Biomaterial-Induced Angiogenesis To Address Peripheral Vascular

Chapter 15

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Biomaterial-Induced Angiogenesis To Address Peripheral Vascular Disease: The Application of Sphere Templated Hydrogels Dale Terasaki,2 Michael Sobel,3 Colleen Irvin,2 Errol Wijelath,3 and Buddy D. Ratner*,1,2 1University

of Washington Engineered Biomaterials (UWEB), University of Washington Seattle, Washington 98195 2Department of Bioengineering, University of Washington, Seattle, Washington 98195 3Department of Surgery, University of Washington, Seattle, Washington 98195 *E-mail: [email protected].

Peripheral arterial disease (PAD) is the atherosclerotic arterial occlusive disease of the legs that leads to muscle ischemia, disability, and limb loss. Therapeutic approaches that can restore circulation to ischemic limbs are needed. Here we explore the potential of a sphere-templated hydrogel scaffold with 30 micron diameter interconnected pores to induce an angiogenic response. A variant of this scaffold with 60 micron diameter parallel channels in addition to the 30 micron spherical pores was studied to determine if larger vessels might form in the larger channels. As a control, expanded polytetrafluoroethylene (PTFE) was studied. The three types of porous materials were implanted in mouse muscle for two weeks. The scaffold with 30 micron spherical pores showed the highest level of angiogenesis. However, only capillary-dimension blood vessels were seen in all materials.

© 2013 American Chemical Society In Tailored Polymer Architectures for Pharmaceutical and Biomedical Applications; Scholz, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Introduction Peripheral arterial disease (PAD) is the atherosclerotic arterial occlusive disease of the legs that leads to muscle ischemia, disability, and limb loss. According to a 1999-2004 study, 12.2% of adults over the age of 60 suffered from PAD (1). Significant risk factors associated with PAD include smoking, hypertension and diabetes mellitus (1). Non-Hispanic black patients exhibited a higher prevalence of 19.2%. The overall prevalence rose to 23.2% for those over 80 years old. The need for effective treatment of PAD will escalate over the next few decades, as the number of patients over the age of 65 is predicted to increase from 35.3 million to 70.2 million by the year 2030 (2). Furthermore, a study that followed a large group of elderly men and women for ten years and assessed the mortality rates of those with PAD reported a 3-fold increase in mortality associated with PAD, even when controlling for age, sex, and other risk factor for cardiovascular disease (3). Although mild to moderate PAD can be managed conservatively, more severe atherosclerotic obstructions of the leg arteries require surgical vascular grafting for relief of ischemia. And while natural graft conduits of autogenous veins work best for replacing these small diameter vessels, autogenous conduits are frequently not available, so prosthetic grafts are required (commonly ePTFE – expanded polytetrafluoroethylene). The critical problem with these small diameter prosthetic grafts is that host tissue ingrowth is severely limited by their structure and form. Without transmural tissue ingrowth and a native endothelium, they are persistently thrombogenic, and stimulate neointimal hyperplasia (4), leading to frequent graft failure. The ideal prosthetic graft material would permit and encourage the transmural ingrowth of host tissue, and the elaboration of a stable, durable endothelium. Angiogenesis is the growth of capillaries from pre-existing blood vessels in the body. It occurs naturally in response to tissue growth and repair, yet it is also involved in various inflammatory diseases such as rheumatoid arthritis, psoriasis, and cancers (5). A related physiological process is arteriogenesis, which is the development of larger, collateral vessels sprouting from pre-existing arteries. It has been observed in patients with vascular diseases that collateral vessels can develop to bypass an obstruction and restore flow. Clinically, there is an association between collateral vessels and higher survival rates after myocardial infarction (6). Researchers have been investigating arteriogenic therapy – the stimulation of arterial growth from preexisting arteries – as an approach to treat peripheral vascular disease. A number of review articles (5, 7–9) have been published that document the progress of this research thrust. The discovery and characterization of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in the late 1970’s and in the 1980’s led to many early efforts to explore angiogenesis and arteriogenesis for therapeutic applications. For example, one study assessed the effects of bFGF on blood flow to an ischemic canine myocardium (10). Dogs that were given bolus injections of bFGF showed a statistically significantly higher degree of collateral flow than those with no injection. This was demonstrated in the rabbit ischemic hind-limb model as well (11). 246 In Tailored Polymer Architectures for Pharmaceutical and Biomedical Applications; Scholz, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Therapeutic arteriogenesis is being explored by using other growth factors and novel modes of delivery. A marked increase in vascular density and vascular branching was observed when an osmotic pump was used to supply an ischemic rabbit hind limbs with VEGF (12). The rabbits treated with VEGF in this study also exhibited new hair growth and restored functionality in their limbs. Monocyte chemoattractant protein-1 (MCP-1) was also explored and evidence was observed of the angiogenic and arteriogenic functionality (13). In another study that utilized a rabbit hind limb ischemia model, plasmid gene therapy was employed to increase platelet derived endothelial growth factor (PD-ECGF) production (14) . Within 30 days of transfection, there was a significant increase in capillary density, arteriolar density, and calf blood pressure. Grundmann et al. (15) used an implantable infusion pump that released granulocyte-macrophage colony stimulating factor (GM-CSF) in a pig vascular occlusion model. Collateral conductance was shown to increase with the added stimulating factor. Biomaterial scaffolds have been of great interest in the field of tissue engineering. The goal is to construct implantable materials of either biologic or synthetic origin to guide the in-growth/formation of native tissue and a native endothelium. Here we explore the use of precision engineered scaffolds that might encourage new blood vessel formation and could be used to construct optimized porous grafts that promote ingrowth and endothelialization. We have applied the process of sphere-templating for creating pro-angiogenic, anti-fibrotic scaffolds. We refer to the manufacture of these scaffolds as the “6S” process: sifting microspheres to a uniform size, sonicating the microspheres to close-pack them, sintering them together, surrounding them with the desired scaffold polymer, and solubilizing away the beads to yield interconnected pores. Marshall et al. discovered that pore size is strongly associated with vascular growth into sphere-templated materials (16). In the Marshall et al. experiments, scaffolds with pores of approximately 35µm diameter yielded the greatest density of blood vessels in a mouse subcutaneous model. The 6S scaffolds have been further enhanced by incorporating 60 µm diameter parallel channels running through the sphere-templated structure (17). These channels might allow larger blood vessels to form and to provide directionality for those vessels.

