineering. Part B, Reviews*, 14(1), pp.105-118.

*Atlas*. Baltimore, MD: Lippincott Williams & Wilkins, pp. 400-433.

scaffolds for delivery of BMP-2 plasmid DNA. *Journal of Controlled Release* 120(1-2),

bisphosphonates and calcitonin. *Journal of Clinical Investigation*, 104(10), pp.1363-

collagen-PCL blends: morphogenesis and mechanical properties. *Tissue Engineering.*

differentiation to neuronal cells on electrospun nanofibrous substrates for nerve

mineralization suitable for biomimetic bone tissue engineering. *Journal of the*

electrospun scaffolds for bone tissue engineering applications. Tissue Engineering.

tendon-to-bone insertion site. *Nano Letters*, 9(7), pp.2763-2768.

*Mechanical Behavior of Biomedical Materials*, 1(3), pp.252-260.

polymer fibers. *Macromolecules*, 35(22), pp.8456-8466.

augmentation. *Tissue Engineering. Part A*, 15(1), pp.115-126.

contraction. *Tissue Engineering. Part A*, 14(4), pp.473-482.

Lippincott Williams & Wilkins. pp.12-14.

porosity. *Tissue Engineering*, 13(9), pp.2249-2257.

tissue engineering. *Biomaterials*, 30(28), pp.4996-5003.

electrospinning. *Nanotechnology*, 7, pp.216-223.

and Company. pp.820-833.

Part C, 16(5), pp.1095-1105.

pp.111-121.

*Part A*, 15(8), pp.2177-2187.

1374.

**17**

*Korea*

**Three-Dimensional Nanofiber**

**Scaffolds for Regenerative Medicine**

*1Institute of Cell & Tissue Engineering, Catholic University,*

Bit Na Lee, Jae Ho Kim, Heung Jae Chun1 and Moon Suk Kim *Department of Molecular Science and Technology, Ajou University, Suwon,*

Tissue engineering is an interdisciplinary technology of the basic concept of bioscience and biotechnology. It is aiming to make tissues that can replace or regenerate diseased tissues and organs after understanding of correlation between the structure and function of normal biological tissues. The regenerated tissues made in such way are improving, reviving, restoring, or substituting the functions of human body as well as maintaining the functions

The three elements of tissue engineering for regenerating the biological tissues are cell, growth factor, and scaffold. When the number of cells has been reduced due to some troubles of cell proliferation in the damaged tissue, cell can regenerate the damaged area by insertion of external cells, and the growth factor controls the growth and differentiation of cells. Scaffold is used to assist the growth and proliferation of cells. Scaffold helps cells growing and functioning in normal condition. If scaffold does not play its role properly, the replace or regenerate diseased tissues and organs must not success. Recent researches, therefore, have been focused on the development of scaffold that is influencing the

Scaffold must have, particularly, the similar form of extracellular matrix that supports cells, which should have the following characteristics.1,2 (1) Scaffold must connect tissue and blood vessel with each other by the appropriate size pores. (2) Scaffold must be able to adjust the biodegradability and bioabsorbability. (3) Scaffold must have the chemical surface where cells can achieve adherence, differentiation, and proliferation. (4) Scaffold must not induce other reverse functions or side effects. (5) Scaffold must be formed in various shapes and sizes, and it must be easy for the penetration or manipulation of various materials

Nanofiber is getting noticed the most intensively among the scaffolds with above characteristics (**Fig.1**).3 Nanofiber is expected to overcome the limitation of conventional materials. So, it will be adopted in the new field with lots of advantages of high surface area per unit volume, high porosity, numerous fibers in the unit area, micro space created between fibers, and its flexibility. Nanofiber can be produced through phase separation, self-assembly method, electrospinning method, etc. Nanofiber can be used in various biomedical application such as high-functional filter and wound healing material, reinforced fiber of composite biomaterials, and scaffold for tissue engineering. Among them, the nanofiber as a scaffold for

**1. Introduction**

inside the scaffold.

after transplanted into human body.1

proliferation and differentiation of cells.

## **Three-Dimensional Nanofiber Scaffolds for Regenerative Medicine**

Bit Na Lee, Jae Ho Kim, Heung Jae Chun1 and Moon Suk Kim *Department of Molecular Science and Technology, Ajou University, Suwon, 1Institute of Cell & Tissue Engineering, Catholic University, Korea*

#### **1. Introduction**

372 Nanofibers – Production, Properties and Functional Applications

Zhou, Y. et al., 2008. Electrospun water-soluble carboxyethyl chitosan/poly(vinyl alcohol)

Ziabari, M., Mottaghitalab, V. & Haghi, A.K., 2010. A new approach for optimization of electrospun nanofiber formation process. *Methodology*, 27(1), pp.340-354.

*Biomacromolecules*, 9(1), pp.349-354.

nanofibrous membrane as potential wound dressing for skin regeneration.

Tissue engineering is an interdisciplinary technology of the basic concept of bioscience and biotechnology. It is aiming to make tissues that can replace or regenerate diseased tissues and organs after understanding of correlation between the structure and function of normal biological tissues. The regenerated tissues made in such way are improving, reviving, restoring, or substituting the functions of human body as well as maintaining the functions after transplanted into human body.1

The three elements of tissue engineering for regenerating the biological tissues are cell, growth factor, and scaffold. When the number of cells has been reduced due to some troubles of cell proliferation in the damaged tissue, cell can regenerate the damaged area by insertion of external cells, and the growth factor controls the growth and differentiation of cells. Scaffold is used to assist the growth and proliferation of cells. Scaffold helps cells growing and functioning in normal condition. If scaffold does not play its role properly, the replace or regenerate diseased tissues and organs must not success. Recent researches, therefore, have been focused on the development of scaffold that is influencing the proliferation and differentiation of cells.

Scaffold must have, particularly, the similar form of extracellular matrix that supports cells, which should have the following characteristics.1,2 (1) Scaffold must connect tissue and blood vessel with each other by the appropriate size pores. (2) Scaffold must be able to adjust the biodegradability and bioabsorbability. (3) Scaffold must have the chemical surface where cells can achieve adherence, differentiation, and proliferation. (4) Scaffold must not induce other reverse functions or side effects. (5) Scaffold must be formed in various shapes and sizes, and it must be easy for the penetration or manipulation of various materials inside the scaffold.

Nanofiber is getting noticed the most intensively among the scaffolds with above characteristics (**Fig.1**).3 Nanofiber is expected to overcome the limitation of conventional materials. So, it will be adopted in the new field with lots of advantages of high surface area per unit volume, high porosity, numerous fibers in the unit area, micro space created between fibers, and its flexibility. Nanofiber can be produced through phase separation, self-assembly method, electrospinning method, etc. Nanofiber can be used in various biomedical application such as high-functional filter and wound healing material, reinforced fiber of composite biomaterials, and scaffold for tissue engineering. Among them, the nanofiber as a scaffold for

Three-Dimensional Nanofiber Scaffolds for Regenerative Medicine 375

Amphiphile (PA). When attaching PAs consisted with dialkyl chain (tail part of hydrophobicity) to the N-α amino group in the end of peptide chain, the peptides become similar to the base sequence of collagen amino acid of human ECM. However, self-assembly method is limited only for several polymer arrays (two block copolymers, three block copolymers, peptides-amphiphilic three block copolymers, and dendrimers). Another problem is that its mass production is not easy because of complicated manufacturing

Electrospinning had been proposed in 1930s together with electrospraying method. But it has not been commercialized since its development as its application was limited (**Fig.2**). Electrospinning method gets spotlighted again later in mid of 1990s when Reneker succeeded in the manufacturing of nanofiber with various polymers after simplifying the electrospinning device.4 The porosity, thickness, and components of nanofiber can be adjusted with simple experimental equipment in the electrospinning method. So it has been able to produce continuous nanofiber not only with polymer but also with ceramics at low process cost. It can produce nonwoven fabric type nanofiber at the same time of spinning, so its spinning time is short and its construction is simpler than general spinning facilities. Also, nanofiber can be produced with various polymers, and the spinning is available with just a little amount of polymer. The biggest feature of electrospinning method is that its nanofiber has the similar structure of ECM in terms of morphology. ECM forming collagen is consisted with micro fibril of 50~100 nm, and the ECM-similar nanofiber can be produced

process and low productivity.

**2.3 Electrospinning method**

by using the electrospinning method.

Fig. 2. Schematic image of the electrospinning method.

tissue engineering can provide similar environment as collagen of the extracellular matrix (ECM). It is advantageous for the cell adhesion when cultivating the cells.

This chapter focuses on three-dimensional nanofiber scaffolds, and highlights their potential applications for regenerative medicine. The first section reviews the fabrication of nanofiber scaffolds that provide an optimal microenvironment for cell proliferation, migration, differentiation, and guidance for the reconstruction or replacement of damaged or diseased tissues and organs. The following section focuses on natural and synthetic biodegradable biomaterials that have been applied as nanofiber scaffolds. The last section focuses on the preclinical applications of nanofiber scaffolds in regenerative medicine.

Fig. 1. Scanning electron micrograph (SEM) image of nanofibers.

#### **2. Nanofiber manufacturing technology**

Nanofiber manufacturing technology can be largely divided into 3 technologies of phase separation, self-assembly method, and electrospinning method. The nanofiber manufactured by phase separation and self-assembly method shows the limitation as a scaffold for the applications for tissue engineering. On the other hand, the nanofiber manufactured by electrospinning method shows various characteristics, which are suitable for the tissue engineering. This section introduces the nanofiber productions by phase separation, self-assembly method, and electrospinning method.

