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Characteristics of Poly-l -Ornithine-coated alginate microcapsules
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Biomaterials 26 (2005) 6846–6852 www.elsevier.com/locate/biomaterials
Characteristics of Poly-L-Ornithine-coated alginate microcapsules Marcus D. Darrabiea,b, William F. Kendall Jr.a,b, Emmanuel C. Oparac, a
Department of Surgery, Duke University Medical Center, Durham, NC, USA b VA Medical Center, Durham, NC, USA c Pritzker Institute of Biomedical Science and Engineering, Illinois Institute of Technology, Chicago, IL, USA Received 29 March 2005; accepted 6 May 2005 Available online 13 June 2005
Abstract Poly-L-Lysine (PLL) is the most widely used biomaterial for providing perm-selectivity in alginate microcapsules for islet transplantation. We had previously reported that Poly-L-Ornithine (PLO) is less immunogenic than PLL, and in the present study, we have compared the physical characteristics of PLO- and PLL-coated hollow alginate microcapsules. Microspheres made with 1.5% alginate were divided into 2 groups that were first coated with either 0.1% PLO or PLL, followed by a second coating with 0.25% alginate. After liquefaction of the inner alginate core with sodium citrate, the microcapsules were washed with saline and used for experiments. Pore size exclusion studies were performed with FITC-labeled lectins incubated with encapsulated pig islets followed by examination for fluorescence activity. Mechanical strength was assessed by an osmotic pressure test and by 36 h of mechanical agitation of microcapsules with inert soda lime beads. The pore size exclusion limit of microcapsules after 20 min of coating was significantly smaller with PLO. While the mean7SEM diameter of PLL-coated microcapsules increased from 718717 to 821717 mm (po0:05) during 14 days incubation at 37 1C, the PLO group did not change in size. Also, PLL group had a higher percentage of broken capsules (52.774.9%) compared to 3.172.05% for PLO capsules (po0:0001; n ¼ 6). We conclude that PLOcoated alginate microcapsules are mechanically stronger and provide better perm-selectivity than PLL-coated microcapsules. r 2005 Elsevier Ltd. All rights reserved. Keywords: Alginate; Microencapsulation; Poly-amino acid membrane; Physical properties
1. Introduction The alginate–polylysine microencapsulation procedure as developed by Lim and Sun has been the dominant protocol for cell microencapsulation for over 20 years [1]. Although some variation has occurred, the procedure essentially involves gelling alginate/islet microspheres with a divalent cationic solution such as BaCl2 or CaCl2, applying a polyamino acid layer, followed by an outer coating with very dilute concentration of alginate [2]. Whereas current transplantation procedures rely on immunosuppressive protocol, the hallmark of the microencapsulated islet is based on its ability to create a permeability barrier between graft Corresponding author. Tel.: +1 312 567 3858; fax: +1 312 567 5707. E-mail address: [email protected] (E.C. Opara).
0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2005.05.009
tissue and the host immune system [3,4]. Successful use of this procedure is dependent on the viability of the microencapsulated islet, which in turn relies on the ability of the alginate microcapsule to prevent exposure of the tissue to immune system components [5,6]. In the field of cell microencapsulation, there is a need to develop more durable capsules that would permit long-term function of encapsulated cells [7]. In addition, biocompatibility of the capsule is essential considering that fibrosis may prevent or restrict the flow of important nutrients into the capsule and result in islet necrosis and graft failure. Low swelling capsules that have increased mechanical strength and biocompatibility are less likely to rupture or invoke inflammatory responses. Studies examining the molecular composition and purification of alginate grades have resulted in the production of more biocompatible microcapsules and longer functioning cell transplants [2]. In addition, the
ARTICLE IN PRESS M.D. Darrabie et al. / Biomaterials 26 (2005) 6846–6852
perm-selective coating with Poly-L-Lysine (PLL) has been found to play a vital role in the encapsulation process as it firmly binds to alginate thereby restricting the permeability of the alginate microcapsule [8]. Although application of PLL has resulted in the reversal of hyperglycemia after encapsulated islet transplantation, some studies have suggested that PLL can increase the likelihood of host inflammatory responses [9,10]. Without application of a final dilute layer of alginate, non-reacted PLL may also invoke immune reactions from the host [8]. While results of islet graft function obtained with PLL have been inconsistent, the benefits of an alternative polycation, such as Poly-L-Ornithine (PLO), remains to be fully explored. We have previously shown that removing Ca++ from the hygroscopic alginate core or replacing it with the monovalent cation, Na+, can benefit the microencpasulation process by reducing the swelling phenomenon and polymorphism seen in alginate-PLL microcapsules [7,10]. However, several studies have suggested that PLO may have some advantages for microencapsulation than the traditional use of PLL for coating alginate microcapsules [10–12], albeit, there is scarcity of data on the physical properties of alginate microcapsules coated with PLO. In response to the pressing need for effective and durable immunoisolation of transplant tissue, our study was designed to address the extent to which PLL and PLO can individually affect perm-selectivity and microbead swelling, which affects the mechanical strength of alginate microcapsules.
2. Materials and methods 2.1. Materials Ultrapurified sodium alginate [EX-8085] (3% w/v) with a high content of mannuronic acid was obtained from Kelco/ Monsanto (San Diego, CA). Liberase PI enzyme blend was obtained from Roche Molecular Biochemicals (Indianapolis, IN). Hanks balanced salt solution was purchased from GIBCO BRL (Grand Island, NY). FITC-Triticum Vulgare (WGA), and FITC-Ricinus Communis I (RCA-I), FITC-Sambucas Nigra (SNA), were obtained from EY Lab Inc. FITC-Maackia Amurensis I was purchased from Vector Laboratories (Burlingame, CA). Trypan Blue, Poly Lysine, Poly-L-Ornithine and all analytical chemicals for solutions were purchased from Sigma Chemical (St. Louis, Mo.) 2.2. Surgery and isolation of porcine islet cells Porcine islets were isolated using a modification of a previously described procedure [13]. Briefly, the pigs were anesthetized with 2% Isoflurane, and following intra-arterial infusion of ice-cold University of Wisconsin solution, a complete pancreatectomy was performed with sterile techniques. The pancreas was weighed and placed in a dish for
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cannulation of the pancreatic duct and infusion of digestion medium, comprising Hanks balanced salt solution containing Liberase PI enzyme blend, 0.5 mg/mL; Deoxyribonuclease I, 10,000 U; and 1 mmol/L Trolox, a water-soluble analogue of vitamin E. After 15 min of incubation on ice, the pancreas was incubated at 37 1C for 45–60 min with slight and periodic agitation. The digested tissue was filtered using a nylon mesh with a pore size of 1000 mm, and the islet cells were purified using an Optiprep density separation procedure [14]. The islet cells were then washed in Hanks balanced salt solution and identified by Dithizone staining.