Experimental Scaffolds Scaffolds Three different groups of rod-shaped implants were used for the mouse implant study. The first group of scaffolds was comprised of poly(2-hydroxyethyl methacrylate)(PHEMA) fashioned to have longitudinal channels interconnected by spherical pores. These samples will be referred to as "channeled" scaffolds. The second group consisted of PHEMA implants with an interconnected porous geometry similar to the first group, but lacking the channels. These samples will be referred to as "porous" scaffolds. The third group was expanded 247 In Tailored Polymer Architectures for Pharmaceutical and Biomedical Applications; Scholz, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


polytetrafluoroethylene (PTFE) with channel-like spaces in between the fibrils of the material. These samples were segments obtained from a commonly used clinical type of PTFE graft. This material was included as a control in order to contrast with the effects of the channeled architecture materials.

Figure 1. A schematic representation of the “6S” process for fabricating scaffolds with uniform, interconnected pores. 248 In Tailored Polymer Architectures for Pharmaceutical and Biomedical Applications; Scholz, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Fabrication of Scaffolds Porous scaffolds and channeled scaffolds were both fabricated using variants of the 6S sphere-templating process, and were composed of crosslinked pHEMA (17, 18). As illustrated in Figure 1, PMMA beads are sieved to attain a uniformly sized fraction, shaken by sonication to ensure close packing, sintered together at >140°C, and surrounded by monomer solution under low vacuum pressure. The monomer mixture (purified HEMA monomer, tetraethylene glycol dimethacrylate crosslinking agent, ethylene glycol-water solvent and Irgacure 651) is then allowed to fully infiltrate the “bead cake” under atmospheric pressure. Upon UV initiation, the bead/polymer complex is immersed in solvents to remove the beads. Interconnected pores of approximately 30 microns in diameter were produced. The channeled scaffolds were fabricated by a process developed at the University of Washington (17), a variant of the 6S process, as illustrated in Figure 2.