#### **2.1 Phase separation**

Phase separation is the porous polymer membrane forming technique used for years. Phase separation can control the pore structure of nanofiber by using two or more materials of different physical characteristics, and porous fiber is obtained when using polymer and highly volatile solvent. So, pore size can be changed by control of the volatility of solvent. Also it is possible to manufacture the nanofiber of which hydrophilic property has been adjusted, as pore structure can be changed by the interaction of solvent and water molecules in the air. However, there happens rapid phase separation between solvent and solute, when using the volatile solvent, due to the radical solidification of polymer with the volatilization of solvent. So it is not easy to control the concentration of polymer solution. It also has a problem that mass production of nanofiber is difficult as it can be applied only for limited numbers of polymer.

#### **2.2 Self-assembly method**

Self-assembly means that each component forms orderly structure voluntarily by the noncovalent bond. The universal method to make nanofiber is synthesizing the Peptide Amphiphile (PA). When attaching PAs consisted with dialkyl chain (tail part of hydrophobicity) to the N-α amino group in the end of peptide chain, the peptides become similar to the base sequence of collagen amino acid of human ECM. However, self-assembly method is limited only for several polymer arrays (two block copolymers, three block copolymers, peptides-amphiphilic three block copolymers, and dendrimers). Another problem is that its mass production is not easy because of complicated manufacturing process and low productivity.

#### **2.3 Electrospinning method**

374 Nanofibers – Production, Properties and Functional Applications

tissue engineering can provide similar environment as collagen of the extracellular matrix

This chapter focuses on three-dimensional nanofiber scaffolds, and highlights their potential applications for regenerative medicine. The first section reviews the fabrication of nanofiber scaffolds that provide an optimal microenvironment for cell proliferation, migration, differentiation, and guidance for the reconstruction or replacement of damaged or diseased tissues and organs. The following section focuses on natural and synthetic biodegradable biomaterials that have been applied as nanofiber scaffolds. The last section focuses on the

Nanofiber manufacturing technology can be largely divided into 3 technologies of phase separation, self-assembly method, and electrospinning method. The nanofiber manufactured by phase separation and self-assembly method shows the limitation as a scaffold for the applications for tissue engineering. On the other hand, the nanofiber manufactured by electrospinning method shows various characteristics, which are suitable for the tissue engineering. This section introduces the nanofiber productions by phase

Phase separation is the porous polymer membrane forming technique used for years. Phase separation can control the pore structure of nanofiber by using two or more materials of different physical characteristics, and porous fiber is obtained when using polymer and highly volatile solvent. So, pore size can be changed by control of the volatility of solvent. Also it is possible to manufacture the nanofiber of which hydrophilic property has been adjusted, as pore structure can be changed by the interaction of solvent and water molecules in the air. However, there happens rapid phase separation between solvent and solute, when using the volatile solvent, due to the radical solidification of polymer with the volatilization of solvent. So it is not easy to control the concentration of polymer solution. It also has a problem that mass production of nanofiber is difficult as it can be applied only for

Self-assembly means that each component forms orderly structure voluntarily by the noncovalent bond. The universal method to make nanofiber is synthesizing the Peptide

(ECM). It is advantageous for the cell adhesion when cultivating the cells.

preclinical applications of nanofiber scaffolds in regenerative medicine.

Fig. 1. Scanning electron micrograph (SEM) image of nanofibers.

separation, self-assembly method, and electrospinning method.

**2. Nanofiber manufacturing technology**

**2.1 Phase separation**

limited numbers of polymer.

**2.2 Self-assembly method**

Electrospinning had been proposed in 1930s together with electrospraying method. But it has not been commercialized since its development as its application was limited (**Fig.2**). Electrospinning method gets spotlighted again later in mid of 1990s when Reneker succeeded in the manufacturing of nanofiber with various polymers after simplifying the electrospinning device.4 The porosity, thickness, and components of nanofiber can be adjusted with simple experimental equipment in the electrospinning method. So it has been able to produce continuous nanofiber not only with polymer but also with ceramics at low process cost. It can produce nonwoven fabric type nanofiber at the same time of spinning, so its spinning time is short and its construction is simpler than general spinning facilities. Also, nanofiber can be produced with various polymers, and the spinning is available with just a little amount of polymer. The biggest feature of electrospinning method is that its nanofiber has the similar structure of ECM in terms of morphology. ECM forming collagen is consisted with micro fibril of 50~100 nm, and the ECM-similar nanofiber can be produced by using the electrospinning method.

Fig. 2. Schematic image of the electrospinning method.

Three-Dimensional Nanofiber Scaffolds for Regenerative Medicine 377

affinity and less immune reaction, moreover, as it contains lots of chemical inducers which are related to the adhesion, proliferation, and differentiation of cells. Collagen is also reported to have effectiveness in improving the physiology such as cell proliferation and organ formation, promotion of wound healing, and so on. It is disclosed that collagen nanofiber is inducing the biological tissue switching effect of nanocomposites in the

Chitosan, a naturally abundant antibacterial polymer, is found in the microbe cell walls and in the exoskeleton of crustaceans. Chitosan is manufactured by removing acetyl out of chitin, a kind of polysaccharide. The chitosan based nanofiber production is under research as it has less stimulation, excellent biocompatibility and biodegradability, and blood coagulating function. Also the chitosan nanofiber is being researched as antibacterial wound

Alginate is one of natural polymers, which is safe without toxicity. Alginate is produced in fiber form and processed in the forms of woven fabric, nonwoven fabric, and composite material for the treatment of wound area. Calcium ion is exchanged with sodium ion in the body fluids, when alginate nanofiber contacts with wound exudation, so it is used widely for the absorbent wound dressing material. As alginate is an electrolyte of very high conductivity and high viscosity, however, it is not easy for electrospinning. So, it must be

Elastin is a major component of elastic tissues such as blood vessel, lungs, ligament, and skin, which plays an important role to maintain the elasticity of tissues. Elastin is being proposed now as elastin nanofiber in 3-dimensional structure to produce excellent bioabsorbability, although it had not been easy to form and process elastin due to its stiff

Synthetic polymer is being developed for over 30 years in various forms of surgical suture or screw form, mesh structure, etc. Synthetic polymer is manufactured according to the unique characteristics of each material, and it is being produced to minimize the immune reactions in the tissue engineering (**Fig.4**). As the biodegradation period of nanofiber made of synthetic polymer can be adjusted, its commercialization is being progressed partially as

**Materials Product names Degradation period**

Polycaprolactone (PCL) MONOCRYL > 20 months Poly L-lactic acid (PLLA) BioScrew, PL-FIX 20~60 months Polydioxanone (PDO) PDS BIOSYN 6 months Polyglycolic acid (PGA) DEXON 1~4 months Poly (lactic-co-glycolic acid) (PLGA) VICRYL (90% Glycolide 10% Lactide) 2 months

connective tissues, accordingly, its applications are getting wider.

electrospinning processed after mixing with water-soluble substances.

**Chitosan**

**Alginate**

**Elastin**

cross-linked structure.

**3.2 Synthetic biomaterials**

a tissue engineering scaffold (**Table 1**).

Table 1. Degradable periods of synthetic polymer.5

dressings in the tissue engineering.

Reviewing the electrospinning principle, polymer fine bubbles are forming the taylor cone by the mutual repulsion of induced electric charges when applying high electric field to the polymer solution hanging on the end of a charged jet. Then, polymer solution is emitted when electric repulsion is higher than surface tension. Solvent is volatilized while emitted solution is in the air, then fibers of 50~100 nm diameter are laminated in 3-dimensional network structure to form the mesh structure. The nanofiber made in this way has high ratio of surface area compared to its volume, and it has high porosity to constitute favorable environment for cells to live in. As complex polymers can be used to produce nanofiber, it has been possible to manufacture the nanofiber that has overcome the limit of properties of existing polymers.

#### **3. Nanofiber manufacturing materials**

The scaffold is transplanted into the living body for treatment, so it is necessary to pay a lot of attention in selecting the materials. Both natural polymer and synthetic polymer have been used for treatment as the highly absorbing materials for over 30 years. It is important in recent tissue engineering to make scaffold that decomposes easily in the body without intermediate products or other side effects. Natural polymer is frail in its property but it is superior in the cell affinity and cell compatibility. Synthetic polymer can adjust the property and degradation period easily, but it is less biocompatible as there are no molecules to which cells can adhere. This section will describe the characteristics of each material applied for the production of nanofiber.

#### **3.1 Natural biomaterials**

As natural polymer has similar structure to the macromolecules in the body, it is used a lot as the biomaterial. For the production of nanofiber, chitosan, alginate, and elastin as well as collagen are used representatively (**Fig.3**).

#### **Collagen**

Collagen occupies 20~30% of total protein in vertebrate animals, which is a main component of ECM. At least no less than 22 kinds of collagens are existing in the human body to keep up the tissues or organs and to maintain the figure of human body. Collagen has higher cell affinity and less immune reaction, moreover, as it contains lots of chemical inducers which are related to the adhesion, proliferation, and differentiation of cells. Collagen is also reported to have effectiveness in improving the physiology such as cell proliferation and organ formation, promotion of wound healing, and so on. It is disclosed that collagen nanofiber is inducing the biological tissue switching effect of nanocomposites in the connective tissues, accordingly, its applications are getting wider.

