Arterial Smooth Muscle Cell Proliferation on a Novel Biomimicking, Biodegradable Vascular Graft Scaffold

JOEL D. STITZEL,1 KRISTIN J. PAWLOWSKI,1 GARY E. WNEK,2 DAVID G. SIMPSON3 AND GARY L. BOWLIN1,*
1

Dept. of Biomedical Engineering, Virginia Commonwealth University, P.O. Box 980694, Richmond, VA 23298-0694 2 Dept. of Chemical Engineering, Virginia Commonwealth University, P.O. Box 843028, Richmond, VA 23298-3028 3 Dept. of Anatomy, Virginia Commonwealth University, P.O. Box 980709, Richmond, VA 23298-0709 INTRODUCTION

CI.D.) vascular grafts are insufficient to ensure long-term success of

urrent approaches to small diameter (<6 mm inner diameter,

the graft in vivo. Part of the reason for this is that the current grafts (synthetic polymers) are permanent fixtures in the body, generating tissue ingrowth and fibrosis, but never degrading. The goal of this research is to develop a small diameter vascular construct whose matrix will degrade as it is replaced in vitro and in vivo by native tissue. Furthermore, the construct is predesigned to mimic the natural structure of an artery so that upon implantation, the cells dwelling in the tissue will have to perform minimal remodeling of the extracellular matrix. The vascular construct being investigated was fabricated with collagen fibers wound on a stainless steel mandrel and covered with biodegradable polymer (100 L polylactic acid) (PLA). The biodegradable polymer serves as

*Author to whom correspondence should be addressed. E-mail: glbowlin@saturn.vcu.edu

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JOURNAL OF BIOMATERIALS APPLICATIONS Volume 16 July 2001

1530-8022/01/01 0022-12 $10.00/0 DOI: 10.1106/U2UU-M9QH-Y0BB-5GYL 2001 Technomic Publishing Co., Inc. Journal Online at http://techpub.metapress.com

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the initial structural components of the graft as well as scaffolds on which to seed arterial smooth muscle cells (SMCs) during the formation of the media component. The collagen fiber and matrix is designed to create a residual stress environment similar to that found in native arteries, which, as presented in this paper, encourages SMC orientation along the principal stress directions of an artery. In the body, SMCs in arteries have been shown to be arranged so as to distribute evenly the principal stresses in the tissue. It has been shown that SMCs in both statically and dynamically stressed tissues will show a bipolar spindle shape due to the strain, with spindles aligned parallel to the direction of strain [1]. Arteries, of course, are organized into three recognizable layers: the tunica interna, the tunica media, and the tunica adventitia. It is the tunica media that makes up most of the thickness of the small arteries and is responsible for its mechanical properties in the physiological range of strain [2]. SMCs in the media of elastic arteries of the body are arranged helically to oppose the stresses exhibited both by blood pressure and the stresses imposed by the architecture of the vasculature. The arrangement of these elastic vessels has been shown to be one of cylindrical layers of SMCs and elastic laminae, with different pitches in different layers of the vessel (anisotropic material) [3]. The high pitches of inner and outer layers of the media function to control vessel length changes, while the low pitches in inner layers serve as a regulator of vessel lumen diameter. Gershon et al. [4] developed an arterial prosthesis by filament winding that resembles the biomimicking graft scaffold being attempted in the current work except for the materials used. Gershon et al. used Lycra fiber with a Pellethane matrix; current work is being attempted with collagen fibers and a biodegradable PLA matrix (composite structure). The advantage of filament winding of a prosthesis is that one can control the material properties in different directions by controlling the pitch of the embedded fibers, creating what is essentially a composite material which mimics the anisotropic nature of an artery. The fiber can also be tensioned as it is fed onto the mandrel, in order to create some residual tension in the wall of the construct. This residual tension mimics the tension seen in a native arterysimilar to the zero-stress state pointed out by Fung and others [5]. When a circular cross section of a native artery is cut open, it unfolds, revealing its zero-stress state and the residual tension existing in the artery before the cut. The underlying mechanism creating the residual tension is different in the scaffold being presented, but the tension itself still creates a tendency for the graft to unfold if a circular ring is cut open. Annis et al. [6] developed a winding technique using electrostatic