2.3. Microencapsulation with 2-channel air jacket microencapsulator Prior to encapsulation, tubes containing isolated pig islets were suspended in fresh RPMI 1640 solution. Alginate was then added to the suspension, which resulted in a 1.5% solution of alginate and islets. Encapsulation was performed with a 2-channel air jacket microencapsulator. Droplet formation was achieved with a blunt, 25 gage needle tip, and alginate or islet/alginate droplets were allowed to gel by crosslinking in 1.1% CaCl2. To ensure saturation of active binding sites, the microbeads were incubated in this solution for 15–30 min prior to washing with normal saline. Two groups of capsules were made; one group contained empty microcapsules (without islet tissue), and the other consisted of microcapsules containing pig islets. After the incubation period, the CaCl2 was removed from both groups and capsules were washed twice with saline. Capsules from both groups were then further divided into 4 subgroups, based on the type and duration of perm-selective coating. The subgroups consisted microcapsules coated with PLL or PLO for either 6 or 20 min. Following PLL and PLO coatings, capsules were subsequently washed with saline, after which, a layer of 0.25% sodium alginate was applied to the outer coating of the capsules. Chelation (liquefaction) of the inner Ca2++-alginate core was achieved by suspension in 55 mM sodium citrate for 7 min. Capsules were then rinsed with saline and treated with 6 mM Na2SO4 for 10 min [7], prior to final washings with saline. The capsule suspensions were gently mixed on a rotor (Clay Adams Nutator, Becton Dickinson, Sparks, MD) during the incubation periods involved in the washings, coatings, and chelation steps.
2.4. Lectin-mediated fluorescence Following the washing procedure, 150 mL of encapsulated pig islets were sorted into appropriate groups and incubated with one of four lectins that varied in molecular weight. Lectin incubation consisted of either incubation with 15 mL (1 mg/mL) of Triticum Vulgare (WGA, MW: 36,000), Maackia Amurensis (MAL-I, m.w: 75,000), Ricinus Communis (RCA-I, m.w: 120,000), or Sambuca Nigra (SNA, m.w: 150,000). Capsules were incubated for 48 h at 4 1C with mixing, after which, they were examined on an Olympus BH2 Fluorescent Microscope for fluorescence activity within the capsules.
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2.5. Analysis of microcapsule size changes Capsules that were coated with PLL or PLO for 6 min were suspended in saline and placed in a 37 1C incubator. Diameter measurements were made using an inverted light microscope (Olympus CK40) that was linked to the UTHSCA Image Tool computer program (University of Texas, Austin). Diameter measurements were performed during the course of a 14-day incubation period.
data was performed using an analysis of variance (ANOVA) computer program (GraphPad, San Diego, CA), and depending on the outcome of ANOVA, the Tukey–Kramer multiplecomparison test was used to assess the significance of differences. In comparisons of the significance of the difference between the means of two groups, the student’s t-test was performed, and in all cases, a value of po0:05 was accepted as significant.
2.6. Induction of mechanical stress using osmotic pressure
3. Results
Osmotic stress was applied using a modification of a previously described procedure [15]. Aliquots of alginate microcapsules coated for 6 min with either PLL or PLO were placed in 10 mL of H2O and incubated at 37 1C for 2 h. Capsules were then washed with saline and stained with 0.5% (w/v) Trypan blue. A subsequent washing with saline was then performed prior to assesment of broken and intact capsules using an inverted light microscope.
3.1. Polycation coating and microcapsule pore exclusion size
2.7. Induction of mechanical stress using bead agitation Bead agitation for mechanical stress was applied using a modification of a previously reported procedure [16]. Groups of 350, 6-min PLL- and PLO-coated capsules were placed into flasks containing approximately 6.5 g of 3 mm inert soda lime beads (VWR Scientfic Products Corporation) and 30 mL of normal saline. Capsules from each group were then subjected to mechanical stress for 36 h by shaking at approximately 300 RPM using a Lab Line Orbital Shaker. The percentage of broken and unbroken capsules were determined manually by visual analysis and handpicking under a stereomicroscope (Swift Instruments). 2.8. Data analysis The diameters of the microspheres were measured on day 0 (control) and they were subsequently incubated in saline at 37 1C for 2 weeks with size assessment every 2 days. Microsphere diameters were determined using Image Tool version 2.00-computer software (UTHSCSA University of Texas Health Science Center in San Antonio). The data were then normalized by calculation of the percent change from control (day 0). These data, expressed as mean7SEM, were used for statistical analysis of changes in sizes. Statistical evaluation of
Experiments were performed to examine the effects of PLL and PLO perm-selective membranes and the duration of coating on microcapsule pore size exclusion by exposing encapsulated islets to various sizes of FITC labeled lectins. In each group the percentage of fluorescent capsules was quantified as shown in Table 1. Upon examination after incubation, unencapsulated naked porcine islets contained 100% fluorescence, while empty PLL- or PLO-coated capsules contained no fluorescence, which indicated that the presence of porcine islet tissue was necessary for fluorescent activity to occur within the microcapsules. Furthermore, encapsulated porcine islets without PLL or PLO coatings also contained 100% fluorescence, which indicated that without perm-selective coating, lectins were able to permeate the alginate microcapsules. With 6-min PLLcoated capsules, no capsules incubated with SNA (m.w ¼ 150 KD) and RCA-I (m.w ¼ 120 KD) fluoresced while 100% of MAL-I (m.w ¼ 75 KD) and WGA (m.w ¼ 36 KD) capsules fluoresced. Microcapsules that were coated for 20 min with PLL showed no change in fluorescence compared to those coated for 6 min with PLL. The 6-min PLO-coated capsules showed the same results as were observed with the capsules coated for 6 and 20 min with PLL. However, in comparison to these groups, a significantly lower level of fluorescence (10.2574.6%) was observed when capsules were coated with PLO for 20 min, as shown in Table 1.
Table 1 Permeability of PLL and PLO-coated alginate microcapsules exposing to FITC lectin permeability n ¼ 6 Lectin
Pig tissue (%)
No PLL/ PLO (%)
Empty PLL/ PLO (%)
6 min. PLL (%)
20 min. PLL (%)
6 min. PLO (%)
20 min. PLO
WGA (36 kD) MAL-I (75 kD) RCA-I (120 kD) SNA (150 kD)
100 100 100 100
100 100 100 100
0 0 0 0
100 100 0 0
100 100 0 0
100 100 0 0
100% 10.25%74.16 0% 0%
Microcapsules containing pig islets were incubated with FITC-lectins of various sizes for either 6 or 20 min, prior to examination for fluorescence activity within the microcapsules.