#### **Chitosan**

376 Nanofibers – Production, Properties and Functional Applications

Reviewing the electrospinning principle, polymer fine bubbles are forming the taylor cone by the mutual repulsion of induced electric charges when applying high electric field to the polymer solution hanging on the end of a charged jet. Then, polymer solution is emitted when electric repulsion is higher than surface tension. Solvent is volatilized while emitted solution is in the air, then fibers of 50~100 nm diameter are laminated in 3-dimensional network structure to form the mesh structure. The nanofiber made in this way has high ratio of surface area compared to its volume, and it has high porosity to constitute favorable environment for cells to live in. As complex polymers can be used to produce nanofiber, it has been possible to manufacture the nanofiber that has overcome the limit of properties of existing polymers.

The scaffold is transplanted into the living body for treatment, so it is necessary to pay a lot of attention in selecting the materials. Both natural polymer and synthetic polymer have been used for treatment as the highly absorbing materials for over 30 years. It is important in recent tissue engineering to make scaffold that decomposes easily in the body without intermediate products or other side effects. Natural polymer is frail in its property but it is superior in the cell affinity and cell compatibility. Synthetic polymer can adjust the property and degradation period easily, but it is less biocompatible as there are no molecules to which cells can adhere. This section will describe the characteristics of each material applied

As natural polymer has similar structure to the macromolecules in the body, it is used a lot as the biomaterial. For the production of nanofiber, chitosan, alginate, and elastin as well as

Collagen occupies 20~30% of total protein in vertebrate animals, which is a main component of ECM. At least no less than 22 kinds of collagens are existing in the human body to keep up the tissues or organs and to maintain the figure of human body. Collagen has higher cell

**3. Nanofiber manufacturing materials**

for the production of nanofiber.

collagen are used representatively (**Fig.3**).

Fig. 3. Structures of natural biomaterials.

**Collagen**

**3.1 Natural biomaterials**

Chitosan, a naturally abundant antibacterial polymer, is found in the microbe cell walls and in the exoskeleton of crustaceans. Chitosan is manufactured by removing acetyl out of chitin, a kind of polysaccharide. The chitosan based nanofiber production is under research as it has less stimulation, excellent biocompatibility and biodegradability, and blood coagulating function. Also the chitosan nanofiber is being researched as antibacterial wound dressings in the tissue engineering.

#### **Alginate**

Alginate is one of natural polymers, which is safe without toxicity. Alginate is produced in fiber form and processed in the forms of woven fabric, nonwoven fabric, and composite material for the treatment of wound area. Calcium ion is exchanged with sodium ion in the body fluids, when alginate nanofiber contacts with wound exudation, so it is used widely for the absorbent wound dressing material. As alginate is an electrolyte of very high conductivity and high viscosity, however, it is not easy for electrospinning. So, it must be electrospinning processed after mixing with water-soluble substances.

#### **Elastin**

Elastin is a major component of elastic tissues such as blood vessel, lungs, ligament, and skin, which plays an important role to maintain the elasticity of tissues. Elastin is being proposed now as elastin nanofiber in 3-dimensional structure to produce excellent bioabsorbability, although it had not been easy to form and process elastin due to its stiff cross-linked structure.

#### **3.2 Synthetic biomaterials**

Synthetic polymer is being developed for over 30 years in various forms of surgical suture or screw form, mesh structure, etc. Synthetic polymer is manufactured according to the unique characteristics of each material, and it is being produced to minimize the immune reactions in the tissue engineering (**Fig.4**). As the biodegradation period of nanofiber made of synthetic polymer can be adjusted, its commercialization is being progressed partially as a tissue engineering scaffold (**Table 1**).

Table 1. Degradable periods of synthetic polymer.5

Three-Dimensional Nanofiber Scaffolds for Regenerative Medicine 379

degradation speed is getting faster. To solve such a problem, researches are progressing for better biocompatibility by improving cell's adhesion capability. The PGA nanofiber is pretreated with acid in order to hydrolyze the ester bond and to expose the carboxylic acid

PLA is a synthetic polymer with steric hindrance, more hydrophobic than PGA, as it contains methyl base. It is highly soluble in the organic solvents and slow in hydrolysis, and its degradation period is as long as 30~50 weeks. There are L-type and D-type stereoisomers in PLA. PLLA (Poly L-lactic acid), which is an L-type stereoisomer, has a merit of excellent mechanical property in the polymerized form of lactic acid that is synthesized in the living body. PLLA nanofiber has problems as it is hydrophobic, fragile, and slow in degradation speed. But it has been proved, as a scaffold imitating collagen, to be highly capable in the

PLGA, which is a copolymer of polyglycolide and polylactide, is a synthetic polymer approved by FDA. It is widely used as the biomaterial for porous scaffold, drug delivery system, etc. in tissue engineering. PLGA is harmless to human body as it is changed to lactic acid and glycolic acid by hydrolysis in the living body and changed to carbon dioxide and water when discharging out of the body. As it is highly biocompatible, biodegradable, and processable, it is used widely in tissue engineering and drug delivery system. PLGA is a synthetic polymer with various degradation periods when the amount of monomers is adjusted. The PLGA nanofiber is being applied the most widely as a scaffold imitating biological tissues or drug delivery system, and biosensors. Especially, PLGA is spreading its applications after controlling its properties by adjusting the amount of PLA and PGA.

PDO is biodegradable polyester of 55% crystallinity, which has been developed originally as a biodegradable surgical suture. Its degradation speed in the body is 6 months, intermediate period between PGA and PLA. PDO is superior in flexibility as it contains ester oxygen in the chains of monomer. PDO nanofiber is suitable for the biomaterial as it carries the property between minimum elasticity coefficient of collagen and maximum elasticity

For the applications of nanofiber scaffold to the human body, it must provide processability and appropriate conditions for adhesion, proliferation, and differentiation of cells in the tissue as well as the properties of conventional scaffolds. As the nanofiber satisfying such requisites is widely applied to the artificial tissues such as skin, blood vessel, bone, etc., wound dressings, and drug delivery system, this section is going to describe the

Skin is the biggest tissue that is covering the surface of human body. It prevents loss of moisture, adjusts the body temperature, blocks bacterial invasion, and protects human body from radiant rays and ultraviolet rays. Skin grafting technology has been developed to

and alcohol base. **Polylactic acid (PLA)**

cell adhesion and differentiation. **Poly (lactic-co-glycolic acid) (PLGA)**

**Polydioxanone (PDO)**

coefficient of elastin.

applications of nanofiber.

**4.1 Artificial skin**

**4. Nanofiber applications for tissue engineering**

Fig. 4. Structures of synthetic biomaterials.

#### **Polycaprolactone (PCL)**

PCL is a polyester polymer of high elasticity without toxicity, which is superior in biocompatibility but its degradation period is rather slow for 1~2 years. It is aliphatic polyester with repeating 5 nonpolar methylene groups and 1 polar ester group, which is hydrophobic as of numerous carbons in its structure. It has disadvantages that initial protein absorption capacity is low, as the surface of PCL nanofiber is hydrophobic, the cell adhesion is slow, and the cell differentiation and tissue regeneration is slow. But, it is used as a good absorbent material for either soft or hard tissues as its properties can be adjusted.

#### **Polyglycolic acid (PGA)**

PGA is biodegradable aliphatic polyester, which had been developed for surgical suture in 1970s. Its biological absorptiveness can be predicted, and it takes 2~4 weeks for degradation as it is hydrophilic in the body. Its strength is reduced by 60%, for the first 2 weeks, also the pH and crystallinity are lowered down due to the generation of hydrolysis. The PGA nanofiber has high strength and elasticity initially, which is biodegraded rapidly by the diffusion of water and hydrolysis at the body temperature. However, it may cause undesirable reactions in the tissue as the pH of topical area is increasing rapidly while degradation speed is getting faster. To solve such a problem, researches are progressing for better biocompatibility by improving cell's adhesion capability. The PGA nanofiber is pretreated with acid in order to hydrolyze the ester bond and to expose the carboxylic acid and alcohol base.

#### **Polylactic acid (PLA)**

378 Nanofibers – Production, Properties and Functional Applications

PCL is a polyester polymer of high elasticity without toxicity, which is superior in biocompatibility but its degradation period is rather slow for 1~2 years. It is aliphatic polyester with repeating 5 nonpolar methylene groups and 1 polar ester group, which is hydrophobic as of numerous carbons in its structure. It has disadvantages that initial protein absorption capacity is low, as the surface of PCL nanofiber is hydrophobic, the cell adhesion is slow, and the cell differentiation and tissue regeneration is slow. But, it is used as a good

PGA is biodegradable aliphatic polyester, which had been developed for surgical suture in 1970s. Its biological absorptiveness can be predicted, and it takes 2~4 weeks for degradation as it is hydrophilic in the body. Its strength is reduced by 60%, for the first 2 weeks, also the pH and crystallinity are lowered down due to the generation of hydrolysis. The PGA nanofiber has high strength and elasticity initially, which is biodegraded rapidly by the diffusion of water and hydrolysis at the body temperature. However, it may cause undesirable reactions in the tissue as the pH of topical area is increasing rapidly while

absorbent material for either soft or hard tissues as its properties can be adjusted.

Fig. 4. Structures of synthetic biomaterials.

