Living Scaffolds (Specialized and Unspecialized ... - ACS Publications

March 2008

Published by the American Chemical Society

Volume 9, Number 3

 Copyright 2008 by the American Chemical Society

Reviews Living Scaffolds (Specialized and Unspecialized) for Regenerative and Therapeutic Medicine Sumathy Arumuganathar and Suwan N. Jayasinghe* Department of Mechanical Engineering, University College London, Torrington Place, London WC1E 7JE, United Kingdom Received November 29, 2007; Revised Manuscript Received December 18, 2007

The physical sciences have increasingly demonstrated a significant influence on the life sciences. Engineering in particular has shown its input through the development of novel medical devices and processes having significance to the biomedical field. This review introduces and discusses several fiber generation protocols, which have recently undergone development and exploration for directly handling living cells from which continuous cell-bearing or living threads to scaffolds and membranes have been fabricated. In doing so these protocols have not only demonstrated their versatility but also opened several unique possibilities for the use of these scaffolds in a plethora of biological and medical applications. In particular, these living fibrous structural units could be explored for regeneration purposes, e.g., from accelerated wound healing to combating a wide range of pathologies when coupled with gene therapy. Thus, “living entities” such as these scaffolds could be most useful in surgery/medicine, including its exploration with stem cells for the preparation of unspecialized living scaffolds and membranes.

1. Introduction Fibers are interesting structural entities having a wide range of applications in our everyday lives (from clothing to filtration applications, etc.). Scientific literature contains reports where collection of these continuously generated fibers can form scaffolds to membranes. Recently, these fibrous morphologies have been shown to have many bleeding edge applications ranging from advanced controlled and targeted drug delivery mechanisms to the forming of tissue engineering scaffold materials, with tremendous applicability to the biomedical field.1–3 There are several routes for forming fibers, for example, non-needle assisted and needle assisted. One such non-needleassisted route employs a rotating disk for spinning fibers by the deposition of a polymer at the center of the rotating disk. Subsequently, by way of centrifugation, the polymer is emitted as fibers (exploiting the viscoelasticity nature of that polymer) from random locations on the rim of the rotating disk.4 Another approach for forming fibers is by the drawing of a fiber out of a resting polymer droplet by picking the edge of the polymer droplet followed by stretching, resulting in the formation of a fiber.5 These techniques are simplistic and have been demon* Corresponding author. E-mail: [email protected].

strated to generate fibers in the micrometer range. However, these methods have two associated problems, namely, process control and scaling-up, which are not feasible from these random and manual routes, respectively. Studies on these fiber-generation methodologies have not demonstrated the ability to draw out composite nanosized fibers containing advanced materials (for example, structural, functional, or biological). Contrary to the non-needle techniques, the needle-assisted routes have demonstrated great advancements in this endeavor. There are essentially three needle-based continuous fiber preparation routes, which have been elucidated for forming advanced composite fibers containing materials. These are electrospinning (ES),6,7 aerodynamically assisted threading (AAT),8,9 and, very recently, pressure-assisted spinning (PAS).10,11 In this review the authors will endeavor to discuss the advantages and disadvantages of each needle-assisted fiber generation method in comparison to each other, followed by a discussion of their applicability to the life sciences.

2. Electrospinning (ES) This is a fiber preparation route that has been explored over many decades.12,13 Briefly, ES derives from electrosprays,14,15

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760 Biomacromolecules, Vol. 9, No. 3, 2008

Arumuganathar and Jayasinghe

Figure 1. (a) Digital image of the cell-electrospinning setup in a laminar flow safety cabinet. (b) A schematic representation of the cell electrospinning process in action.

hence exploring a charged needle having an applied voltage in the thousands of volts with respect to a grounded electrode placed centrally and in-line with the needle exit (a similar setup is explored for electrospray studies). Figure 1 demonstrates a modified setup for electrospinning that employs a coaxial or concentric needle arrangement, which promotes the encapsulation of materials.16,17 Electrospinning, unlike electrosprays, explores the applied electric field for drawing out a viscoelastic medium that subsequently forms a continuous uniaxial fiber. The latter forms a jet, which shortly undergoes instabilities forming charged droplets. Both these electrified jetting and threading protocols have been demonstrated for forming microto nanosized residues with great versatility.18–22 Although ES has been carried out for several decades, recent reinvestigations reviving this technique have produced some excellent improvements to fiber modifications such as the ability to form composite (containing a wide range of nonconducting and conducting materials), porous fibers to those having a controlled orientation.23–27 Having said that, in 2006 Jayasinghe et al.,28,29 pioneered the ability to directly electrospin living cell containing threads from which biologically active scaffolds to membranes were derived (Figure 2). This discovery, the ability to directly

electrospin living cells, is now referred to as “cell electrospinning”. Although cell electrospinning is a unique protocol for generating cell-bearing fibrous structures, it should be noted that the media properties such as viscosity and electrical conductivity play a pivotal role. Hence, follow-up developmental studies are currently investigating the control of these properties to the cell behavior when in suspension as a function of time, which have demonstrated to have an affect on the generated fibers showing a nonuniformity of cell placement along threads. In addition the authors are simultaneously studying the post-treated cells in comparison to controls at a genetic, genomic to physiological level. In parallel with these recent developments electrosprays were investigated with a specially tailored living siloxane sol.30–32 These studies have enabled electrosprays to compete with electrospinning in the fiber generation race. Interestingly one advantage that electrospray-based fiber generation has over electrospinning is the ability to site the fibers in a desired location. If this is required from electrospinning, a prefabricated template to control conducting plate arrangement would be required. Although both protocols have been demonstrated to promote the fabrication of micro- to nanosized fibers allowing a wide range of applications, one must note that the applied