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spinning of a polyurethane solution onto a rotating mandrel. Electrostatic spinning of a solution has the advantage of a rather simple setup, and fiber diameters may be produced down to the nanometer size, smaller than is possible with most other fiber spinning techniques. The electrospinning of biodegradable polymers (i.e., PLA) can be attempted as easily as with polyurethane in solution by controlling some of the parameters involved in the process, with polymer concentration, surface tension, and viscosity being some of the major ones. Morphology and porosity can also be controlled, as well as composition. In electrospinning, polymer solutions or melts are deposited as fibrous mats, taking advantage of the polymer chain entanglements which exist in sufficiently high polymer concentrations in solution to produce continuous fibers. The basic setup required includes a high voltage power supply, a ground and source electrode, and a polymer solution to be spun. The polymer solution is then spun from a nozzle, such as a pipette tip, with controlled delivery, such as through a syringe pump, and splaying of the initial jet leads to the formation of very thin fibers. The fibers are collected, in the case of a vascular graft scaffold, onto a stationary or rotating mandrel, though any grounded metal surface will collect fibers. Examples of some polymers that have been electrospun include poly(hydroxybutyrate-co-valerate) [7], poly(ethyleneoxide) [8,9], DNA [10], and poly(tetrafluoroethylene) [11]. The purpose of the current research was to create a biomimicking graft scaffold with collagen fibers for a reinforcement material and a biodegradable matrix material (composite structure). The collagen fibers are wound using a winding apparatus for variable pitch mimicking the SMC structure in an artery, and a biodegradable PLA matrix is spun around this fiber for SMC seeding and to hold the fibers in place during tissue regeneration. The construct was seeded with human aortic SMCs, and the resulting graft was analyzed using scanning electron microscopy (SEM) and histology to determine whether SMCs responded to the residual stress environment they were placed in when seeded on the scaffolding. A control scaffold with no fiber reinforcement (and therefore no residual stress) is included to study the difference, if any, between general SMC alignment and orientation in the control vs. the composite scaffold. SEM analysis was also performed to examine the ultrastructure of the graft resulting from the fabrication process of layering collagen fiber and PLA using a fiber winding apparatus and electrostatic spinning.
MATERIALS AND METHODS

The current approach involves mimicking the architecture, to an

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extent, of the media of an elastic artery. Since SMCs in the vascular wall are arranged helically to oppose the stresses (circumferential and radial) imposed by the circulation of blood and the constant longitudinal tension arteries are under in the body, we created a preliminary graft scaffold (final scaffold will be multi-layered) with a single helical wind of collagen fibers (100250 m diameter; Orion, Brazil) embedded in biodegradable matrix. The biomimicking mechanical environment of the graft is engineered to be favorable for appropriate tissue regeneration and remodeling. Human aortic SMCs (passage 810) were then seeded onto the graft. To create the preliminary graft scaffolds, a layer of 100L PLA biodegradable polymer (Alkermes Medisorb, Cincinnati, OH) dissolved in chloroform at 14 w/v% (0.14 g/mL) was electrospun onto a 4 mm diameter stainless steel mandrel. The mandrel was then placed into the winding apparatus in Figure 1, which consists of a fixture for rotating a mandrel and a cylindrical linear actuator guide that are each turned by a stepper motor. The motors have independent speed control, so by altering the winding speed and traverse speed virtually any pitch (175 from radial) of the fiber can be wound [12]. Collagen fibers were then wound helically onto the mandrel, and the

Figure 1. Fiber winding apparatus showing the 4 mm stainless steel mandrel being wound with a silk suture.