**Polycaprolactone (PCL)**

**Polyglycolic acid (PGA)**

PLA is a synthetic polymer with steric hindrance, more hydrophobic than PGA, as it contains methyl base. It is highly soluble in the organic solvents and slow in hydrolysis, and its degradation period is as long as 30~50 weeks. There are L-type and D-type stereoisomers in PLA. PLLA (Poly L-lactic acid), which is an L-type stereoisomer, has a merit of excellent mechanical property in the polymerized form of lactic acid that is synthesized in the living body. PLLA nanofiber has problems as it is hydrophobic, fragile, and slow in degradation speed. But it has been proved, as a scaffold imitating collagen, to be highly capable in the cell adhesion and differentiation.

#### **Poly (lactic-co-glycolic acid) (PLGA)**

PLGA, which is a copolymer of polyglycolide and polylactide, is a synthetic polymer approved by FDA. It is widely used as the biomaterial for porous scaffold, drug delivery system, etc. in tissue engineering. PLGA is harmless to human body as it is changed to lactic acid and glycolic acid by hydrolysis in the living body and changed to carbon dioxide and water when discharging out of the body. As it is highly biocompatible, biodegradable, and processable, it is used widely in tissue engineering and drug delivery system. PLGA is a synthetic polymer with various degradation periods when the amount of monomers is adjusted. The PLGA nanofiber is being applied the most widely as a scaffold imitating biological tissues or drug delivery system, and biosensors. Especially, PLGA is spreading its applications after controlling its properties by adjusting the amount of PLA and PGA.

#### **Polydioxanone (PDO)**

PDO is biodegradable polyester of 55% crystallinity, which has been developed originally as a biodegradable surgical suture. Its degradation speed in the body is 6 months, intermediate period between PGA and PLA. PDO is superior in flexibility as it contains ester oxygen in the chains of monomer. PDO nanofiber is suitable for the biomaterial as it carries the property between minimum elasticity coefficient of collagen and maximum elasticity coefficient of elastin.

#### **4. Nanofiber applications for tissue engineering**

For the applications of nanofiber scaffold to the human body, it must provide processability and appropriate conditions for adhesion, proliferation, and differentiation of cells in the tissue as well as the properties of conventional scaffolds. As the nanofiber satisfying such requisites is widely applied to the artificial tissues such as skin, blood vessel, bone, etc., wound dressings, and drug delivery system, this section is going to describe the applications of nanofiber.

#### **4.1 Artificial skin**

Skin is the biggest tissue that is covering the surface of human body. It prevents loss of moisture, adjusts the body temperature, blocks bacterial invasion, and protects human body from radiant rays and ultraviolet rays. Skin grafting technology has been developed to

Three-Dimensional Nanofiber Scaffolds for Regenerative Medicine 381

or serum out of the wound is penetrating the wound dressings. If the wound dressing is constituted with nanofiber of micro diameter, the body fluids such as blood cannot penetrate the nanofiber, and so blood flow stays in the wound and coagulated. As there is no blood coagulation in the nanofiber, therefore, it is easy to remove the nanofiber. Also it is easier to exchange the oxygen and evaporate the water-vapor between the wound surface and air when porosity is higher. As nanofiber is a mat of very tiny diameter and pores with high specific surface area, its moisturization and breathability is good enough, it protects wound from germs and prevents body fluids from penetrating, and it is easy to remove. So

Drug delivery system is being developed to reduce the number of medications, to enhance the drug effectiveness and stability by adjusting the initial emissions through continuous emission of constant amount of drug to the medicating area. Numbers of researches are reported recently for using nanofiber produced with biodegradable polymers in the drug delivery systems. The drug delivery system made of nanofiber carries out the drug delivery function with fewer side effects by installing physical barriers using its wide surface area. Also the fibrosis of biocompatible polymers is easy and it does not need to raise the temperature when electrospinning. It is advantageous as drug is not decomposed in the

Nanofiber made of biomaterials in various methods is getting noticed for its very wide application potential as a scaffold imitating the ECM. Nanofiber shows significant effect in the adhesion, proliferation, and differentiation of cells as of its wide surface area and high porosity. Moreover, nanofiber is researched for the scaffold for the musculoskeletal tissues such as bone, spine, ligament, and skeletal muscle and for the tissues of skin, nerves, and blood vessel. It is also expanding its applications up to adjusting the delivery of drug, protein, and DNA. It is expected obviously that nanofiber will be used as an important scaffold in tissue engineering based on these researches. However, researches must be accompanied simultaneously for productivity and safety of nanofiber and securing application safety. It is also for easy biological application of nanofiber and for assessment of long-term regeneration capability in long-term view. Nevertheless, interdisciplinary researches of the experts in the fields of nanotechnology, biomaterials, medicines, and clinics

[1] A Atala, R Lanza, JA Thomson, R Nerem, Principles of Regenerative Medicine, 2nd Ed.,

[2] WM Saltzman, Tissue Engineering: Engineering Principles for the Design of Replacement Organs and Tissues, Oxford University Press, Oxford, 2004. [3] K Gonsalves, C Halberstadt, CT Laurencin, L Nair, Biomedical Nanostructures, Wiley-

[4] DH Reneker, I Chun, Nanometre diameter fibres of polymer produced by

are currently progressing so as to apply nanofiber clinically in the near future.

Academic Press, San Diego, 2010.

Interscience, New Jersey, 2008.

electrospinning, Nanotechnology, 7, 216, 1996.

its application for various wound dressings is also being reviewed actively.

**4.6 Drug delivery system**

**5. Conclusion and prospect**

nanofiber.

**6. References**

regenerate the skin tissue when it had been damaged by a burn, external wound, carcinoma resection, skin disease, etc. It is proved that nanofiber is greatly effective as a tegaderm in the adhesion and proliferation of human skin cells, and it has been reported that nanofiber can be used as an artificial skin.

#### **4.2 Artificial blood vessel**

Scaffold of excellent biocompatibility must be used in the vascular grafting, and it must satisfy several specific requirements such as mechanical elasticity and durability that can put up with repeated inflations and compressions. The nanofiber produced by electrospinning can imitate similarly the component, structure, and mechanical characteristics of blood vessel. It is reported that the adhesion, proliferation, and differentiation of cells have been improved when cultivating the nonstriated muscle cells with the scaffold made of copolymer P(LLA-CL) of PLLA and PCL. Its result has shown that P(LLA-CL) nanofiber is potential to be an ideal artificial blood vessel.

#### **4.3 Artificial bone**

In order to regenerate the bone in tissue engineering, the human regeneration mechanism promoting the bone formation and restoration should be increased or the tissue similar to the bone in living body must be developed. For the past several decades, autograft or allograft has been used for the damaged bones by disease or traumatic injury. However, there was limitation in such bone transplantations. So bioactive materials such as hydroxyapatite or tricalcium phosphate have been used for its substitution, or glass and ceramic have been used in dentistry or in orthopedics as alternative bones. Bioactivity, tissue integration, and mechanical strength are requested for such materials. Various nanofiber scaffolds have been developed recently as bone substitutions to fulfill such requirements. Ramakrishna et al. has made the scaffold by electrospinning of collagen, which has similar structure of extracellular matrix, and hydroxyapatite at the mixing ratio of 1:1. It is proved, when osteoblast was cultured on the nanofiber that had been electrospinning processed with hydroxyapatite, that it has been mineralized as of high differentiation of cells and high concentration of calcium and phosphorus.

#### **4.4 Artificial cartilage**

Peculiarly, there is no blood vessel, nerves and lymphoid tissue in the articular cartilage. There is no inflammation reaction when damaged, therefore, and it is difficult to supplement the cells to recover the damage. So, it is highly limited to recover or regenerate the articular cartilage when it has been damaged. Nanofiber is applied to make hyaline articular cartilage tissue, therefore, by cultivating articular cartilage cells or adipose-derived stem cells in the appropriate 3-dimensional scaffold. Nanofiber scaffold promotes the generation of ECM by the transplanted cells. The nanofiber is also able to adjust the chondrogenesis of human cartilage cells and that nanofiber can be applied widely in the chondral resurfacing by adjusting the shape and size of nanofiber.

#### **4.5 Wound dressing**

Dermis is exposed as epithelial tissue is peeled off in the affected part of skin tissue by wound such as external injury, burns, diabetes, and clogging of venous blood flow. Wound dressing is the first-aid kit to stop bleeding, which promotes the injury protection as blood or serum out of the wound is penetrating the wound dressings. If the wound dressing is constituted with nanofiber of micro diameter, the body fluids such as blood cannot penetrate the nanofiber, and so blood flow stays in the wound and coagulated. As there is no blood coagulation in the nanofiber, therefore, it is easy to remove the nanofiber. Also it is easier to exchange the oxygen and evaporate the water-vapor between the wound surface and air when porosity is higher. As nanofiber is a mat of very tiny diameter and pores with high specific surface area, its moisturization and breathability is good enough, it protects wound from germs and prevents body fluids from penetrating, and it is easy to remove. So its application for various wound dressings is also being reviewed actively.

