Islet transplantation represents a potential method for curing Type I diabetes, and in recent years, its viability was boosted with the improvement of the immunosuppressive protocol used in conjunction with transplantation.1 However, major problems still exist that hamper widespread application of this advanced technique in clinical treatment.2 A limited number of donors, low graft survival rate because of immune rejection, and side effects of immunosuppressive drugs are the main obstacles. Microencapsulation, whereby islets are protected from the immune system within an ultrathin coat of a biocompatible material, provides an option that could alleviate the need for immunosupression and open the doors to an unlimited supply of xenograft tissue.3,4 Although the general concept looks attractive and there are some successful examples,5 most outcomes from both animal experiments and clinical trials remain disappointing.6 A main reason for the failure is overgrowth of fibrotic tissue around the encapsulated islets.7–9 To achieve the desired clinical results with transplanted encapsulated islets, at least two goals must be reached for a breakthrough to occur: better biocompatibility of encapsulation materials and elucidation of the ideal transplantation site.
Alginate-poly-l-lysine (PLL) has been used as a primary encapsulation material for decades but is limited by its relatively poor biocompatibility. Enormous efforts have been made to find new biomaterials that meet the needs of islet encapsulation.10 Available materials include 2-hydroxyethyl methacrylate (pHEMA),11 agarose,12 N-isopropylacrylamide,13 poly(ethylene glycol) (PEG),14 poly(methyl methacrylate) (PMMA) grafted with PEG,15 cellulose sulfate,16 biodritin,17 as well as living cells.18 PEG, which appears very promising, has been used widely in clinical treatments and proven to be highly biocompatible.19–25 It has gained Food and Drug Administration (FDA) approval for application in the manufacture of medicines and surface modification of implanted medical devices. PEG is probably the most common polymer in tissue engineering applications. It is hydrophilic, uncharged and forms highly hydrated polymer coils on its surface. Energy is required to displace water molecules from the hydrophilic coils, making protein adsorption energetically unfavorable. These properties may potentially contribute, at least in part, to the better biocompatibility of PEG. However, questions still remain as to whether the PEG exhibits better biocompatibility over various species and anatomical locations. Our laboratory has dedicated considerable effort to improve PEG encapsulation techniques, such as interfacial polymerization.26,27 In this project, we examined the biocompatibility of PEG in comparison with PLL in multiple anatomical locations in the Lewis rat model.
Methods and Materials
Empty PEG capsules were generated based on the method described by Sawhney et al.14 with modifications. Briefly, a solution containing 225 mM triethanolamine (Fluka Chemie, Buchs, Switzerland), 20% PEG diacrylate (MW 575, Sigma-Aldrich, St. Louis, MO), and 37 mM 1-vinyl-2-pyrrolidinone (Sigma-Aldrich, St. Louis, MO) was adjusted to pH 8 using hydrochloride (1 N) and filtered using a 0.2 μm Teflon filter. Eosin Y was added at a final concentration of 1.25 mM before capsules were made. A 1 ml syringe with a 30-G needle was used to expel droplets onto the surface of heavy mineral oil in a Petri dish. The suspended droplets were kept under ultraviolet (UV) light for 1 hour, while polymerization occurred, and the resulting capsules were then transferred onto nylon mesh with 100 μm pore size. The capsules were washed extensively with warm phosphate-buffered saline (PBS) to remove residual oil and then hand picked and placed in a microcentrifuge tube containing PBS. Only capsules smaller than 800 μm in diameter were used.
Alginate capsules were produced using 1.5% alginate in encapsulation buffer (25 mM HEPES, 118 mM NaCl, 5.6 mM KCl, 2.5 mM MgCl2), which was expelled by an air jacketing technique28 through a 30-G needle attached to a 1 ml syringe into a 100 mM CaCl2 solution. Capsules were sterilized by UV exposure for 1 hour and then washed with PBS and coated with PLL as previously described.29 All materials used in the generation of capsules were sterilized before use. All capsules were produced fresh before implantation.
Animal experiments were performed under the supervision of the Institutional Animal Care and Use Committee (IACUC) of the University of Chicago. Animal protocol 71505 was approved for this particular project. Pre- and postsurgical care according to regulations and requirements was used.
