RESULTADO N° 01.1 (En relación al objetivo específico N° 01):

Th e scaff old architecture at the macro, micro, and nanoscale provides the necessary cues to a successful system, which can then be implanted to repair or regenerate lost tissues. Several fabrication techniques have been used to create highly complex 3D networks for nerve tissue engineering applications. At the macroscale, t he s caff olds h ave to p rovide ide al p ore a rchitectures a nd p orosities, me anwhile at t he microscale allow easy cell penetration, pore interconnectivity, and even allow media infl ux into the 3D spaces. More importantly, it has been understood that the native nerve is composed of oriented collagen fi brils that are 66 nm in diameter,¹¹³ which enhances the surface area for easier cell migration, attach-ment, and other cell surface interactions. Th e potential of nanotechnology-based approaches stems from the similarity at the length scales as well as the ability to functionalize substrates at the cellular microen-vironment. Despite several promising approaches and results at the microscale, several researchers have shown that scaff olds containing nanoscale features promote better nerve regeneration by enhancing the cell material contact, allowing more cells to attach per unit surface area and also show enhanced func-tionalization with growth factors and ECM proteins.

10.3.1 Nanoscale Topographies for Peripheral Nerve Regeneration

Nanoscale topographies on the surface have been shown to enhance the activity of support cells (glial cells) as well provide a suitable path for oriented or guided neuron outgrowth.^2,114 Th ere have been sev-eral approaches to the creation of scaff olds with nanoscale topographies for periphsev-eral nerve regenera-tion (Table 10.1). Some of the most common approaches are electrospinning, micro and nanopatterning, photolithography, e-beam lithography, and self-assembly based approaches.

One of the very fi rst methods to create nanoscale features for peripheral nerve regeneration includes chemical and physical etching. Etching using suitable reagents were used to create patterns on 2D substrates, with feature sizes ranging from 2 to 800 nm,¹¹⁵ based on exposure time and the concentra-tion of the etchant. Some of the most common etchants used are hydrofl uoric acid¹¹⁶ and sodium hydrox-ide. However, most of the systems created using this technique were applicable only to 2D p latforms and have not ye t been totally successful in creating patterns in 3D. Also, owing to t he nature of the technique, it is very diffi cult to create dense patterns on the surfaces and most patterns tend to be either nano or microscale grooves, with high order of repeatability in terms of pitch and feature sizes.

Fan et al., showed the fabrication of nanotopography or surface roughness in the range of 2–800 nm.

A Fluoric acid-based etchant was used to create the nanotopographies on the surface of silicon chips.¹¹⁷ Th e depth and feature size were controlled by the duration of exposure to the etchant. Th ey also reported that a feature in the range of 25 nm produces two orders of magnitude increase in neuronal diff erentia-tion, as compared to feature sizes that are much larger. It was also noted that as the size of the patterns increased (100–200 nm) and even at the microscale (2–5 μm) the response of the neuronal cells reduced indicating the eff ectiveness of the nanotopography to infl uence neuronal cell performance.

Another extension of the above mentioned technique is the application of electrochemical moieties to create specifi c nanoscale patterns on 2D substrates. Most of the substrates prepared by this technique are microscale or nanoscale pores on silicon or ceramic substrates. Moxon et al.,¹¹⁸ showed the ability to create microporous substrates using the above technique on silicon and studied PC12 cell adhesion and advantages of similar features on surfaces. However, this technique is limited to 2D substrates and not yet explored for 3D applications.