#### **4.6 Drug delivery system**

380 Nanofibers – Production, Properties and Functional Applications

regenerate the skin tissue when it had been damaged by a burn, external wound, carcinoma resection, skin disease, etc. It is proved that nanofiber is greatly effective as a tegaderm in the adhesion and proliferation of human skin cells, and it has been reported that nanofiber

Scaffold of excellent biocompatibility must be used in the vascular grafting, and it must satisfy several specific requirements such as mechanical elasticity and durability that can put up with repeated inflations and compressions. The nanofiber produced by electrospinning can imitate similarly the component, structure, and mechanical characteristics of blood vessel. It is reported that the adhesion, proliferation, and differentiation of cells have been improved when cultivating the nonstriated muscle cells with the scaffold made of copolymer P(LLA-CL) of PLLA and PCL. Its result has shown that

In order to regenerate the bone in tissue engineering, the human regeneration mechanism promoting the bone formation and restoration should be increased or the tissue similar to the bone in living body must be developed. For the past several decades, autograft or allograft has been used for the damaged bones by disease or traumatic injury. However, there was limitation in such bone transplantations. So bioactive materials such as hydroxyapatite or tricalcium phosphate have been used for its substitution, or glass and ceramic have been used in dentistry or in orthopedics as alternative bones. Bioactivity, tissue integration, and mechanical strength are requested for such materials. Various nanofiber scaffolds have been developed recently as bone substitutions to fulfill such requirements. Ramakrishna et al. has made the scaffold by electrospinning of collagen, which has similar structure of extracellular matrix, and hydroxyapatite at the mixing ratio of 1:1. It is proved, when osteoblast was cultured on the nanofiber that had been electrospinning processed with hydroxyapatite, that it has been mineralized as of high

Peculiarly, there is no blood vessel, nerves and lymphoid tissue in the articular cartilage. There is no inflammation reaction when damaged, therefore, and it is difficult to supplement the cells to recover the damage. So, it is highly limited to recover or regenerate the articular cartilage when it has been damaged. Nanofiber is applied to make hyaline articular cartilage tissue, therefore, by cultivating articular cartilage cells or adipose-derived stem cells in the appropriate 3-dimensional scaffold. Nanofiber scaffold promotes the generation of ECM by the transplanted cells. The nanofiber is also able to adjust the chondrogenesis of human cartilage cells and that nanofiber can be applied widely in the

Dermis is exposed as epithelial tissue is peeled off in the affected part of skin tissue by wound such as external injury, burns, diabetes, and clogging of venous blood flow. Wound dressing is the first-aid kit to stop bleeding, which promotes the injury protection as blood

P(LLA-CL) nanofiber is potential to be an ideal artificial blood vessel.

differentiation of cells and high concentration of calcium and phosphorus.

chondral resurfacing by adjusting the shape and size of nanofiber.

can be used as an artificial skin.

**4.2 Artificial blood vessel**

**4.3 Artificial bone**

**4.4 Artificial cartilage**

**4.5 Wound dressing**

Drug delivery system is being developed to reduce the number of medications, to enhance the drug effectiveness and stability by adjusting the initial emissions through continuous emission of constant amount of drug to the medicating area. Numbers of researches are reported recently for using nanofiber produced with biodegradable polymers in the drug delivery systems. The drug delivery system made of nanofiber carries out the drug delivery function with fewer side effects by installing physical barriers using its wide surface area. Also the fibrosis of biocompatible polymers is easy and it does not need to raise the temperature when electrospinning. It is advantageous as drug is not decomposed in the nanofiber.

#### **5. Conclusion and prospect**

Nanofiber made of biomaterials in various methods is getting noticed for its very wide application potential as a scaffold imitating the ECM. Nanofiber shows significant effect in the adhesion, proliferation, and differentiation of cells as of its wide surface area and high porosity. Moreover, nanofiber is researched for the scaffold for the musculoskeletal tissues such as bone, spine, ligament, and skeletal muscle and for the tissues of skin, nerves, and blood vessel. It is also expanding its applications up to adjusting the delivery of drug, protein, and DNA. It is expected obviously that nanofiber will be used as an important scaffold in tissue engineering based on these researches. However, researches must be accompanied simultaneously for productivity and safety of nanofiber and securing application safety. It is also for easy biological application of nanofiber and for assessment of long-term regeneration capability in long-term view. Nevertheless, interdisciplinary researches of the experts in the fields of nanotechnology, biomaterials, medicines, and clinics are currently progressing so as to apply nanofiber clinically in the near future.

#### **6. References**

**18**

*USA*

Michael Hadjiargyrou *Stony Brook University*

**Incorporation of DNA into Electrospun**

**Characterization Studies and Gene Delivery**

The ability to successfully deliver exogenous DNA into cells has provided the scientific community with a plethora of opportunities into the realm of basic, clinical, and translational research. It has afforded us the means to study the structure and function of genes and their products (i.e. proteins), correct genetic or acquired disorders, and generate tremendous amounts of data which researchers around the world continuously work to build upon in order to generate the next "magic bullet". Despite such progress, gene delivery continues to suffer from various limitations, predominantly, low transfection efficiencies when using non-viral approaches (i.e. naked DNA, cationic lipids/liposomes, synthetic and natural polymers, etc.) (Al-Dosari & Gao, 2009) and unacceptable levels of toxicity and immunogenicity when using the more traditional viral vectors. To address these limitations, researchers have focused on a number of innovative alternatives, one of which involves the incorporation of DNA into electrospun nanofibrous, non-woven and

Electrospinning is a variation of the electrospray process and enables the experimenter to formulate scaffolds with specified mechanical, biological, chemical and kinetic properties, all readily controlled by alterations of polymer solution composition (i.e. polymer molecular weight and concentration, viscosity, salt concentration, conductivity, surface tension) and processing parameters (i.e. temperature, humidity, electric field strength, distance between spinneret and collector, feed rate and flow rate) (Chiu et al., 2005; Bhardwaj & Kundu, 2010). The resulting nanofibrous scaffolds possess desirable attributes such as high surface area to volume ratio and numerous interconnected pores that mimic the topology of the extracellular matrix (ECM). Obviously these features are a requirement for the transport of oxygen, nutrients, and wastes through the scaffold, as well as supporting robust cell adhesion and viability. Further, the flexible nanofibers and their pliability is beneficial for cell migration throughout the 3D scaffold. As such, electrospinning represents a truly rational and versatile approach for the construction of custom-tailored tissue engineering scaffolds, especially those capable of supporting cell/tissue growth, as well as deliver a range of bioactive molecules, including, DNA, proteins, drugs, etc. These "functionalized" or "biomimetic" scaffolds represent a new paradigm in the design and implementation of

**1. Introduction**

biodegradable scaffolds.

tissue engineering strategies.

**Nanofibrous Scaffolds: Fundamental**

[5] PA Walker, KR Aroom, F Jimenez, SK Shah, MT Harting, BS Gill, CS Jr Cox. Advances in Progenitor Cell Therapy Using Scaffolding Constructs for Central Nervous System Injury, Stem Cell Rev., 5, 283-300, 2009.

### **Incorporation of DNA into Electrospun Nanofibrous Scaffolds: Fundamental Characterization Studies and Gene Delivery**

Michael Hadjiargyrou *Stony Brook University USA*

#### **1. Introduction**

382 Nanofibers – Production, Properties and Functional Applications

[5] PA Walker, KR Aroom, F Jimenez, SK Shah, MT Harting, BS Gill, CS Jr Cox. Advances

System Injury, Stem Cell Rev., 5, 283-300, 2009.

in Progenitor Cell Therapy Using Scaffolding Constructs for Central Nervous

The ability to successfully deliver exogenous DNA into cells has provided the scientific community with a plethora of opportunities into the realm of basic, clinical, and translational research. It has afforded us the means to study the structure and function of genes and their products (i.e. proteins), correct genetic or acquired disorders, and generate tremendous amounts of data which researchers around the world continuously work to build upon in order to generate the next "magic bullet". Despite such progress, gene delivery continues to suffer from various limitations, predominantly, low transfection efficiencies when using non-viral approaches (i.e. naked DNA, cationic lipids/liposomes, synthetic and natural polymers, etc.) (Al-Dosari & Gao, 2009) and unacceptable levels of toxicity and immunogenicity when using the more traditional viral vectors. To address these limitations, researchers have focused on a number of innovative alternatives, one of which involves the incorporation of DNA into electrospun nanofibrous, non-woven and biodegradable scaffolds.

Electrospinning is a variation of the electrospray process and enables the experimenter to formulate scaffolds with specified mechanical, biological, chemical and kinetic properties, all readily controlled by alterations of polymer solution composition (i.e. polymer molecular weight and concentration, viscosity, salt concentration, conductivity, surface tension) and processing parameters (i.e. temperature, humidity, electric field strength, distance between spinneret and collector, feed rate and flow rate) (Chiu et al., 2005; Bhardwaj & Kundu, 2010). The resulting nanofibrous scaffolds possess desirable attributes such as high surface area to volume ratio and numerous interconnected pores that mimic the topology of the extracellular matrix (ECM). Obviously these features are a requirement for the transport of oxygen, nutrients, and wastes through the scaffold, as well as supporting robust cell adhesion and viability. Further, the flexible nanofibers and their pliability is beneficial for cell migration throughout the 3D scaffold. As such, electrospinning represents a truly rational and versatile approach for the construction of custom-tailored tissue engineering scaffolds, especially those capable of supporting cell/tissue growth, as well as deliver a range of bioactive molecules, including, DNA, proteins, drugs, etc. These "functionalized" or "biomimetic" scaffolds represent a new paradigm in the design and implementation of tissue engineering strategies.

Incorporation of DNA into Electrospun Nanofibrous Scaffolds:

solution while in the electrospinning jet.

Society.

of self-assembled DNA devices.