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resulting incomplete construct was overlaid with electrospun PLA. The resulting matrix (Figure 2) was composed of fine fibers (~10 m diameter) of electrostatically deposited PLA that are ideal for endothelial (neointimal) and SMC seeding and also serve as the matrix portion of a fiber/matrix composite. The composite structures were then wetted with 100% ethanol for 30 minutes, rinsed once with sterile water, and placed in fresh sterile water for 30 minutes. The wetting procedure allows effective seeding of the interstices of the matrix as well as simultaneously sterilizing the matrix/fiber structure. It has been demonstrated that biodegradable foams of hydrophobic polymers which are normally resistant to infiltration by water into their pores can be effectively wetted by immersion in ethanol followed by immersion in water [13]. This results in a final material which has over 90% of its void volume sterilized and filled with water. This is a necessary condition if one is to seed cells on and/or within the fibrous structure. After wetting, the grafts were soaked in 100 g/mL human fibronectin (Collaborative Biomedical Products, Bedford, MA) in phosphate buffered saline for 20 minutes, and seeded with human aortic SMCs in a small volume (~1.5 mL at 1.5 106 SMC/mL) of culture medium contained in a 1.5 mL cryovial (horizontal) for 1 hour turning at 1/8 rpm. The seeded scaffolds

Figure 2. Illustration of the random electrospun PLA structure (matrix) of the graft scaffold before cell seeding.

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were then cultured under static conditions in a T-25 culture flask on end containing 10 mL of culture media. The culture media was changed every 2 days and was composed of 66% DMEM (GibcoBRL), 22% F-12 HAM (GibcoBRL), 9% fetal bovine serum (FBS, GibcoBRL, Rockville, MD) and a 2% Penicillin-Streptomycin-Fungizone mixture (BioWhittaker, Walkersville, MD), composed of 10,000 Units penicillin/mL, 10,000 Units streptomycin/mL, and 25 UG amphotericinB/mL. The seeded scaffolds at set time periods were removed from culture and placed in 10% formalin and processed appropriately for light [Hematoxylin and Eosin (H&E) staining] and scanning electron microscopy (JSM-820; JEOL, Ltd).
RESULTS AND DISCUSSION

A scanning electron micrograph of the fibrous structures resulting from this preliminary work is shown in Figure 2. This is a close-up of the fibrous structure resulting from electrospinning the 14 w/v% (0.14 g/mL) 100L PLA in chloroform. Since the electrospinning was performed onto a stationary mandrel, the resulting matrix fiber structure is highly random. The nearly constant 10 m diameter of the fibers

Figure 3. Cross-sectional view of the biodegradable fibrous matrix and collagen fiber contained within composite structure.

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is also apparent. The voids in the fibrous structure vary anywhere from 12 micrometers to a hundred or more micrometers. Future studies will examine the porosity of the resultant scaffolds as a function of electrospinning process parameters. The layered architecture of the electrospun PLA and a single collagen fiber can be seen in Figure 3. The single luminal layer can be seen at the top of the micrograph, followed by a single wind of the collagen fiber (~200 micron diameter) and multiple layers of electrospun PLA on top of that (toward the bottom of the micrograph). The separation between layers is due to the momentary stopping of the electrospinning procedure in order to clean the nozzle delivering the polymer solution, and the wide gaps between layers are seen only because of processing the sample for SEM. It is evident that the thickness of the layers can be controlled by varying the spinning time. Again, the fibrous structure is very randomly oriented, with the same uniform fiber diameter seen previously. Speculation by the authors is that this layering may be another key to developing a successful small diameter tissue engineered vascular prosthetic, due to formation of mimicking laminae instead of one continuous media layer. A cross section of a graft (Figure 4) created with a PGA/PLA (50:50 copolymer, Boehringer Ingelheim, Germany) matrix nevertheless

Figure 4. SEM illustrating the tendency of collagen fibers to spring outward and uncoil if not properly encased in matrix material. This is shown to demonstrate the residual stress of the fibers after the winding process.