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3.2. Polycation coating and microbead swelling We examined if PLO coating would affect the phenomenon of bead swelling that is commonly seen in PLL-coated alginate microcapsules. Batches of alginate microcapsules coated for 6 min with either PLL or PLO and incubated in normal saline were assessed over a 14-day period for changes in size using measurements of their diameters. As illustrated in
PLL vs. PLO Bead Swelling
900
Diameter (Micrometers)
Days vs PLL Days vs PLO 850
800
750
700
650 0
2
4
6 8 10 Time (Days)
12
14
16
Fig. 1. Changes in the diameter of alginate microcapsules during 14day saline incubation at 37 1C. Capsules were coated with either PLL or PLO. Data represent mean7SEM (n ¼ 20 from each of 3 separate microcapsule preparations).
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Figs. 1 and 2, there was a significant increase in the diameters of PLL-coated capsules. In contrast, PLOcoated capsules showed no statistically significant change in size over the incubation period. 3.3. Polycation coating and mechanical strength In initial studies of the mechanical strength of microcapsules, we compared the effects of PLL and PLO coatings on the mechanical strength of microcapsules that were exposed to a hypotonic environment. Within minutes of incubation in H2O, microcapsules from both groups rapidly increased in size. As illustrated in Fig. 3, the resultant osmotic stress in the presence of Trypan blue staining, showed that a majority of the capsules in the PLL group ruptured as demonstrated by large fissures on the surface of the capsule. Although microcapsules in both groups were prone to rupture, those coated with PLL had significantly higher breakage when compared to PLO-treated capsules. To simulate in vivo shear stress, additional studies were performed using 3 mm beads mixed with microcapsules and incubated for 36 h with shaking. Capsules coated with PLL experienced 52.774.92% breakage following subjection to 36 h of agitation in the presence of soda lime beads. In contrast, only a negligible percentage of the PLO-coated microcapsules (3.0572.05%) were found to be broken, as illustrated in Fig. 4. Assessment of the effects of both osmotic pressure and bead agitation procedures indicated that
Fig. 2. Visual image of size changes in alginate microcapsules. During the incubation period, PLL-coated capsules (upper panel) increased significantly while PLO-coated capsules (lower panel) showed no significant change in size.
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PLO coating resulted in alginate microcapsules that were approximately 50% less likely to break when compared to PLL treatment using simulated methods of induced mechanical stress. See Fig. 4.
4. Discussion The effectiveness of immunoisolation by microencapsulation is strongly linked to the permeability of the
Fig. 3. Effect of PLL and PLO coatings on microcapsules response to osmotic pressure. Following exposure to osmotic pressure, capsules from PLL and PLO groups were suspended in saline and observed at 4 and 10 magnification under an inverted light microscope.
microcapsule by immune cells and cytotoxic antibodies [17]. Increasing the time of interaction between the polycation and alginate microcapsules should allow for increased binding of the polycation on the capsule surface [18–20]. In contrast to previous reports, in the present study, 20-min PLL coating of alginate microcapsules showed no reduction in the microcapsule pore size when compared to 6-min duration of coating. However, it is possible that the pore size was reduced for the molecular size range of 120 and 150 KD during the 20-min duration, but was not detected by our method of assessment. In addition to decreased lectin permeability, we also found that PLO coating nearly eliminated bead swelling while significantly increasing the mechanical strength of the alginate microcapsule. The significance of this finding is vital in that breakage or rupture of the alginate membrane should correspondingly be reduced in PLO-coated alginate microcapsules. Reduction in capsule swelling will also result in lower shear stress, increased biocompatibility, and increased islet viability [21]. Long-term swelling of alginate microcapsules leads to capsule polymorphism and may also lead to a gradual increase in microcapsule pore size and permeability [22]. Previous studies have indicated that membrane thickness is an indicator of membrane strength and permeability [18,19]. The amino acid monomer of PLO is shorter in structure than that of PLL by one methyl group [23]. This difference in structure likely allows PLO to bind more efficiently to the alginate membrane, which in turn would result in increased membrane thickness and strength.
Fig. 4. Effect of PLL and PLO coatings on microcapsule mechanical strength. (A) Ruptured capsules were quantified following subjection to induced osmotic stress. (B) Following 36 h of bead agitation, broken capsules in both PLL- and PLO-coated capsules were quantified. Data represent mean7SEM (n ¼ 6).
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Apart from such factors as gelling cation, alginate grade, and alginate purity, the alginate–polycation complex plays an important role in the stability of alginate microcapsules [24,25]. Using PLL for coating alginate microcapsules of transplanted islets has resulted in promising but limited success. Previous studies have shown inflammatory reactions to transplanted alginate microcapsules [26,27]. A recent study has indicated that certain concentrations of PLL have potent necrosis inducing properties on encapsulated islet transplants [28]. It remains to be determined if further investigations into the use of other membrane polymers, which include, agarose, polyethylene glycol, polyacrylates and AN69 hydrogel may yield greater opportunities for immunoisolation of islets [29–32]. Some studies performed in rodents have suggested that an uncoated alginate microcapsule by itself would be suitable for transplantation [33,34]. However, transplantation of uncoated encapsulated islets would require low dose immunosuppression, and in addition, our study and many others have indicated that a 150 KD immune system component, such as IgG, would be fully capable of traversing the capsule [20,28,35], thus exposing the encapsulated islets to destruction by these cytotoxic molecules. The immunological effects of PLO on cell viability are still unclear, however, previous groups have successfully reversed hyperglycemia for extended periods with PLO-coated alginate microcapsules [11,12], which along with our present data on the superior physical characteristics of PLO-coated alginate microcapsules, suggest that it may be a better alternative to PLL-coated microcapsules. In conclusion, we have shown that PLO coating can reduce swelling and increase the mechanical strength of alginate microcapsules when compared to PLL coating. We also found that PLO coating more effectively restricts higher molecular weight components than PLL from permeating the microcapsules. The advantages provided in terms of capsule resistance and molecular weight exclusion should result in alginate microcapsules that have increased structural integrity and biocompatibility.
Acknowledgment We thank Spencer Bridges and Yusuf Neville for invaluable technical assistance. References [1] Lim F, Sun AM. Microencapsulated islets as bioartificial endocrine pancreas. Science 1980;210:908–10. [2] Kendall WF, Collins BH, Opara EC. Islet cell transplantation for the treatment of diabetes mellitus. Expert Opin Biol Ther 2001;2:503–11.