10.3.1.1 Micro and Nanopatterning Using Poly(Dimethylsiloxane) (PDMS) Stamps Over the last decade, several other techniques have evolved to create microscale and nanoscale patterns on 3D sub strates. O ne o f t he e arliest te chniques w as t he m icropatterning-based app roach to c reate channels or protein containing patterns on 3D surfaces. In such applications, fi rst a master mold is pre-pared t hat has t he pat tern which can be t ransferred or i mprinted to su rfaces, either i n 2D o r 3D.¹¹⁹ Photolithography-based te chniques, c ommon to s emiconductor app lications, a re u sed to c reate t he master. Common materials used for the fabrication of the master are poly(dimethylsiloxane) (PDMS).^120,121 Th is material is photo-cross-linkable and upon exposure to light, at specifi c wavelengths, through spe-cifi c patterns, it creates patterns, otherwise called stamps. Based on current technology, distinct and clear patterns can be formed with feature size ranging from 50 nm to 20 μm in width.

Miller et al., created microscale patterns on PLLA-based scaff olds prepared by phase sep aration.

Using the adsorption-based technique, they showed the localization of laminin into the microgrooves and studied dorsal root ganglion (DRG) neurite outgrowth in the patterns. Th ey also studied Schwann cell attachment and alignment into the grooves with and without laminin. Th e authors reported that in the presence of oriented Schwann cells and laminin, an enhanced neurite outgrowth was observed, as compared to m icrogrooves without Schwann cells or laminin. Th ey observed oriented neurites in the same direction as the microgrooves, even in the case of shallow micropatterns. Similar results were also reported by Th ompson et al.,¹²² where bovine serum albumin (BSA) was coupled to laminin and cross-linked to microgrooves that were 20 μm wide. Th ey reported that 94% of the Schwann cells oriented along the microgrooves aft er 24 h aft er seeding.

TABLE 10.1 Nanoscale Fabrication for Neural Tissue Engineering Fabrication

Method Feature Sizes Advantages Disadvantages

Phase separation Pore sizes ≥1 nm Good for creating porous scaff olds.

Porosity is easily controlled. Simple, no special equipment necessary

Organized patterning not possible

Polymer demixing Vertical: ≥13 nm Simple, fast, and inexpensive Only sample features can be created (e.g., islands, pits, ribbons) Chemical etching Dependent on etchant

used, time, and many other factors

Simple, fast, and no special

equipment needed Specifi c feature geometries not possible

(near-UV) ≥0.5 nm Can create precise geometries and

patterns Expensive equipment, feature

size is the largest of reviewed methods

Can create aligned fi brous meshes.

Ability to be used with biological polymers such as collagen

10.3.1.2 Electron Beam Lithography

Recent developments in science have led to t he use of electron beam lithography-based techniques to create na nopatterns on surfaces. E-beam lithography creates na noscale features using a h igh energy focused ele ctron b eam b y pat terning o n to ph oto-resist-like m aterials, w hich u ndergo physical a nd chemical changes upon exposure to an electron beam. Th e cross-linking, or changes in physical and chemical attributes is marked by molecular resistance to movement through interlocking of molecules that happens when they come into contact with the e-beam, leads to pat tern formation. Unlike other traditional light-based techniques, this technique does not need a mask to create the pattern. However, a programmable unit is needed to control the beam location on the substrate.

E-beam l ithography a llows t he c reation of pat terns t hat a re 3 –5 nm i n si ze, at a v ery h igh den sity.

However, this technique is not very applicable in cases bulk 3D substrates owing to the limitations of pen-etration depth of the electron beam. Krsko et al.,¹²³ showed the fabrication of nanoscale patterns on a sili-con substrate coated w ith polyethylene g lycol (PEG). Th e PEG i s c ross-linked a s t he ele ctron b eam i s restored on the surface, and the un-cross-linked PEG can be released from the surface by washing in water.

Th e density and the mechanical properties of the gels can be controlled based on the dosage and time of exposure.¹²⁴ Krsko et al., reported that the stiff er the gels were, the greater the attachment and interaction with the cells. Th ey also used an amine terminated PEG and showed the creation of functional surfaces by crosslinking laminin to t he nanoscale patterns.¹²⁵ DRG neurite attachment and neurite extension were studied on the substrate and with increasing pattern density neurite outgrowth reduced. It was observed that growth cones migrated into the scaff olds, especially at regions where no hydrogels were present.