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displays the tendency of the fibers embedded in the matrix to exert an outward force on the matrix and, hence, protrude out of the scaffold. For this reason, the initial layer (Figure 3) of electrospun matrix was placed on the mandrel prior to collagen fiber winding to anchor the fiber within the matrix. The outer layers of the PLA must take up the force exerted by the fiber, resulting in a matrix structure that is stressed simply by its enclosure of the coiled collagen fiber. The SEM in Figure 4 demonstrates an inherent static property of the grafts fabricated in this fashion. Without the PLA matrix to hold the fiber in place, the fibers would unwind to relieve the tension imposed during winding and have a tendency always to uncoil in place, as evidenced by the behavior of the single uncovered strand in Figure 4. A preliminary SMC seeded scaffold held under static culture conditions is pictured in Figure 5. The graft was fixed in formalin after 10 days of culture, but, already, SMCs exhibit their response to the stress created by the fiber. One can see the bipolar spindle shaped SMCs aligned approximately along the direction of the large collagen fiber and proliferated across the outer surface of the graft. Though the alignment is not perfectly along the fiber, a distinct regularity in alignment of the SMCs is apparent. It is the outward force exerted by the fiber that is thought to contribute to the alignment and generally regular pattern of SMCs found on the surface of the graft. A similar morphology can be observed on the luminal surface of the scaffold as well (not pictured). The bulging of the fiber within the matrix is partially due to its size and also due to the outward force exerted by the fiber. Histological cross sections of the graft demonstrate some cell infiltration and a monolayer of cells are evident on the outer surface of the graft. Figure 6 shows an SEM of the same surface, at higher magnification. The orientation and alignment of SMCs is more readily apparent, with the fiber direction from top left to bottom right as shown in Figure 5. Note: the separation or gaps between the cells on the micrographs are most likely an artifact of the drying process for SEM analysis. Figure 7 shows the random orientation of SMCs when there is no large collagen fiber to exert a force on the PLA matrix structure. The matrix structure photographed was fabricated with no reinforcing fiber material and is essentially a tube with walls composed of PLA fibers of the characteristic 10 m diameter and random orientation. This matrix was seeded with SMCs and cultured for 30 days under static conditions. The smooth muscle cells have clearly invaded some of the porous structure of the matrix; however, their orientation is random. In comparison, the SMCs are much less organized and aligned in this state than those seen on the composite scaffold (Figures 5 and 6).

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Figure 5. External surface view of smooth muscle cell proliferation/orientation (10-day incubation period) on the fiber-reinforced biodegradable scaffold.

Figure 6. SEM demonstrating the smooth muscle cell proliferation/orientation (10-day incubation period) on the matrix scaffold reinforced with fiber.

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Figure 7. SEM illustrating the smooth muscle cell proliferation/orientation (30-day incubation period) on randomly oriented matrix scaffold (with no reinforcing collagen fiber contained within).

CONCLUSIONS

From the preliminary SEM analysis, it has been shown that PLA can be deposited onto helically wound collagen fibers, and the resulting composite structure has mechanical integrity. An initial prototype graft scaffold (composite), like a native artery, exhibits an opening angle when a circular cross section is sliced open. The residual stress which exists before the section is cut is a possible indicator of favorable initial mechanical properties prior to implantation and exposure to physiologic stresses. This hypothesis is based on the fact that the stress state of an artery affects SMC orientation. By creating a matrix architecture that mimics that of a native artery out of biodegradable materials, one can positively influence the alignment of SMCs before implantation, improving the speed with which the graft can be incorporated into the vasculature (minimal tissue reorganization) and consequently the probability of long-term success of the graft. This residual stress found in the composite scaffold mimics that resulting from a static pressurization of the graft or the stress state which exists in a closed section of artery. However, the graft does not have to be pressurized during incubation,

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since the fiber creates the stress. After 10 days of incubation, the seeded scaffold shows extensive (a monolayer of 100% confluence) SMC proliferation and general orientation along the principal stress lines. Histology shows an even SMC distribution within the scaffold matrix wall with the majority of cells found on the luminal and external surfaces. A control graft scaffold with unoriented PLA matrix fibers, but no reinforcement collagen fiber, exhibits a much more random orientation of SMCs than the fiber/matrix composite graft scaffold. Further variations in construct processing (structure and composition), numerical modeling, and mechanical testing will be performed utilizing the basic premise described in the paper to reach the end goal of a viable small diameter (<6 mm I.D.) arterial vascular graft.
ACKNOWLEDGMENTS

The authors would like to thank the A. D. Williams Fund and the Whitaker Foundation (RG-98-0465) for their support of this research.
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