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[3] Schrezenmeir J, Faust P, Laue C, Kirchgessner J, Stallmach T, Muntefering H, Beyer J. Immunoprotection by biocompatible membrane permits transplantation of piscine islets to rats. Transplant Proc 1989;21:2730–5. [4] Lanza RP, Borland RM, Staruk JE, Appel MC, Solomon MA, Chick WL. Transplantation of encapsulated canine islets into spontaneously diabetic BB/W or rats without immunosuppresion. Endocrinology 1992;131:637–42. [5] Ricker A, Stockberger S. Hyperimmune response to microencapsulated xenogenic tissue in NOD mice. Diabetes 1986;35:61A. [6] Basta G, Sarchielli P, Luca G, Racanicchi L, Nastruzzi C, Guido L, Mancuso F, Macchiarulo G, Calabrese G, Brunetti P, Calafiore R. Optimized parameters for microencapsulation of pancreatic islet cells: an in vitro study clueing on islet graft immunoprotection in type 1 diabetes mellitus. Transplant Immunol 2004;13:289–96. [7] Darrabie M, Freeman BK, Kendall Jr. WF, Hobbs HA, Opara EC. Durability of sodium sulfate-treated polylysine-alginate microcapsules. J Biomed Mater Res 2001;54:396–9. [8] Thu B, Bruheim P, Espevik T, Smidsrod O, Soon-Shiong P, Skjak-Braek G. Alginate polycation microcapsules I. Interaction between alginate and polycation. Biomaterials 1996;17: 1031–40. [9] Strand BL, Gaserod O, Kulseng B, Espevik T, Skjak-Braek G. Alginate–polylysine–alginate microcapsules: effect of size reduction on capsule properties. J Microencapsul 2002;19:615–30. [10] Hobbs HA, Kendall WF, Darrabie M, Collins B, Bridges S, Opara E. Substitution of polyornithine for polylysine in alginate microcapsules. Diabetes 2000;49(Suppl. 1):A111. [11] Brunetti P, Basta G, Falorni A, Calcinaro F, Pietropaolo M, Calafiore R. Immunoprotection of pancreatic islet grafts within artificial microcapsules. Int J Artif Organs 1991;14:789–91. [12] Calafiore R, Basta G, Boselli C, Bufalari A, Giustozzi CM, Luca G, Tortoioli C, Brunetti P. Effects of alginate/polyaminoacidic coherent microcapsulse transplantation in adult pigs. Transplant Proc 1997;29:2126–7. [13] Ching CD, Harland RC, Collins BH, Kendall W, Hobbs H, Opara EC. A reliable method for isolation of viable porcine islet cells. Arch Surg 2001;136:276–9. [14] Krickhahn M, Meyer T, Buhler C, Thiede A, Ulrichs K. Highly efficient isolation of porcine islets of Langerhans for xenotransplantion: numbers, purity, yield and in vitro function. Ann Transplant 2001;6:48–54. [15] Van Raamsdonk JM, Chang PL. Osmotic pressure test: a simple, quantitative method to assess the mechanical stability of alginate microcapsules. J Biomed Mater Res 2001:264–71. [16] Leblond FA, Tessier J, Halle´ J. Quantitative method for the evaluation of biomicrocapsule resistance to mechanical stress. Biomaterials 1996;17:2097–102. [17] De Vos P, De Haan BJ, Wolters GHJ, Strubbe JH, Van Schilfgaarde R. Improved biocompatibility but limited graft survival after purification of alginate for microencapsulation of pancreatic islets. Diabetologia 1997;40:262–70. [18] King GA, Daugulis AJ, Faulkner P, Goosen MF. Alginate–polylysine microcapsules of controlled membrane molecular weight cutoff for mammalian cell culture enginnering. Biotechnol Prog 1987;3:231–40. [19] Vandenbossche GMR, Van Oostveldt PV, Demeester J, Remon JP. The molecular weight cut-off of microcapsulses is determined by the reaction between alginate and polylysine. Biotechnol Bioeng 1993;42:381–6. [20] Brissova M, Petro M, Lacik I, Powers AC, Wang T. Evaluation of microcapsule permeability via inverse size exclusion chromatography. Anal Biochem 1996;242:104–11. [21] Thu B, Bruheim P, Espevik T, Smidsrod O, Soon-Shiong P, Skjak-Braek G. Alginate polycation microcapsules II. Some functional properties. Biomaterials 1996;17:1069–79.
ARTICLE IN PRESS 6852
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[22] Hobbs HA, Kendall Jr WF, Darrabie M, Opara EC. Prevention of morphological changes in alginate microcapsules for islet xenotransplantation. J Invest Med 2001;49:572–5. [23] Inaki Y, Tohnai N, Miyabayashi K, Miyata M. Isopoly Lornithine derivative as nucleic acid model. Nucleic Acids Symp Ser 1997;37:25–6. [24] Dusseault J, Leblond FA, Robitaille R, Jourdan G, Tessier J, Menard M, Henley N, Halle J-P. Microencapsulation of living cells in semi-permeable membranes with covalently cross-linked layers. Biomaterials 2005;26:1515–22. [25] De Vos P, De Haan B, Van Schilfgaarde R. Effect of the alginate composition on the biocompatibility of alginate–polylysine microcapsules. Biomaterials 1997;18:273–8. [26] Horcher A, Zekorn T, Siebers U, Klock G, Frank H, Houben R, Bretzel RG, Zimmermann U, Federlin K. Transplantation of microencapsulated islets in rats: evidence for induction of fibrotic overgrowth by islet alloantigens released from microcapsules. Transplant Proc 1994;26:784–6. [27] King A, Sandler S, Andersson A. The effect of host factors and capsule composition on the cellular overgrowth on implanted alginate capsules. J Biomed Mater Res 2001;57:374–83. [28] Strand BL, Ryan TL, In’t Veld P, Kulseng B, Rokstad AM, Skjak-Brek G, Espevik T. Poly L-Lysine induces fibrosis on alginate microcapsules via the induction of cytokines. Cell Transplant 2001;10:263–75.