Sixteen male Lewis rats (Charles River Laboratories, Portage, MA) weighing 200–300 grams were used in the experiment. All surgeries were performed in the operating room of the animal facility at the University of Chicago. Rats were anesthetized by inhalation of isoflurane. Fifty capsules were implanted using a pipette tip into each target site [subcutaneous (SC) flank, intramuscular (IM) upper rear leg, and intra-epididymis (IE)] (Figure 1). Proper suture techniques were used to close the wounds. Tissues where capsules were implanted were extracted 14 and 28 days after surgery and fixed with 10% formalin for morphological and immunological analysis.
Histological Analysis of Fibrotic Overgrowth
Formalin-fixed tissue blocks were sectioned at 6 μm and both Masson's trichrome (Newcomer Supply, Middleton, WI) staining and Sirius red (Sigma-Aldrich, MO) staining30 were performed to analyze fibrosis induction. Sirius red specifically stains collagen fibers with a bright yellow or orange color under the bipolarized microscope,31 whereas Masson's Trichrome staining was used as a parallel reference to confirm the result as well as for morphological observation. Staining procedures provided by the manufacturing companies were followed. Sirius red-stained sections were visualized under a bipolarized microscope (Axioskop, Imaging Core in University of Chicago). Ten sections from each tissue sample were stained and 20 random images were taken for quantitative analysis using the ImageJ software program (NIH). Two variables were used for calculations: the intensity of stain color and the thickness of the stained area surrounding the capsules. The relative fibrotic overgrowth was calculated as average intensity multiplied by average thickness.
The results were statistically analyzed with either unpaired t test, to compare capsules within an implantation site, or analysis of variance (ANOVA), to compare among implantation sites. A p-value <0.05 was considered statistically significant.
Postimplantation, all animals showed no symptoms or signs of negative response to surgery. On daily observation, no local inflammation around incision sites was seen. All animals recovered from the wounds within 5 days of surgery. No noticeable difference in activity was observed post-versus presurgery. On retrieval, almost all capsules remained intact and spherical in shape, indicating high stability of both materials over the duration of implantation.
Fibrotic Overgrowth Analysis
Animals were randomly separated into two groups after surgery. Fibrotic overgrowth was quantitatively evaluated using ImageJ software (NIH) at either 14 or 28-day postsurgery. Ten histological sections from each tissue sample were stained with Sirius red from which at least 20 images were taken from randomly chosen capsules with a bipolarized microscope. The color intensity and average thickness of the stained area around each capsule were used to calculate relative fibrotic overgrowth for each capsule. The results show that there were significant differences in fibrosis between the PEG and PLL capsules in two of three implantation sites, i.e., SC flank and IE, (p < 0.001) at 14-day postsurgery (Figure 2 and Table 1). The same result was observed at the SC site 28-day postsurgery. However, PEG capsules in the IE site caused significantly higher fibrosis than alginate-PLL capsules at the 28-day time point, contradicting the results obtained at the 14-day time point.
Interestingly, capsules of either material, implanted at IM sites, induced the least amount of fibrotic overgrowth in comparison with the other two locations. No statistical difference in fibrotic overgrowth was observed between the two materials at either time point.
Uniformity of fibrotic overgrowth among the randomly selected capsules and experimental animals was also evaluated. We observed that for both PEG and alginate-PLL capsules, the degree of fibrotic overgrowth varied in thickness among capsules and showed no correlation with the size of capsules among individual animals and anatomical locations (Figure 3 and Table 1).
In this project, we measured fibrotic overgrowth induced by PLL or PEG in the Lewis rat model by implanting capsules in the same animal in three different anatomical locations.