Fundamental Characterization Studies and Gene Delivery 385

The authors found that the DNA length vs. time yielded a time constant ranging from 2.1 - 19 seconds, thus adding new knowledge on the dynamic behaviour of the DNA in the PEO

Fig. 1. Fluorescently labelled DNA/PEO nanofibers. Images a-c show the presence of the fluorescently labelled DNA within the PEO nanofibers. Image d shows control nanofibers without any DNA. Reprinted with permission from Bellan et al., 2005, American Chemical

The same group also electrospun fluorescently labelled DNA and deposited it over photolithographically patterned trenches and gold electrodes and found that the nanofibers displayed diameters of ~27 nm (Bellan et al., 2007). Further, they imaged the nanofibers via a number of imaging techniques, including fluorescence microscopy, scanning electron microscopy (SEM), and AFM. Using AFM, the Young's modulus of a single suspended electrospun DNA fiber was determined by a three point bending test to be 15 ± 2 GPa. In their conclusion, the authors suggest that this approach could conceivably be used to manufacture nanosensors based on such DNA nanofibers, as well as aid in the development

An additional study was reported in the literature that utilized a mixture of DNA/PEO to electrospin nanofibers (Liu et al, 2007). The authors extensively evaluated the basic electrospinning solution properties such as conductivity, surface tension and viscosity (Table 1) and determined that the ionic conductivity of the solution increased significantly with the addition of DNA. In contrast, there was only a slight increase in the ionic conductivity with increasing amounts of PEO, which also decreased the surface tension. On the other hand, the viscosity increased with the addition of either DNA or PEO and as such the authors concluded that solutions containing both DNA and PEO had ideal properties for electrospinning. The electrospun DNA/PEO nanofibers displayed diameters of 50–250 nm

Since the initial study of Fang & Renneker (1997), demonstrating the possibility of electrospinning pure DNA nanofibers, the use of this technique for a variety of applications has truly exploded. Research has been reported in areas as diverse as scaffolds for tissue regeneration/regenerative medicine (Martins et al., 2007), drug delivery systems (Hadjiargyrou & Chiu, 2008), wound dressing materials (Khil et al., 2003; Kang et al., 2010; Chen & Chian, 2010; Cai et al., 2010), biocatalysis (Nair et al., 2007), cardiovascular grafts (Sell et al., 2009), prevention of post-surgical adhesions (Zong et al., 2004; Bölgen et al., 2007), filtration (Kattamuri et al., 2005), energy and environmental applications (Thavasi et al., 2008), biosensors (Manesh et al., 2007; Kowalczyk et al., 2008; Ding et al., 2010), and protective clothing (Gibson et al., 1998 ; Seungsin & Obendorf, 2006). In fact, there are over 1,650 reports indexed in PubMed for a keyword search of "electrospun or electrospinning". As many of these electrospinning applications are covered in other parts of this book, this chapter will strictly focus on the current literature and latest research on the use of electrospun polymeric scaffolds for fundamental characterization studies on the incorporation and behavior of DNA as well as their application as gene delivery systems.

#### **2. Fundamental studies: Incorporation and characterization of DNA into polymeric nanofibers**

The initial and simple study of electrospinning DNA into nanofibers was reported in 1997 by Fang and Reneker, who used commercially available calf thymus genomic DNA that was diluted in a range of 0.3 – 1.5% in a mixture of 70% water and 30% ethanol and electrospun at 12-14 kV onto porous copper grids. The resulting pure DNA fibers had diameters as small as 30 nm that spanned the 50 m pores of the grids. In addition, the DNA fibers contained beads with diameters from 80 – 300 nm that were a few microns apart on each DNA nanofiber, resulting from DNA retraction into droplets. Lastly, the authors presented a high magnification image of a single DNA fiber that was 62 nm in diameter and speculated that ~600 DNA molecules could pass through each cross section to form the fiber (Fang and Reneker, 1997).

A similar study was conducted using salmon testis DNA with a range of 200-90 kb fragments that was diluted in a range of 0.5 – 2.5 wt% solution in a mixture of 70% water and 30% ethanol and electrospun using a range of voltages (15 – 30 kV) onto mica substrates (Takahashi et al., 2005). The resulting DNA fibers were examined with atomic force microscopy (AFM) and the authors reported the presence of four different types of fibers; twisted shaped, twisted and stretched, curved and straight. Further, the height and length of the fibers ranged from 1.8 nm and 1 m, respectively, but these slightly varied with the different % DNA solutions and voltages used. The authors concluded that the optimum parameter values for generating ultrathin nanofibers were 1.5 wt% for DNA concentration and 20 kV for the applied voltage.

Aside from electrospinning pure DNA in solution, other investigations were carried out using DNA mixed with various synthetic polymers. For example, one study mixed fluorescently labelled bacteriophage lambda DNA molecules with polyethylene oxide (PEO) and produced polymer nanofibers that contained isolated and stretched DNA molecules (Bellan et al., 2005). These DNA nanofibers had diameters that ranged between 100-350 nm and lengths between 1 and 19 m, as measured by AFM (Fig. 1). Making use of the fluorescently labelled DNA, this study also examined the relaxation time of the DNA in a viscous solution (bulk PEO) in order to understand the fluid dynamic nature of the DNA.

Since the initial study of Fang & Renneker (1997), demonstrating the possibility of electrospinning pure DNA nanofibers, the use of this technique for a variety of applications has truly exploded. Research has been reported in areas as diverse as scaffolds for tissue regeneration/regenerative medicine (Martins et al., 2007), drug delivery systems (Hadjiargyrou & Chiu, 2008), wound dressing materials (Khil et al., 2003; Kang et al., 2010; Chen & Chian, 2010; Cai et al., 2010), biocatalysis (Nair et al., 2007), cardiovascular grafts (Sell et al., 2009), prevention of post-surgical adhesions (Zong et al., 2004; Bölgen et al., 2007), filtration (Kattamuri et al., 2005), energy and environmental applications (Thavasi et al., 2008), biosensors (Manesh et al., 2007; Kowalczyk et al., 2008; Ding et al., 2010), and protective clothing (Gibson et al., 1998 ; Seungsin & Obendorf, 2006). In fact, there are over 1,650 reports indexed in PubMed for a keyword search of "electrospun or electrospinning". As many of these electrospinning applications are covered in other parts of this book, this chapter will strictly focus on the current literature and latest research on the use of electrospun polymeric scaffolds for fundamental characterization studies on the incorporation and behavior of DNA as well as their application as gene delivery systems.

**2. Fundamental studies: Incorporation and characterization of DNA into**

The initial and simple study of electrospinning DNA into nanofibers was reported in 1997 by Fang and Reneker, who used commercially available calf thymus genomic DNA that was diluted in a range of 0.3 – 1.5% in a mixture of 70% water and 30% ethanol and electrospun at 12-14 kV onto porous copper grids. The resulting pure DNA fibers had diameters as small as 30 nm that spanned the 50 m pores of the grids. In addition, the DNA fibers contained beads with diameters from 80 – 300 nm that were a few microns apart on each DNA nanofiber, resulting from DNA retraction into droplets. Lastly, the authors presented a high magnification image of a single DNA fiber that was 62 nm in diameter and speculated that ~600 DNA molecules could pass through each cross section to form the fiber (Fang and

A similar study was conducted using salmon testis DNA with a range of 200-90 kb fragments that was diluted in a range of 0.5 – 2.5 wt% solution in a mixture of 70% water and 30% ethanol and electrospun using a range of voltages (15 – 30 kV) onto mica substrates (Takahashi et al., 2005). The resulting DNA fibers were examined with atomic force microscopy (AFM) and the authors reported the presence of four different types of fibers; twisted shaped, twisted and stretched, curved and straight. Further, the height and length of the fibers ranged from 1.8 nm and 1 m, respectively, but these slightly varied with the different % DNA solutions and voltages used. The authors concluded that the optimum parameter values for generating ultrathin nanofibers were 1.5 wt% for DNA concentration

Aside from electrospinning pure DNA in solution, other investigations were carried out using DNA mixed with various synthetic polymers. For example, one study mixed fluorescently labelled bacteriophage lambda DNA molecules with polyethylene oxide (PEO) and produced polymer nanofibers that contained isolated and stretched DNA molecules (Bellan et al., 2005). These DNA nanofibers had diameters that ranged between 100-350 nm and lengths between 1 and 19 m, as measured by AFM (Fig. 1). Making use of the fluorescently labelled DNA, this study also examined the relaxation time of the DNA in a viscous solution (bulk PEO) in order to understand the fluid dynamic nature of the DNA.

**polymeric nanofibers**

Reneker, 1997).

and 20 kV for the applied voltage.

The authors found that the DNA length vs. time yielded a time constant ranging from 2.1 - 19 seconds, thus adding new knowledge on the dynamic behaviour of the DNA in the PEO solution while in the electrospinning jet.

Fig. 1. Fluorescently labelled DNA/PEO nanofibers. Images a-c show the presence of the fluorescently labelled DNA within the PEO nanofibers. Image d shows control nanofibers without any DNA. Reprinted with permission from Bellan et al., 2005, American Chemical Society.

The same group also electrospun fluorescently labelled DNA and deposited it over photolithographically patterned trenches and gold electrodes and found that the nanofibers displayed diameters of ~27 nm (Bellan et al., 2007). Further, they imaged the nanofibers via a number of imaging techniques, including fluorescence microscopy, scanning electron microscopy (SEM), and AFM. Using AFM, the Young's modulus of a single suspended electrospun DNA fiber was determined by a three point bending test to be 15 ± 2 GPa. In their conclusion, the authors suggest that this approach could conceivably be used to manufacture nanosensors based on such DNA nanofibers, as well as aid in the development of self-assembled DNA devices.