[29] Iwata H, Amemiya H, Matsuda T, Takano H, Hayashi R, Akutsu T. Evaluation of microencapsulated islets in agarose gel as bioartificial pancreas by studies of hormone secretion in culture and by xenotransplantation. Diabetes 1989;38(Suppl 1): 224–5. [30] Matthew HW, Salley SO, Peterson WD, Klein MD. Complex coacervate microcapsules for mammalian cell culture and artificial organ development. Biotechnol Prog 1993;9:510–9. [31] Sawhney AS, Hubbell JA. Polyethylene oxide-graft-poly L-lysine) copolymers to enhance the biocompatibility of poly L-lysinealginate microcapsule membranes. Biomaterials 1992;13:863–70. [32] Prevost P, Flori S, Collier C, Muscat E, Rolland E. Application of AN69 hydrogel to islet encapsulation. Evaluation in streptozotocin-induced diabetic rat model. Ann NY Acad Sci 1997;831: 344–9. [33] Lanza RP, Kuhtreiber WM, Ecker D, Staruk JE, Marsh J, Chick WL. A simple method for transplanting discordant islets into rats using alginate gel spheres. Transplantation 1995;59: 1486–7. [34] Yang H, O’Hali W, Kearns H, Wright Jr JR. Long-term function of fish islet xenografts in mice by alginate encapsulation. Transplantation 1997;64:28–32. [35] Powers AC, Brissova M, Lacik I, Anilkumar AV, Shahrokhi K, Wang TG. Permeability assessment of capsules for islet transplantation. Ann NY Acad Sci 1997;831:208–16.
Biomaterials 26 (2005) 6846–6852 www.elsevier.com/locate/biomaterials
Characteristics of Poly-L-Ornithine-coated alginate microcapsules Marcus D. Darrabiea,b, William F. Kendall Jr.a,b, Emmanuel C. Oparac, a
Department of Surgery, Duke University Medical Center, Durham, NC, USA b VA Medical Center, Durham, NC, USA c Pritzker Institute of Biomedical Science and Engineering, Illinois Institute of Technology, Chicago, IL, USA Received 29 March 2005; accepted 6 May 2005 Available online 13 June 2005
Abstract Poly-L-Lysine (PLL) is the most widely used biomaterial for providing perm-selectivity in alginate microcapsules for islet transplantation. We had previously reported that Poly-L-Ornithine (PLO) is less immunogenic than PLL, and in the present study, we have compared the physical characteristics of PLO- and PLL-coated hollow alginate microcapsules. Microspheres made with 1.5% alginate were divided into 2 groups that were first coated with either 0.1% PLO or PLL, followed by a second coating with 0.25% alginate. After liquefaction of the inner alginate core with sodium citrate, the microcapsules were washed with saline and used for experiments. Pore size exclusion studies were performed with FITC-labeled lectins incubated with encapsulated pig islets followed by examination for fluorescence activity. Mechanical strength was assessed by an osmotic pressure test and by 36 h of mechanical agitation of microcapsules with inert soda lime beads. The pore size exclusion limit of microcapsules after 20 min of coating was significantly smaller with PLO. While the mean7SEM diameter of PLL-coated microcapsules increased from 718717 to 821717 mm (po0:05) during 14 days incubation at 37 1C, the PLO group did not change in size. Also, PLL group had a higher percentage of broken capsules (52.774.9%) compared to 3.172.05% for PLO capsules (po0:0001; n ¼ 6). We conclude that PLOcoated alginate microcapsules are mechanically stronger and provide better perm-selectivity than PLL-coated microcapsules. r 2005 Elsevier Ltd. All rights reserved. Keywords: Alginate; Microencapsulation; Poly-amino acid membrane; Physical properties
1. Introduction The alginate–polylysine microencapsulation procedure as developed by Lim and Sun has been the dominant protocol for cell microencapsulation for over 20 years [1]. Although some variation has occurred, the procedure essentially involves gelling alginate/islet microspheres with a divalent cationic solution such as BaCl2 or CaCl2, applying a polyamino acid layer, followed by an outer coating with very dilute concentration of alginate [2]. Whereas current transplantation procedures rely on immunosuppressive protocol, the hallmark of the microencapsulated islet is based on its ability to create a permeability barrier between graft Corresponding author. Tel.: +1 312 567 3858; fax: +1 312 567 5707. E-mail address: [email protected] (E.C. Opara).
0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2005.05.009
tissue and the host immune system [3,4]. Successful use of this procedure is dependent on the viability of the microencapsulated islet, which in turn relies on the ability of the alginate microcapsule to prevent exposure of the tissue to immune system components [5,6]. In the field of cell microencapsulation, there is a need to develop more durable capsules that would permit long-term function of encapsulated cells [7]. In addition, biocompatibility of the capsule is essential considering that fibrosis may prevent or restrict the flow of important nutrients into the capsule and result in islet necrosis and graft failure. Low swelling capsules that have increased mechanical strength and biocompatibility are less likely to rupture or invoke inflammatory responses. Studies examining the molecular composition and purification of alginate grades have resulted in the production of more biocompatible microcapsules and longer functioning cell transplants [2]. In addition, the
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perm-selective coating with Poly-L-Lysine (PLL) has been found to play a vital role in the encapsulation process as it firmly binds to alginate thereby restricting the permeability of the alginate microcapsule [8]. Although application of PLL has resulted in the reversal of hyperglycemia after encapsulated islet transplantation, some studies have suggested that PLL can increase the likelihood of host inflammatory responses [9,10]. Without application of a final dilute layer of alginate, non-reacted PLL may also invoke immune reactions from the host [8]. While results of islet graft function obtained with PLL have been inconsistent, the benefits of an alternative polycation, such as Poly-L-Ornithine (PLO), remains to be fully explored. We have previously shown that removing Ca++ from the hygroscopic alginate core or replacing it with the monovalent cation, Na+, can benefit the microencpasulation process by reducing the swelling phenomenon and polymorphism seen in alginate-PLL microcapsules [7,10]. However, several studies have suggested that PLO may have some advantages for microencapsulation than the traditional use of PLL for coating alginate microcapsules [10–12], albeit, there is scarcity of data on the physical properties of alginate microcapsules coated with PLO. In response to the pressing need for effective and durable immunoisolation of transplant tissue, our study was designed to address the extent to which PLL and PLO can individually affect perm-selectivity and microbead swelling, which affects the mechanical strength of alginate microcapsules.