10.3.1.3 Peptide-Based Nanofi bers Produced by Self-Assembly

Peptide and protein-based scaff olds have recently been explored to generate 3D scaff olds for peripheral nerve regeneration. Th e building blo cks a re u sually small p eptides, w hich s elf a ssemble to f orm 3D hydrogel-like constructs. Usually the peptides chosen have a h ydrophilic backbone and hydrophobic heads. Specifi c peptides sequences can be selected to create hydrogels based on given cells or organ of interest. Specifi cally, for PNS applications, a p eptide sequence composed of isoleucine–lysine–valine–

alanine–valine (IKVAV) has shown the ability to promote axonal elongation.

Silva e t a l., s howed t he f abrication o f o ne suc h h ydrogel s ystem, w herein t he he ad g roups w ere composed of IKVAV peptide sequence. Th ey showed that self-assembly takes place when the peptides are introduced into a cell suspension at 37°C owing to ionic or Van der Waals interaction between the peptides.¹²⁶ Th e peptides self-assemble to form a hydrogel-like 3D structure composed of nanofi bers that are few microns in length and a have diameter of 5–8 nm. Th ese hydrogels showed enhanced diff erentia-tion and neurite outgrowth as compared to c ontrol laminin coated controls. Th ey also showed faster rates of diff erentiation as compared to the control scaff olds.

10.3.1.4 Electrospun Nanofi brous Scaffolds for Peripheral Nerve Regeneration

Over the last decade, electrospinning has been regarded as one of the most suitable approaches to fabri-cate scaff olds f or t issue eng ineering app lications. Th e nanofi bers produced t hrough e lectrospinning mimic nat ive EC M, a nd h ave en hanced su rface a rea to p romote c ell at tachment, proliferation, a nd migration.¹²⁷ E lectrospinning i s a te chnique w herein na nofi bers a re p roduced b y t he app lication o f high voltage to a fl owing viscous solution of any polymer. Owing to the fl exibility of the system, scaf-folds c an b e c reated w ith v arying d iameter, o rientations, a nd t hickness o f t he g raft b y v arying t he viscosity of the solution used, fl ow-rate of the process, and even the voltage used. Th e other important feature of electrospinning is the fact that almost all polymers either water soluble or organic solvent soluble can be electrospun. In addition, the electrospinning setup can be modifi ed to collect various fi ber morphologies ranging from braided scaff olds, aligned nanofi bers, random nanofi bers, and even porous nanofi bers. It is also versatile enough to spin fi bers that contain biological compounds such as drugs,¹²⁸ growth factors,¹²⁹ ECM proteins,¹³⁰ and even cells.¹³¹

Gupta et al.,¹³² fabricated aligned and random electrospun PCL/gelatin scaff olds for peripheral nerve tissue engineering applications. Th e nanofi bers hence produced were 240 nm in diameter a nd had a porosity of about 90%, which closely matches the porosity of native nerve tissues. Th ey also observed that on blending gelatin with PCL, they had enhanced mechanical properties as compared to pure PCL-based scaff olds. Th ey studied Schwann cell attachment and proliferation on the scaff olds and reported an enhanced cell attachment and proliferation on gelatin-containing scaff olds as compared to pure PCL-based scaff olds. From the scanning electron micrographs, it was evident that the Schwann cells oriented along the direction of the nanofi bers, in the case of aligned scaff olds and no signifi cant orienta-tion was observed in the case of random nanofi bers.

Koh e t a l.,¹³³ e xperimented w ith t he ele ctrospinning o f l aminin f unctionalized PLLA na nofi bers.