An additional study was reported in the literature that utilized a mixture of DNA/PEO to electrospin nanofibers (Liu et al, 2007). The authors extensively evaluated the basic electrospinning solution properties such as conductivity, surface tension and viscosity (Table 1) and determined that the ionic conductivity of the solution increased significantly with the addition of DNA. In contrast, there was only a slight increase in the ionic conductivity with increasing amounts of PEO, which also decreased the surface tension. On the other hand, the viscosity increased with the addition of either DNA or PEO and as such the authors concluded that solutions containing both DNA and PEO had ideal properties for electrospinning. The electrospun DNA/PEO nanofibers displayed diameters of 50–250 nm

Incorporation of DNA into Electrospun Nanofibrous Scaffolds:

**3. Gene delivery: Transfection efficiency**

for the 10 and 12% triblock, respectively) (Fig. 2).

models.

**3.1** *In vitro* **studies**

ultimate stress (by ~1 MPa).

transfection.

Fundamental Characterization Studies and Gene Delivery 387

The aforementioned fundamental studies all dealt mostly with processing parameters and characterization of the DNA nanofibers, either electrospun on with pure DNA or in a mixture with various polymers. Moving past the stage of initial characterization and translating into biological feasibility, the issue of transfection efficiency is key for nonviral gene delivery. This was addressed in a number of studies reported in the literature. Thus, I have divided these manuscripts that focus strictly in generating electrospun scaffolds as gene delivery systems into two categories; those that focus on *in vitro* cell based studies and those that report on implanted scaffolds *in vivo* using various animal

Beyond the critical fundamental studies on DNA and polymer interactions, the first demonstration of the ability of electrospun scaffolds to serve as non-viral gene delivery systems using plasmid DNA (pDNA) was conducted in my laboratory in 2003. We prepared polymer solutions of poly-lactide-co-glycolide (PLGA) mixed with either, 10, 12 or 15% of a poly(D,L-lactide)-poly(ethylene glycol) (PLA-PEG-PLA) triblock copolymer and with pDNA, electrospun multiple scaffolds and evaluated the morphology, material properties, pDNA incorporation and release, and transfection efficiency (Luu et al., 2003). These studies showed that increasing the concentration of tri-block copolymer resulted in the formation of larger diameter (~2.5 – 5.0 m with the 15% triblock) electrospun fibers in comparison to those observed with lower concentrations of block copolymer (250-875 nm and 375-1.1 m

Despite the variation in fiber diameters, no significant differences in material properties were detected. On average these electrospun scaffolds displayed a tensile modulus of about 35 MPa, 45% tensile strength and ultimate stress of 3.3 MPa, matching those of skin and cartilage. We also showed that the addition of pDNA to the fibers did not change the ultimate strain but did significantly increase both the tensile modulus (by ~10 MPa) and

Characterizing the incorporation and release of pDNA showed that ~68-80% of the pDNA that was incorporated, was released in two phases over the 20 day study-period. The first phase constituted of an initial burst, where ~18-36% of the pDNA was released within 15 min of immersion of the scaffold into a saline buffer solution and the second phase was a slow and sustained release over the next 20 days. The initial release burst of pDNA indicated that a substantial amount of the electrospun DNA remained on the surface of the nanofibers with the rest embedded within the nanofibers. The integrity of the electrospun pDNA was demonstrated via agarose gel electrophoresis which revealed that the pDNA was indeed structurally intact and more importantly, its bioactivity was demonstrated by directed transfection in cell culture. Sections of the pDNA containing scaffolds placed in culture medium above a monolayer of pre-osteoblastic (MC3T3) cells were able to release active pDNA and transfect cells with the encoded reporter gene ( galactosidase). The transgene was found to be expressed, albeit, at a very low efficiency in comparison to lipid mediated transfection (Fig. 3). Interestingly, when the released pDNA was complexed with a lipid mediator (Fugene6), the transfection efficiency only reached 20% that of the positive control (Fig. 3), but even 20% is considered a low level of

that became thinner with decreased feed rate (tested a range between 50-200 L/min), increased tip-to-collector distance (10-25 cm range) and thicker with low voltage (5-20 kv range) applied during electrospinning (Liu et al., 2007). As a result of these analyses, it was concluded that these DNA/PEO ultrathin fibers create a 3D porous structure with future potential applications in biosensing as well as cell culturing.

aDetermined by a model 20 pH/conductivity meter (Denver Instrument).

bDetermined by a KSV contact angle analyzer (goniometer, KSV instruments Ltd).

cDetermined by a DV-II viscometer, shear rate using 20 s-1 for viscosity testing.

Reprinted with permission from Liu et al., 2007, Elsevier.

Table 1. Properties of aqueous solutions (100 mL) containing DNA/PEO.

Taking further advantage of the unique optical and electronic properties of DNA and thinking along the lines of using DNA as a candidate for device applications (electronics, filtration, sensors), Ner and colleagues (2008), complexed salmon sperm DNA (molecular weight [MW] 500 kDa) with a cationic surfactant (cetyltrimethylammonium chloride, CTMA), as well as a hemicyanine chromophore and electrospun nanofibers. These nanofibers had an average diameter between 250-350 nm and demonstrated amplified emission of the chromophore when compared to thin films of identical composition. When the material properties of these nonwoven DNA-CTMA fiber meshes were tested, it was found that the average initial modulus was 1.44 MPa with a breaking strength of 37.4 kPa, orders of magnitude lower than those of electrospun fluorescently labelled DNA (15 ± 2 GPa), as previously reported (Bellan et al., 2007). The reason for this difference maybe the presence of the CTMA in the DNA fibers, which were capable of only 11% elongation prior to failure, indicating stress-stiffening that may be due to the particular alignment of the DNA strands along the stress axis or the lateral inter-laminar sliding within the fibers. Although the incorporation of dye did not affect the fibers, it did enhance fluorescence that resulted from both the fiber morphology (5–6-fold amplification) and specific interactions (groove-binding) between the chromophore and DNA (18–21-fold amplification). Based on their data the authors suggest that electrospun DNA nanofibers can serve as advanced functional biomaterial for molecular electronics (Ner et al., 2008).

#### **3. Gene delivery: Transfection efficiency**

The aforementioned fundamental studies all dealt mostly with processing parameters and characterization of the DNA nanofibers, either electrospun on with pure DNA or in a mixture with various polymers. Moving past the stage of initial characterization and translating into biological feasibility, the issue of transfection efficiency is key for nonviral gene delivery. This was addressed in a number of studies reported in the literature. Thus, I have divided these manuscripts that focus strictly in generating electrospun scaffolds as gene delivery systems into two categories; those that focus on *in vitro* cell based studies and those that report on implanted scaffolds *in vivo* using various animal models.

#### **3.1** *In vitro* **studies**

386 Nanofibers – Production, Properties and Functional Applications

that became thinner with decreased feed rate (tested a range between 50-200 L/min), increased tip-to-collector distance (10-25 cm range) and thicker with low voltage (5-20 kv range) applied during electrospinning (Liu et al., 2007). As a result of these analyses, it was concluded that these DNA/PEO ultrathin fibers create a 3D porous structure with future

potential applications in biosensing as well as cell culturing.

aDetermined by a model 20 pH/conductivity meter (Denver Instrument). bDetermined by a KSV contact angle analyzer (goniometer, KSV instruments Ltd). cDetermined by a DV-II viscometer, shear rate using 20 s-1 for viscosity testing.

functional biomaterial for molecular electronics (Ner et al., 2008).

Table 1. Properties of aqueous solutions (100 mL) containing DNA/PEO.

Taking further advantage of the unique optical and electronic properties of DNA and thinking along the lines of using DNA as a candidate for device applications (electronics, filtration, sensors), Ner and colleagues (2008), complexed salmon sperm DNA (molecular weight [MW] 500 kDa) with a cationic surfactant (cetyltrimethylammonium chloride, CTMA), as well as a hemicyanine chromophore and electrospun nanofibers. These nanofibers had an average diameter between 250-350 nm and demonstrated amplified emission of the chromophore when compared to thin films of identical composition. When the material properties of these nonwoven DNA-CTMA fiber meshes were tested, it was found that the average initial modulus was 1.44 MPa with a breaking strength of 37.4 kPa, orders of magnitude lower than those of electrospun fluorescently labelled DNA (15 ± 2 GPa), as previously reported (Bellan et al., 2007). The reason for this difference maybe the presence of the CTMA in the DNA fibers, which were capable of only 11% elongation prior to failure, indicating stress-stiffening that may be due to the particular alignment of the DNA strands along the stress axis or the lateral inter-laminar sliding within the fibers. Although the incorporation of dye did not affect the fibers, it did enhance fluorescence that resulted from both the fiber morphology (5–6-fold amplification) and specific interactions (groove-binding) between the chromophore and DNA (18–21-fold amplification). Based on their data the authors suggest that electrospun DNA nanofibers can serve as advanced

Reprinted with permission from Liu et al., 2007, Elsevier.