2. Materials and methods 2.1. Materials Ultrapurified sodium alginate [EX-8085] (3% w/v) with a high content of mannuronic acid was obtained from Kelco/ Monsanto (San Diego, CA). Liberase PI enzyme blend was obtained from Roche Molecular Biochemicals (Indianapolis, IN). Hanks balanced salt solution was purchased from GIBCO BRL (Grand Island, NY). FITC-Triticum Vulgare (WGA), and FITC-Ricinus Communis I (RCA-I), FITC-Sambucas Nigra (SNA), were obtained from EY Lab Inc. FITC-Maackia Amurensis I was purchased from Vector Laboratories (Burlingame, CA). Trypan Blue, Poly Lysine, Poly-L-Ornithine and all analytical chemicals for solutions were purchased from Sigma Chemical (St. Louis, Mo.) 2.2. Surgery and isolation of porcine islet cells Porcine islets were isolated using a modification of a previously described procedure [13]. Briefly, the pigs were anesthetized with 2% Isoflurane, and following intra-arterial infusion of ice-cold University of Wisconsin solution, a complete pancreatectomy was performed with sterile techniques. The pancreas was weighed and placed in a dish for
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cannulation of the pancreatic duct and infusion of digestion medium, comprising Hanks balanced salt solution containing Liberase PI enzyme blend, 0.5 mg/mL; Deoxyribonuclease I, 10,000 U; and 1 mmol/L Trolox, a water-soluble analogue of vitamin E. After 15 min of incubation on ice, the pancreas was incubated at 37 1C for 45–60 min with slight and periodic agitation. The digested tissue was filtered using a nylon mesh with a pore size of 1000 mm, and the islet cells were purified using an Optiprep density separation procedure [14]. The islet cells were then washed in Hanks balanced salt solution and identified by Dithizone staining.
2.3. Microencapsulation with 2-channel air jacket microencapsulator Prior to encapsulation, tubes containing isolated pig islets were suspended in fresh RPMI 1640 solution. Alginate was then added to the suspension, which resulted in a 1.5% solution of alginate and islets. Encapsulation was performed with a 2-channel air jacket microencapsulator. Droplet formation was achieved with a blunt, 25 gage needle tip, and alginate or islet/alginate droplets were allowed to gel by crosslinking in 1.1% CaCl2. To ensure saturation of active binding sites, the microbeads were incubated in this solution for 15–30 min prior to washing with normal saline. Two groups of capsules were made; one group contained empty microcapsules (without islet tissue), and the other consisted of microcapsules containing pig islets. After the incubation period, the CaCl2 was removed from both groups and capsules were washed twice with saline. Capsules from both groups were then further divided into 4 subgroups, based on the type and duration of perm-selective coating. The subgroups consisted microcapsules coated with PLL or PLO for either 6 or 20 min. Following PLL and PLO coatings, capsules were subsequently washed with saline, after which, a layer of 0.25% sodium alginate was applied to the outer coating of the capsules. Chelation (liquefaction) of the inner Ca2++-alginate core was achieved by suspension in 55 mM sodium citrate for 7 min. Capsules were then rinsed with saline and treated with 6 mM Na2SO4 for 10 min [7], prior to final washings with saline. The capsule suspensions were gently mixed on a rotor (Clay Adams Nutator, Becton Dickinson, Sparks, MD) during the incubation periods involved in the washings, coatings, and chelation steps.
2.4. Lectin-mediated fluorescence Following the washing procedure, 150 mL of encapsulated pig islets were sorted into appropriate groups and incubated with one of four lectins that varied in molecular weight. Lectin incubation consisted of either incubation with 15 mL (1 mg/mL) of Triticum Vulgare (WGA, MW: 36,000), Maackia Amurensis (MAL-I, m.w: 75,000), Ricinus Communis (RCA-I, m.w: 120,000), or Sambuca Nigra (SNA, m.w: 150,000). Capsules were incubated for 48 h at 4 1C with mixing, after which, they were examined on an Olympus BH2 Fluorescent Microscope for fluorescence activity within the capsules.
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2.5. Analysis of microcapsule size changes Capsules that were coated with PLL or PLO for 6 min were suspended in saline and placed in a 37 1C incubator. Diameter measurements were made using an inverted light microscope (Olympus CK40) that was linked to the UTHSCA Image Tool computer program (University of Texas, Austin). Diameter measurements were performed during the course of a 14-day incubation period.
data was performed using an analysis of variance (ANOVA) computer program (GraphPad, San Diego, CA), and depending on the outcome of ANOVA, the Tukey–Kramer multiplecomparison test was used to assess the significance of differences. In comparisons of the significance of the difference between the means of two groups, the student’s t-test was performed, and in all cases, a value of po0:05 was accepted as significant.
2.6. Induction of mechanical stress using osmotic pressure
3. Results
Osmotic stress was applied using a modification of a previously described procedure [15]. Aliquots of alginate microcapsules coated for 6 min with either PLL or PLO were placed in 10 mL of H2O and incubated at 37 1C for 2 h. Capsules were then washed with saline and stained with 0.5% (w/v) Trypan blue. A subsequent washing with saline was then performed prior to assesment of broken and intact capsules using an inverted light microscope.
3.1. Polycation coating and microcapsule pore exclusion size
2.7. Induction of mechanical stress using bead agitation Bead agitation for mechanical stress was applied using a modification of a previously reported procedure [16]. Groups of 350, 6-min PLL- and PLO-coated capsules were placed into flasks containing approximately 6.5 g of 3 mm inert soda lime beads (VWR Scientfic Products Corporation) and 30 mL of normal saline. Capsules from each group were then subjected to mechanical stress for 36 h by shaking at approximately 300 RPM using a Lab Line Orbital Shaker. The percentage of broken and unbroken capsules were determined manually by visual analysis and handpicking under a stereomicroscope (Swift Instruments). 2.8. Data analysis The diameters of the microspheres were measured on day 0 (control) and they were subsequently incubated in saline at 37 1C for 2 weeks with size assessment every 2 days. Microsphere diameters were determined using Image Tool version 2.00-computer software (UTHSCSA University of Texas Health Science Center in San Antonio). The data were then normalized by calculation of the percent change from control (day 0). These data, expressed as mean7SEM, were used for statistical analysis of changes in sizes. Statistical evaluation of
Experiments were performed to examine the effects of PLL and PLO perm-selective membranes and the duration of coating on microcapsule pore size exclusion by exposing encapsulated islets to various sizes of FITC labeled lectins. In each group the percentage of fluorescent capsules was quantified as shown in Table 1. Upon examination after incubation, unencapsulated naked porcine islets contained 100% fluorescence, while empty PLL- or PLO-coated capsules contained no fluorescence, which indicated that the presence of porcine islet tissue was necessary for fluorescent activity to occur within the microcapsules. Furthermore, encapsulated porcine islets without PLL or PLO coatings also contained 100% fluorescence, which indicated that without perm-selective coating, lectins were able to permeate the alginate microcapsules. With 6-min PLLcoated capsules, no capsules incubated with SNA (m.w ¼ 150 KD) and RCA-I (m.w ¼ 120 KD) fluoresced while 100% of MAL-I (m.w ¼ 75 KD) and WGA (m.w ¼ 36 KD) capsules fluoresced. Microcapsules that were coated for 20 min with PLL showed no change in fluorescence compared to those coated for 6 min with PLL. The 6-min PLO-coated capsules showed the same results as were observed with the capsules coated for 6 and 20 min with PLL. However, in comparison to these groups, a significantly lower level of fluorescence (10.2574.6%) was observed when capsules were coated with PLO for 20 min, as shown in Table 1.