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Biomacromolecules, Vol. 9, No. 3, 2008 761

Figure 2. Characteristic (a) optical and (b) florescent microscope images of the generated cell-bearing scaffolds/membranes prepared by way of cell electrospinning.

voltage, which is generally in the thousands of volts, could be considered a hazard to the operator (Table 1).

3. Aerodynamically Assisted Threading (AAT) AAT is a process where a pressure differential between a pressurized chamber and the surrounding atmosphere provides the aerodynamic forces for drawing out a viscoelastic medium

through an exit orifice, thus forming a continuous thread to scaffold. The process has been explored in a coaxial form for forming compound threads with liquid–liquid, liquid–gas systems and very recently with suspensions.8,33 A closely related technique to AAT, aerodynamically assisted jetting, was recently demonstrated for the process fabrication of structural and functional particulate materials from suspensions.34–37 Notably this process was later adopted for process handling living cells



Table 1. Characteristics of the Direct Cell Threading Protocols Discussed in This Reviewa protocol cell electrospinning/ electrospraying coaxial aerodynamically assisted biothreading pressure-assisted cell spinning a

driving mechanism electric field aerodynamic flow field applied pressure

hazards

processable cell density, cells/mL

mechanism for controlling fiber orientation

processable biopolymer viscosity, mPa s

electric field

∼107

electric field

g12500

–

∼107

mechanical

∼10000

–

∼10

mechanical

∼10000

7

N.B. The reader should note that this is only a guide and these characteristics could vary as a function of the cell type or types (as they vary in

diameter) to the biopolymer explored.

Figure 3. Typical digital images of (a) the coaxial aerodynamically assisted bio-threading equipment set-up in a class II safety cabinet, (b) a close-up image of the coaxial aerodynamically assisted bio-threading device and (c) a schematic representation of the threading process.

in both single (aerodynamically assisted bio-jetting) and coaxial needle (Figure 3) configurations.38,39 This technique subsequently in its coaxial configuration was coupled with a medical grade viscoelastic medium for encapsulating living primary cells for forming continuous active fibers to scaffolds and membranes (Figure 4). This methodology is known as “coaxial aerodynamically assisted bio-threading”.40 In comparison to both electrified threading techniques this process removes the hazardous high voltage and applied electric field. It is noteworthy that unlike in the case of ES, in these AAT studies the rheological properties

play a prominent role while the electrical conductivity of the media has no influence on this threading protocol. However this process has difficulties in conceiving the generation of nanofibers (Table 1).

4. Pressure-Assisted Spinning (PAS) Pressure-assisted spinning evolves from its jetting counterpart, which has been previously explored in a wide range of pneumatic applications.41 It should be noted that the inner or the outer needle


Figure 4. Representative florescent image depicting the cell-bearing scaffold/membrane prepared by way of coaxial aerodynamically assisted bio-threading.


could hold the flow of the applied pressure while the other held the flow of the jetting or threading medium. Here fibers are generated by the modulation of the media’s rheological properties. It is important right at the onset to distinguish that this jetting or threading protocol requires two needles for jetting or threading a single or multiphase medium.10,11 For encapsulation the process requires three concentric needles42 (Figure 5) in which either the innermost or the outermost accommodates the applied pressure while the encapsulating or driving medium is in the outer needle. This setup has been shown to promote the formation of composite fibers from which structures from miscible and immiscible media have been generated (Figure 6). Much like both ES and AAT, the process not only has been explored for its ability for handling structural and functional micro- to nanoparticulates in suspension but also has been extended for handling living primary cells for forming biologically viable scaffolds to membranes (Figure 7).43 This technique has similarities and advantages with and over AAT as its driving mechanism is an applied pressure; however it does not require a pressurized chamber (Table 1).

Figure 5. Characteristic digital images of (a) the pressure-assisted cell spinning equipment setup within a laminar flow safety cabinet and (b) a close-up of the three concentric needle pressure assisted device.



Figure 6. Typical (a) digital image of a prepared composite scaffold/membrane and (b) a scanning electron micrograph of the composite fibrous structure.

Figure 7. Representative florescent micrograph depicting the cellbearing scaffold prepared by way of pressure-assisted cell spinning.

to the simultaneous threading of several primary cell types with a biopolymer — giving birth to a novel methodology for directly forming biologically viable microenvironments. These posttreated cells have been assessed for their cellular viability by means of flow cytometry which demonstrated a significant population of viable cells. However, these post-treated cells are currently undergoing investigation from a make-up level upward with comparison to controls over both short and long time periods. The authors see that these protocols as applied to healthcare technologies will have tremendous implications for a wide range of biomedical applications, for example, exploration of these novel biomicrofabrication techniques in addressing problems in the area of personalized medicine (e.g., regenerative and therapeutic medicine to medical diagnostics). Interestingly, these techniques remove a fabrication stage required for directly forming biological microenvironments containing living cells as previously explored in tissue engineering studies.