Multiple factors may affect the biocompatibility of alginate, including purification procedure and chemical composition.32–34 The chemical composition, especially the proportion of guluronic and mannuronic acid residues of alginate, determines its physical biocompatibility.29 Furthermore, composition of alginate can also influence cell function and activity of encapsulated cells.35 It is difficult to predict the degree of immune response to a particular batch of alginate without previous testing, and researchers usually experience poor reproducibility of experiments. Alginate capsules alone are not stable under physiologic conditions, and PLL must be used to coat the surface of alginate capsules to increase stability. PLL, however, also reduces biocompatibility of the capsules. The hydrophilic, uncharged nature of PEG, combined with its higher stability under physiologic conditions, contributes to its potential as a more highly biocompatible material than PLL-alginate. Highly-hydrated polymer coils form on the capsule surface, making protein adsorption energetically unfavorable, as energy is required to displace water molecules from the hydrophilic coils, which may contribute, at least in part, to the higher biocompatibility of PEG.20 It was found that protein adsorption capacity is also related to the size of PEG molecules. A significant decrease in protein absorption is seen in PEG with a molar mass above 4000 g/mol.36 When PEG with high molecular weight (15,000–30,000 g/mol) was coated on the surface of PLL capsules, the biocompatibility was dramatically improved.37 However, the permeability of the capsules is also decided by the size of the PEG molecule, where control of pore size is an important consideration in developing immunoisolation materials. We have used a much smaller PEG molecule (575 g/mol) in this study, which may have contributed to the observed amount of fibrotic overgrowth.
Fibrotic overgrowth is a widely accepted metric of biocompatibility evaluation of materials; however, there is no convincing methodology for quantification. Several published studies have used the positively stained percentage of analyzed area to determine the degree of fibrotic overgrowth.30 We used relative intensity of Sirius red staining because more densely packed collagen fibers stain with a brighter color and indicate a higher degree of fibrotic deposition than do more loosely packed, lighter stained fibers. We also used the average thickness of fibrotic material instead of area percentage as method to eliminate the influence of capsule size on results.
Transplantation site has been reported to be an important factor related to the degree of fibrotic overgrowth of encapsulated islets observed.30 Intraportal (hepatic) and renal sub-capsular sites may not be ideal implantation sites for encapsulated islets that tend to have higher total volume than naked islets. Earlier investigations looked at the peritoneal cavity as a transplant site,32,38,39 but strong fibrotic overgrowth and poor performance of transplanted islets limit this approach. SC and IM sites have the significant advantage of providing sufficient space for transplantation. Grafts can be implanted in multiple locations and are retrievable and replaceable if need be. In this study, we chose to implant into three anatomical locations and give comparisons among sites in the same animal, thus avoiding the bias of differences among animals in response to the tested materials.
We noticed that there is a high degree of variation within most of the experimental groups, especially in the groups that induced heavy fibrotic overgrowth. Possible reasons for these observations include that the capsules were implanted as a bulk graft, as opposed to implanting each capsule individually, into the space between tissues, and that those capsules close to or in direct contact with tissue may have heavier fibrotic overgrowth than capsules in the middle of the bulk. We found that fibrotic tissue growth was more pronounced between capsules and tissue than that between capsules. Another potential reason for variation lies in the surgery itself. Although we did not document the severity of bleeding, which could have been used to compare results among sites or animals, local hemorrhage may contribute to fibrosis. Finally, endotoxin contamination is a possibility, although all materials were kept sterile throughout all procedures. It is known that the presence of endotoxin has been proven to induce fibrosis, and we cannot rule out this possibility because endotoxin assays were not performed before implantation.
Our experiments revealed the interesting finding that PEG capsules implanted in the IE site showed heavier fibrotic overgrowth than alginate-PLL capsules at 28-day postsurgery. Capsules implanted in the same site and retrieved after 14-day postsurgery showed no significant differences in lymphocyte or macrophage infiltration between the materials. Further investigation is needed to elucidate the mechanisms behind this observation, and several possibilities are proposed. PEG could cause a stronger reaction in delayed, as compared with immediate, immune response. Local immune response toward PEG may vary among implantation sites. Epididymis is considered an immune privileged tissue, and when the epididymal barrier is broken, the host immune system may demonstrate stronger delayed immune response than other non-immune privileged locations.
One limitation of this project was the use of empty capsules instead of encapsulated islets. Although the data represent real characteristics of both biomaterials, which were the primary goal of the study, it is preliminary to link the result with clinical reality. Transplanted islets secrete hormones, cytokines, and cell metabolites that may affect fibrosis. It is reasonable to speculate that a different immune response to encapsulated islets may be observed in comparison with empty capsules. Furthermore, encapsulated islet allo- or xeno-grafts may result in differences in immune response and fibrosis induction. Further studies will be necessary to investigate these questions.
This work is supported by American Diabetes Association.
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