Table 1 Permeability of PLL and PLO-coated alginate microcapsules exposing to FITC lectin permeability n ¼ 6 Lectin
Pig tissue (%)
No PLL/ PLO (%)
Empty PLL/ PLO (%)
6 min. PLL (%)
20 min. PLL (%)
6 min. PLO (%)
20 min. PLO
WGA (36 kD) MAL-I (75 kD) RCA-I (120 kD) SNA (150 kD)
100 100 100 100
100 100 100 100
0 0 0 0
100 100 0 0
100 100 0 0
100 100 0 0
100% 10.25%74.16 0% 0%
Microcapsules containing pig islets were incubated with FITC-lectins of various sizes for either 6 or 20 min, prior to examination for fluorescence activity within the microcapsules.
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3.2. Polycation coating and microbead swelling We examined if PLO coating would affect the phenomenon of bead swelling that is commonly seen in PLL-coated alginate microcapsules. Batches of alginate microcapsules coated for 6 min with either PLL or PLO and incubated in normal saline were assessed over a 14-day period for changes in size using measurements of their diameters. As illustrated in
PLL vs. PLO Bead Swelling
900
Diameter (Micrometers)
Days vs PLL Days vs PLO 850
800
750
700
650 0
2
4
6 8 10 Time (Days)
12
14
16
Fig. 1. Changes in the diameter of alginate microcapsules during 14day saline incubation at 37 1C. Capsules were coated with either PLL or PLO. Data represent mean7SEM (n ¼ 20 from each of 3 separate microcapsule preparations).
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Figs. 1 and 2, there was a significant increase in the diameters of PLL-coated capsules. In contrast, PLOcoated capsules showed no statistically significant change in size over the incubation period. 3.3. Polycation coating and mechanical strength In initial studies of the mechanical strength of microcapsules, we compared the effects of PLL and PLO coatings on the mechanical strength of microcapsules that were exposed to a hypotonic environment. Within minutes of incubation in H2O, microcapsules from both groups rapidly increased in size. As illustrated in Fig. 3, the resultant osmotic stress in the presence of Trypan blue staining, showed that a majority of the capsules in the PLL group ruptured as demonstrated by large fissures on the surface of the capsule. Although microcapsules in both groups were prone to rupture, those coated with PLL had significantly higher breakage when compared to PLO-treated capsules. To simulate in vivo shear stress, additional studies were performed using 3 mm beads mixed with microcapsules and incubated for 36 h with shaking. Capsules coated with PLL experienced 52.774.92% breakage following subjection to 36 h of agitation in the presence of soda lime beads. In contrast, only a negligible percentage of the PLO-coated microcapsules (3.0572.05%) were found to be broken, as illustrated in Fig. 4. Assessment of the effects of both osmotic pressure and bead agitation procedures indicated that
Fig. 2. Visual image of size changes in alginate microcapsules. During the incubation period, PLL-coated capsules (upper panel) increased significantly while PLO-coated capsules (lower panel) showed no significant change in size.
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PLO coating resulted in alginate microcapsules that were approximately 50% less likely to break when compared to PLL treatment using simulated methods of induced mechanical stress. See Fig. 4.
4. Discussion The effectiveness of immunoisolation by microencapsulation is strongly linked to the permeability of the
Fig. 3. Effect of PLL and PLO coatings on microcapsules response to osmotic pressure. Following exposure to osmotic pressure, capsules from PLL and PLO groups were suspended in saline and observed at 4 and 10 magnification under an inverted light microscope.
microcapsule by immune cells and cytotoxic antibodies [17]. Increasing the time of interaction between the polycation and alginate microcapsules should allow for increased binding of the polycation on the capsule surface [18–20]. In contrast to previous reports, in the present study, 20-min PLL coating of alginate microcapsules showed no reduction in the microcapsule pore size when compared to 6-min duration of coating. However, it is possible that the pore size was reduced for the molecular size range of 120 and 150 KD during the 20-min duration, but was not detected by our method of assessment. In addition to decreased lectin permeability, we also found that PLO coating nearly eliminated bead swelling while significantly increasing the mechanical strength of the alginate microcapsule. The significance of this finding is vital in that breakage or rupture of the alginate membrane should correspondingly be reduced in PLO-coated alginate microcapsules. Reduction in capsule swelling will also result in lower shear stress, increased biocompatibility, and increased islet viability [21]. Long-term swelling of alginate microcapsules leads to capsule polymorphism and may also lead to a gradual increase in microcapsule pore size and permeability [22]. Previous studies have indicated that membrane thickness is an indicator of membrane strength and permeability [18,19]. The amino acid monomer of PLO is shorter in structure than that of PLL by one methyl group [23]. This difference in structure likely allows PLO to bind more efficiently to the alginate membrane, which in turn would result in increased membrane thickness and strength.
Fig. 4. Effect of PLL and PLO coatings on microcapsule mechanical strength. (A) Ruptured capsules were quantified following subjection to induced osmotic stress. (B) Following 36 h of bead agitation, broken capsules in both PLL- and PLO-coated capsules were quantified. Data represent mean7SEM (n ¼ 6).
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Apart from such factors as gelling cation, alginate grade, and alginate purity, the alginate–polycation complex plays an important role in the stability of alginate microcapsules [24,25]. Using PLL for coating alginate microcapsules of transplanted islets has resulted in promising but limited success. Previous studies have shown inflammatory reactions to transplanted alginate microcapsules [26,27]. A recent study has indicated that certain concentrations of PLL have potent necrosis inducing properties on encapsulated islet transplants [28]. It remains to be determined if further investigations into the use of other membrane polymers, which include, agarose, polyethylene glycol, polyacrylates and AN69 hydrogel may yield greater opportunities for immunoisolation of islets [29–32]. Some studies performed in rodents have suggested that an uncoated alginate microcapsule by itself would be suitable for transplantation [33,34]. However, transplantation of uncoated encapsulated islets would require low dose immunosuppression, and in addition, our study and many others have indicated that a 150 KD immune system component, such as IgG, would be fully capable of traversing the capsule [20,28,35], thus exposing the encapsulated islets to destruction by these cytotoxic molecules. The immunological effects of PLO on cell viability are still unclear, however, previous groups have successfully reversed hyperglycemia for extended periods with PLO-coated alginate microcapsules [11,12], which along with our present data on the superior physical characteristics of PLO-coated alginate microcapsules, suggest that it may be a better alternative to PLL-coated microcapsules. In conclusion, we have shown that PLO coating can reduce swelling and increase the mechanical strength of alginate microcapsules when compared to PLL coating. We also found that PLO coating more effectively restricts higher molecular weight components than PLL from permeating the microcapsules. The advantages provided in terms of capsule resistance and molecular weight exclusion should result in alginate microcapsules that have increased structural integrity and biocompatibility.