5. Possible Applications in Healthcare In a biological standpoint it is interesting to note that in processing living primary cells in either of these protocols we have found a cellular viability of g70% in comparison to controls by way of flow cytometry. Hence, our developmental studies to date with these three bio-threading protocols not only have been carried out with the threading of a biopolymer and a single primary cell type in suspension but have been extended

5.1. Regenerative Medicine. Tissue engineering44,45 and regenerative medicine are areas that recently not only received much press coverage within the scientific community but also has reached the public’s attention. The reason for this is directly due to the steeply rising demand in donor tissue, which is required for biorepair in surgery. The current demand for such biomaterials is approximately three-fold greater than the supply


and rising. Hence addressing such a demand could be tackled through protocols that possess the ability to build such cellular structures. Therefore, protocols such as those highlighted in this article present some interesting possibilities for the direct in vivo fabrication of cell-bearing biologically viable tissues. These protocols to some extent have already been shown to have the ability to directly form scaffolds/membrane containing specialist cells hence giving rise to a specialized tissue. The association of these threading protocols with stem cells could prove to be not only challenging but an exciting prospect where unspecialized tissue could be directly derived. These investigations are currently well underway where all the biological and engineering aspects are being investigated in great detail for assessing cell differentiation to the formation of cellular networks while cells are in scaffold. Such structural entities could also be investigated for the development, screening, and discovery of drugs. It should be noted that these processes will take time to establish; having said that the promises these protocols offer tissue engineering and regenerative medicine are truly exciting.46 5.2. Medical Therapeutics. Along with the rising demand in tissue for regenerative purposes, medical therapeutic applications are gaining an equal amount of interest as this addresses a wide range of therapeutics47,48 for combating pathologies from the cellular level upward. The techniques discussed in this review demonstrate the fabrication of cell-bearing scaffolds to membranes by way of encapsulating cells containing a bioluminescent, for, e.g., GFP (green florescent protein). This brings out an interesting point, which promotes the possibility of coupling gene therapy with these threading protocols, hence implying that these scaffolds could have a therapeutic element. Therefore addressing a specialized tissue pathology could be carried out by simultaneously threading multiple cell types which could all be put through gene therapy first by transfecting with that respective vector subsequently fabricated into a living scaffold, which could be applied to that tissue site. In coda, these protocols offer some unique possibilities for tackling a wide range of cellular and tissue pathologies through these swiftly emerging novel therapeutic protocols.46

6. Future Challenges Although the discussed threading protocols have some exciting possibilities for regenerative and therapeutic medicinal areas of research, there is much investigation that needs to be carried out before these protocols are taken to the next stage. The authors’ believe that these protocols must follow the listed path for truly assessing their potential in a clinical setting. These could be categorically segregated as biological and physical investigations as follows: The biological investigations will need to fully assess the cellular viability of threaded cells from a genetic, genomic to physiological level through studies such as gene expression, spectral karyotyping to many assay-based type assessment protocols where the post-treated cellular make-up is investigated in close comparison to several controls (untreated and those that have been passed through the respective devices without the application of their driving mechanisms) over a function of time. It is first important to assess cellular viability as this will address the most important issue with respect to whether these protocols promote any cellular ailments to deterioration posttreatment (for example, such as cancer, etc.). If these studies prove that those post-treated cells in comparison to their controls are failing to demonstrate any cellular ailments from a makeup level upward, engineering aspects could then be initiated.


The engineering or physical science part will need to develop fine control of the cellular suspension properties, which will give rise to uniform placement of encapsulated cells along the length of the fabricated thread. This is most important in a therapeutic sense, as those scaffolds prepared for addressing cellular/tissue ailments will require the release of such therapeutic payloads in a targeted and precisely controlled manner. Hence, the threads to scaffold/membranes should be free of any cellular clusters or cell agglomerates. The encapsulating biopolymer could also possess features such as the ability to act as a polymeric barrier, which contains the living cells while efficiently maintaining their intricate metabolisms. This will be modulated by the barrier allowing only selected molecules (nutrients) to enter while cells’ waste is diffused through the barrier. Furthermore, the polymer employed could also have the ability to disintegrate at a known time point, which will enable the effective release of therapeutic agents through these transfected cells to that respective site. If all these studies prove to be a success, these protocols will enter exploration with a wide range of stem cells for the direct in vivo generation of unspecialized tissues for both tissue regeneration and cell/tissue therapeutics. Acknowledgment. S.N.J. specially thanks the Royal Society (through both seed-corn research and Wolfson Laboratory Refurbishment grants) and the Engineering and Physical Sciences Research Council (EP/D506964/1, funding a research assistantship for S.A.) both of the U.K. for funding this research at UCL.

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Living Scaffolds (Specialized and Unspecialized ... - ACS Publications

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