Figure 2. Channeled scaffold fabrication: 1) Polycarbonate fibers with PMMA cladding are spun onto a reel. 2) Fiber bundles are inserted into shrink tubing. 3) Tubing is heated and compresses the fibers together. 4) Tubing is sectioned into smaller cylinders for ease of handling. 5) The ends of cylinders are glued to hold rods in place. 6) Shrink tubing is then cut away. 7) The PMMA cladding is dissolved away with xylenes. 8) Void spaces are filled with PMMA microbeads, which are then fused together by heating (see Figure 1). 9) Cylinders are infiltrated with HEMA monomer and allowed to polymerize. 10) PMMA and polycarbonate are dissolved away using as series of dichloromethane, acetone, ethanol, and water washes. The ePTFE materials were 0.6 mm wall thickness,with a porosity of 60 micron internodal distance (obtained from Atrium Medical, Hudson, NH). They were steam sterilized for 4 min before use. All samples were cut into approximately 5mm long rods with 1mm x 1mm square cross sections. These sizes were estimates in that the hydrogel samples exhibited varying swelling properties depending on the media. Implantation/Study Details An MTT cytotoxicity test and a gel-clot endotoxin assay were performed on all samples to ensure freedom from endotoxin and cytotoxic leachables. 249 In Tailored Polymer Architectures for Pharmaceutical and Biomedical Applications; Scholz, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Five male C57BL/6 mice were purchased from Charles River Laboratories (http://www.criver.com/). Mouse implantations were performed under aseptic conditions under the guidelines of the University of Washington IACUC. Ten scaffolds were implanted in 5 mice. Bilateral bicep femoris muscle implants of a single sample per muscle were performed on each mouse using Ketamine-Xylazine anesthesia cocktail. Each muscle incision was closed with a suture. Skin incisions were closed using wound clips. One dose of Buprenorphine was given per mouse for post surgical analgesia. Implants were explanted at two weeks and fixed in a zinc fixative. Non-specific stains included Trichrome, Picrosirius Red, and 4′,6-diamidino-2-phenylindole (DAPI). Immunohistochemical analysis consisted of MECA-32 staining. Not all implants could be found after excision and slide processing. Hence, there are fewer ePTFE samples than other scaffolds. Each explanted sample led to 12 or 30 sequential slides for the various stains desired.

Histological Analysis All paraffin-embedded tissue samples were sectioned into 5µm thick slices and placed in duplicate on slides. All histological images were acquired using a Nikon E800 Upright Microscope and Metamorph Version 6.3r7 software unless otherwise specified.

Figure 3. A DAPI-stained image of a section of the porous scaffold. Scale Bar = 200 micron. 250 In Tailored Polymer Architectures for Pharmaceutical and Biomedical Applications; Scholz, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Slides of each region of each scaffold group were stained with DAPI, and 10x micrographs were acquired. Using Adobe Photoshop CS4, an area of the image within the bounds of the scaffold was isolated as shown in Figure 3. Next, the blue nuclei were selected and the pixel area was recorded. The inverse, black pixel area was also recorded such that the ratio of blue nuclei pixel area to total pixel area could be calculated. MECA-32 is a mouse panendothelial cell antigen. For counting MECA-32 positively stained cells, images at 20x magnification were selected in a staggered fashion from sections at each region of each implant. Next, a grid of 300 μm x 200 μm was overlaid in the Metamorph software (Figure 4). Positively stained cells were counted using a software-specific tool and these counts were exported to an Excel spreadsheet for further analysis. A positive cell was deemed to be noticeably stained (unstained cells exist in the image) and have a well-defined nuclei.

Figure 4. An example of a MECA-32 stained slide: 20x magnified channeled scaffold with 300 μm x 200 μm grid. The “1’s” indicate positive cell counts. The arrow points to a vessel lumen.

MECA-32 lumen widths were calculated using calipers in the Metamorph software. Again, images from each region – if available – of each section were included in the analysis. A lumen was defined by the inner space between two long, parallel endothelial cells, or by the space within a circular endothelial “donut” shape. A well defined lumen is seen at the top of the image in Figure 4. 251 In Tailored Polymer Architectures for Pharmaceutical and Biomedical Applications; Scholz, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.

Results In Vivo Study


Prior to implantation, a MTT cytotoxicity assay was performed. The implant samples showed equal or greater levels of cellular proliferation relative to the positive control. None of the samples tested positive for endotoxins using the gelclot endotoxin assay. After explantation, DAPI staining and MECA-32 staining was performed. Figure 5 compares DAPI staining and MECA-32 staining for the PTFE and the porous scaffolds. Cells are dispersed more uniformly in the porous scaffold and in tissue adjacent to the porous scaffold. In contrast, the PTFE shows a heavy accumulation of inflammatory cells in the adjacent tissue compared to in the channels.

Figure 5. DAPI and MECA-32 staining of the porous scaffold and the ePTFE material after 2 week implantation in mice. Scale bars are 100 microns.

DAPI staining allows us to obtain information about general cellular infiltration within the scaffolds. The ratios of positively stained pixel area to total area for each scaffold type was determined using Adobe Photoshop CS3. These data are presented in Figure 6. There were statistically significant differences between the channeled and porous scaffolds, the channeled and PTFE scaffolds, and the porous and PTFE scaffolds (p-value

Biomaterial-Induced Angiogenesis To Address Peripheral Vascular

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