Th ey studied three diff erent methods to i nclude laminin in the nanofi bers such as electrospinning a blend of the polymer with laminin, adsorption of laminin on the nanofi ber template, and covalent bind-ing of laminin usbind-ing basic carbodiimide chemistry. Th e nanofi bers fabricated were in the range of 100–

500 nm and the authors suggested that no signifi cant diff erence in the fi ber diameter was observed when laminin was electrospun w ith PLLA as a blend . On t he evaluation of laminin-loaded content in t he nanofi bers using the three treatments, the author reports that the blended scaff old had a sig nifi cantly higher loading effi ciency as compared to the other two techniques. Th ey also observed that inclusion of laminin i nto t he na nofi bers en hanced P C12 c ells at tachment, p roliferation, a nd d iff erentiation. An increased neurite outgrowth was observed in the case of laminin-blended PLLA scaff olds as compared to other scaff old types, perhaps due to the increased laminin loading in such fi bers. However, no signifi -cant diff erence in cell proliferation was observed between all other scaff old types, incorporated with laminin or even control PLLA scaff olds without laminin.

Bini et al.,¹³⁴ showed the fabrication of braided scaff olds produced by electrospinning aligned PLGA based nanofi bers. Th e nanofi ber system was tested with C 17.2 cells and showed good attachment and proliferation of the cells. However, the authors observed that post diff erentiation, the cells oriented in random directions and did not follow the orientation of the nanofi bers.

Bellamkonda et al.,¹³⁵ showed the fabrication of scaff olds composed of aligned nanofi bers inside a polysolfone nerve guidance channel. Th ey fabricated aligned electrospun nanofi bers from polyacry-lonitrile methacrylate (PAN-MA) that were 400–600 nm in diameter. Th ey also fabricated randomly oriented PAN-MA nanofi bers as controls. Th ese scaff olds were fi rst tested in vitro using primary DRG cells. From their data, it was evident that the neurites as well as the Schwann cells that migrated from the DRGs, elongated in the direction of orientation. In the case of random nanofi ber no signifi cant direction w as ob served. I n t he c ase o f a ligned na nofi brous scaff olds, a lo nger neu rite leng th w as observed as compared to scaff olds with random nanofi bers. Th ey also reported the application of the same type of scaff old in v ivo. It w as ob servable t hat t he s caff olds t hat h ad t he a ligned na no fi bers showed improved regeneration at the proximal and distal ends. Meanwhile the scaff olds that had the random nanofi bers showed regeneration only closer to the proximal ends and minimal regeneration at the distal end.

Chew et a l.,¹³⁶ s howed t he i mportance o f a lignment o n g lial c ell at tachment, p roliferation, a nd maturation. E lectrospun P CL fi bers, w ith either a ligned or r andom orientation were f abricated a nd evaluated for Schwann cell attachment, proliferation, and gene expression. Even though the fi bers they produced had a diameter around 1 μm, the eff ect of fi ber orientation on Schwann cell maturation was apparent. From their data, it is evident that on aligned and random fi ber scaff olds the expression of vital factors such as NGF, GDNF, and MAP were higher as compared to thin fi lm-based scaff olds.

From the previous examples, it is understandable that electrospinning can yield scaff olds that have a substrate that mimics native ECM in terms of alignment, architecture, and even in terms of porosity and mechanical properties. However, it is extremely challenging to create 3D scaff olds f rom electrospun nanofi bers with optimal mechanical properties. In order to m itigate this drawback and also enhance surface areas available for cell attachment, spiral shaped scaff olds were developed by coating aligned or random nanofi bers onto PLGA microsphere-based sintered scaff olds.³⁹ Th e sintered scaff olds provide

necessary mechanical stability (tensile properties) to p revent collapse of t he scaff old, meanwhile t he spiral shape enhances surface area. Th e open scaff old architecture allows media infl ux into and waste removal from the scaff old.

Th ere are several other references indicating the applicability of the electrospinning process to neural tissue eng ineering applications, especially for f unctionalization, g rowth factor release, DNA release, which have been discussed in the appropriate sections in the text below.

10.4 Nanotechnology Platforms for Controlled Release