Beyond the critical fundamental studies on DNA and polymer interactions, the first demonstration of the ability of electrospun scaffolds to serve as non-viral gene delivery systems using plasmid DNA (pDNA) was conducted in my laboratory in 2003. We prepared polymer solutions of poly-lactide-co-glycolide (PLGA) mixed with either, 10, 12 or 15% of a poly(D,L-lactide)-poly(ethylene glycol) (PLA-PEG-PLA) triblock copolymer and with pDNA, electrospun multiple scaffolds and evaluated the morphology, material properties, pDNA incorporation and release, and transfection efficiency (Luu et al., 2003). These studies showed that increasing the concentration of tri-block copolymer resulted in the formation of larger diameter (~2.5 – 5.0 m with the 15% triblock) electrospun fibers in comparison to those observed with lower concentrations of block copolymer (250-875 nm and 375-1.1 m for the 10 and 12% triblock, respectively) (Fig. 2).

Despite the variation in fiber diameters, no significant differences in material properties were detected. On average these electrospun scaffolds displayed a tensile modulus of about 35 MPa, 45% tensile strength and ultimate stress of 3.3 MPa, matching those of skin and cartilage. We also showed that the addition of pDNA to the fibers did not change the ultimate strain but did significantly increase both the tensile modulus (by ~10 MPa) and ultimate stress (by ~1 MPa).

Characterizing the incorporation and release of pDNA showed that ~68-80% of the pDNA that was incorporated, was released in two phases over the 20 day study-period. The first phase constituted of an initial burst, where ~18-36% of the pDNA was released within 15 min of immersion of the scaffold into a saline buffer solution and the second phase was a slow and sustained release over the next 20 days. The initial release burst of pDNA indicated that a substantial amount of the electrospun DNA remained on the surface of the nanofibers with the rest embedded within the nanofibers. The integrity of the electrospun pDNA was demonstrated via agarose gel electrophoresis which revealed that the pDNA was indeed structurally intact and more importantly, its bioactivity was demonstrated by directed transfection in cell culture. Sections of the pDNA containing scaffolds placed in culture medium above a monolayer of pre-osteoblastic (MC3T3) cells were able to release active pDNA and transfect cells with the encoded reporter gene ( galactosidase). The transgene was found to be expressed, albeit, at a very low efficiency in comparison to lipid mediated transfection (Fig. 3). Interestingly, when the released pDNA was complexed with a lipid mediator (Fugene6), the transfection efficiency only reached 20% that of the positive control (Fig. 3), but even 20% is considered a low level of transfection.

Incorporation of DNA into Electrospun Nanofibrous Scaffolds:

to be greater than 65%.

Fundamental Characterization Studies and Gene Delivery 389

In subsequent experiments we were able to increase the transfection efficiency substantially by plating the cells directly on the electrospun pDNA/PLGA/PLA-PEG-PLA scaffolds (Fig. 4, Liang et al., 2005). This increase in transfection efficiency probably occurred because the cells were physically connected to the surface of the nanofibers where a substantial amount of pDNA was present. As a result, the degradation of the pDNA that probably occurred in our previous study (Luu et al., 2003) where the scaffolds were placed in the culture medium floating above the cells and the pDNA was released into the medium, was eliminated. Unfortunately, we did not conduct a confocal analysis of the scaffolds and thus we were not able to determine the exact transfection efficiency (transfected cells/total cells). Regardless, from the cells on surface shown in these images (Fig. 4), the transfection efficiency appears

Fig. 4. Transfection of MC3T3 cells by nanofibrous electrospun scaffolds. Panels A-D show the increased transfection by plating MC3T3 cells directly on GFP pDNA/PLGA/10% block copolymer scaffolds. (A) Fluorescent image of cells 24 hr post-plating. Arrowheads indicate representative transfected GFP-expressing cells and arrows indicate non-transfected cells. (B) Light micrograph of cells (shown in A) stained with nuclear fast red. Arrowheads and arrows indicate identical cells in A. (C) Fluorescent image of cells on a control scaffold containing no DNA and indicating no fluorescent (green) cells (arrow). (D) Light

micrograph of cells (shown in C) stained with nuclear fast red. Arrow indicates identical cells in C. This transfection efficiency reflected that of cells present only on the exterior surface of the scaffold and not for all cells within the scaffold. Scale bar: 10 m. Reprinted

We also investigated the interactions between the polymers and pDNA using laser light scattering (LLS). Specifically, we determined that in a solution of N,N-dimethylformamide (DMF), the organic solvent used to dissolve the PLGA polymer, pDNA condenses into sphere-like structures and in the presence of the triblock copolymer, the formation of a micellar structure occurs with DNA being encapsulated by a shell made from the PLA-PEG-PLA triblock copolymer. When electrospun, these micelles are deposited randomly on and

with permission from Liang et al., 2005, Oxford University Press.

Fig. 2. Morphology of electrospun scaffolds. Highly porous scaffolds shown under low magnification on left, and high magnification on right. 10% block copolymer formulations (a, b) exhibited thin, fairly uniform fibers (250 to 875 nm). 12% block copolymer formulations (c, d) showed larger fibers and a larger distribution (375 nm to 1.1 mm). 15% block copolymer solutions (e, f) displayed thick, plate-like fibers of roughly 2.5 to 5 mm diameter at the junctions. Scale bar for (a), (c), (e) (left) 10 mm and for (b), (d), (f) (right) 1 m. Reprinted with permission from Luu et al., 2003, Elsevier.

Fig. 3. Bioactivity of released DNA. (a) Naked DNA (2 g) added directly to cell medium. (b) Cells transfected with complexed control DNA (2 g) with a lipid-based transfection reagent (Fugene6). (c) Two 1.5 x 1cm sections of scaffold were directly incubated with cells for 4 h, then removed. (d) Released DNA from scaffold (2 g) complexed with Fugene6. Following transfection (48 hr), cells were fixed and stained for -galactosidase. Scale bar 100 m. Reprinted with permission from Luu et al., 2003, Elsevier.

Fig. 2. Morphology of electrospun scaffolds. Highly porous scaffolds shown under low magnification on left, and high magnification on right. 10% block copolymer formulations

formulations (c, d) showed larger fibers and a larger distribution (375 nm to 1.1 mm). 15% block copolymer solutions (e, f) displayed thick, plate-like fibers of roughly 2.5 to 5 mm diameter at the junctions. Scale bar for (a), (c), (e) (left) 10 mm and for (b), (d), (f) (right) 1

Fig. 3. Bioactivity of released DNA. (a) Naked DNA (2 g) added directly to cell medium. (b) Cells transfected with complexed control DNA (2 g) with a lipid-based transfection reagent (Fugene6). (c) Two 1.5 x 1cm sections of scaffold were directly incubated with cells for 4 h, then removed. (d) Released DNA from scaffold (2 g) complexed with Fugene6. Following transfection (48 hr), cells were fixed and stained for -galactosidase. Scale bar 100 m.

(a, b) exhibited thin, fairly uniform fibers (250 to 875 nm). 12% block copolymer

m. Reprinted with permission from Luu et al., 2003, Elsevier.

Reprinted with permission from Luu et al., 2003, Elsevier.

In subsequent experiments we were able to increase the transfection efficiency substantially by plating the cells directly on the electrospun pDNA/PLGA/PLA-PEG-PLA scaffolds (Fig. 4, Liang et al., 2005). This increase in transfection efficiency probably occurred because the cells were physically connected to the surface of the nanofibers where a substantial amount of pDNA was present. As a result, the degradation of the pDNA that probably occurred in our previous study (Luu et al., 2003) where the scaffolds were placed in the culture medium floating above the cells and the pDNA was released into the medium, was eliminated. Unfortunately, we did not conduct a confocal analysis of the scaffolds and thus we were not able to determine the exact transfection efficiency (transfected cells/total cells). Regardless, from the cells on surface shown in these images (Fig. 4), the transfection efficiency appears to be greater than 65%.

Fig. 4. Transfection of MC3T3 cells by nanofibrous electrospun scaffolds. Panels A-D show the increased transfection by plating MC3T3 cells directly on GFP pDNA/PLGA/10% block copolymer scaffolds. (A) Fluorescent image of cells 24 hr post-plating. Arrowheads indicate representative transfected GFP-expressing cells and arrows indicate non-transfected cells. (B) Light micrograph of cells (shown in A) stained with nuclear fast red. Arrowheads and arrows indicate identical cells in A. (C) Fluorescent image of cells on a control scaffold containing no DNA and indicating no fluorescent (green) cells (arrow). (D) Light micrograph of cells (shown in C) stained with nuclear fast red. Arrow indicates identical cells in C. This transfection efficiency reflected that of cells present only on the exterior surface of the scaffold and not for all cells within the scaffold. Scale bar: 10 m. Reprinted with permission from Liang et al., 2005, Oxford University Press.

We also investigated the interactions between the polymers and pDNA using laser light scattering (LLS). Specifically, we determined that in a solution of N,N-dimethylformamide (DMF), the organic solvent used to dissolve the PLGA polymer, pDNA condenses into sphere-like structures and in the presence of the triblock copolymer, the formation of a micellar structure occurs with DNA being encapsulated by a shell made from the PLA-PEG-PLA triblock copolymer. When electrospun, these micelles are deposited randomly on and

Incorporation of DNA into Electrospun Nanofibrous Scaffolds:

and Wang, 2007, Elsevier.

as an ideal gene delivery system.

Fundamental Characterization Studies and Gene Delivery 391

Fig. 6. The morphology observed in fiber cross sections containing pDNA/chitosan nanoparticles (left panel) and pDNA/chitosan nanoparticles mixed with a PLGA/HAp solution (right panel). This image illustrates clearly the encapsulated pDNA/chitosan nanoparticles (arrows) at the cut section of the fiber. Reprinted with permission from Nie