Acknowledgment We thank Spencer Bridges and Yusuf Neville for invaluable technical assistance. References [1] Lim F, Sun AM. Microencapsulated islets as bioartificial endocrine pancreas. Science 1980;210:908–10. [2] Kendall WF, Collins BH, Opara EC. Islet cell transplantation for the treatment of diabetes mellitus. Expert Opin Biol Ther 2001;2:503–11.
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[3] Schrezenmeir J, Faust P, Laue C, Kirchgessner J, Stallmach T, Muntefering H, Beyer J. Immunoprotection by biocompatible membrane permits transplantation of piscine islets to rats. Transplant Proc 1989;21:2730–5. [4] Lanza RP, Borland RM, Staruk JE, Appel MC, Solomon MA, Chick WL. Transplantation of encapsulated canine islets into spontaneously diabetic BB/W or rats without immunosuppresion. Endocrinology 1992;131:637–42. [5] Ricker A, Stockberger S. Hyperimmune response to microencapsulated xenogenic tissue in NOD mice. Diabetes 1986;35:61A. [6] Basta G, Sarchielli P, Luca G, Racanicchi L, Nastruzzi C, Guido L, Mancuso F, Macchiarulo G, Calabrese G, Brunetti P, Calafiore R. Optimized parameters for microencapsulation of pancreatic islet cells: an in vitro study clueing on islet graft immunoprotection in type 1 diabetes mellitus. Transplant Immunol 2004;13:289–96. [7] Darrabie M, Freeman BK, Kendall Jr. WF, Hobbs HA, Opara EC. Durability of sodium sulfate-treated polylysine-alginate microcapsules. J Biomed Mater Res 2001;54:396–9. [8] Thu B, Bruheim P, Espevik T, Smidsrod O, Soon-Shiong P, Skjak-Braek G. Alginate polycation microcapsules I. Interaction between alginate and polycation. Biomaterials 1996;17: 1031–40. [9] Strand BL, Gaserod O, Kulseng B, Espevik T, Skjak-Braek G. Alginate–polylysine–alginate microcapsules: effect of size reduction on capsule properties. J Microencapsul 2002;19:615–30. [10] Hobbs HA, Kendall WF, Darrabie M, Collins B, Bridges S, Opara E. Substitution of polyornithine for polylysine in alginate microcapsules. Diabetes 2000;49(Suppl. 1):A111. [11] Brunetti P, Basta G, Falorni A, Calcinaro F, Pietropaolo M, Calafiore R. Immunoprotection of pancreatic islet grafts within artificial microcapsules. Int J Artif Organs 1991;14:789–91. [12] Calafiore R, Basta G, Boselli C, Bufalari A, Giustozzi CM, Luca G, Tortoioli C, Brunetti P. Effects of alginate/polyaminoacidic coherent microcapsulse transplantation in adult pigs. Transplant Proc 1997;29:2126–7. [13] Ching CD, Harland RC, Collins BH, Kendall W, Hobbs H, Opara EC. A reliable method for isolation of viable porcine islet cells. Arch Surg 2001;136:276–9. [14] Krickhahn M, Meyer T, Buhler C, Thiede A, Ulrichs K. Highly efficient isolation of porcine islets of Langerhans for xenotransplantion: numbers, purity, yield and in vitro function. Ann Transplant 2001;6:48–54. [15] Van Raamsdonk JM, Chang PL. Osmotic pressure test: a simple, quantitative method to assess the mechanical stability of alginate microcapsules. J Biomed Mater Res 2001:264–71. [16] Leblond FA, Tessier J, Halle´ J. Quantitative method for the evaluation of biomicrocapsule resistance to mechanical stress. Biomaterials 1996;17:2097–102. [17] De Vos P, De Haan BJ, Wolters GHJ, Strubbe JH, Van Schilfgaarde R. Improved biocompatibility but limited graft survival after purification of alginate for microencapsulation of pancreatic islets. Diabetologia 1997;40:262–70. [18] King GA, Daugulis AJ, Faulkner P, Goosen MF. Alginate–polylysine microcapsules of controlled membrane molecular weight cutoff for mammalian cell culture enginnering. Biotechnol Prog 1987;3:231–40. [19] Vandenbossche GMR, Van Oostveldt PV, Demeester J, Remon JP. The molecular weight cut-off of microcapsulses is determined by the reaction between alginate and polylysine. Biotechnol Bioeng 1993;42:381–6. [20] Brissova M, Petro M, Lacik I, Powers AC, Wang T. Evaluation of microcapsule permeability via inverse size exclusion chromatography. Anal Biochem 1996;242:104–11. [21] Thu B, Bruheim P, Espevik T, Smidsrod O, Soon-Shiong P, Skjak-Braek G. Alginate polycation microcapsules II. Some functional properties. Biomaterials 1996;17:1069–79.
ARTICLE IN PRESS 6852
M.D. Darrabie et al. / Biomaterials 26 (2005) 6846–6852
[22] Hobbs HA, Kendall Jr WF, Darrabie M, Opara EC. Prevention of morphological changes in alginate microcapsules for islet xenotransplantation. J Invest Med 2001;49:572–5. [23] Inaki Y, Tohnai N, Miyabayashi K, Miyata M. Isopoly Lornithine derivative as nucleic acid model. Nucleic Acids Symp Ser 1997;37:25–6. [24] Dusseault J, Leblond FA, Robitaille R, Jourdan G, Tessier J, Menard M, Henley N, Halle J-P. Microencapsulation of living cells in semi-permeable membranes with covalently cross-linked layers. Biomaterials 2005;26:1515–22. [25] De Vos P, De Haan B, Van Schilfgaarde R. Effect of the alginate composition on the biocompatibility of alginate–polylysine microcapsules. Biomaterials 1997;18:273–8. [26] Horcher A, Zekorn T, Siebers U, Klock G, Frank H, Houben R, Bretzel RG, Zimmermann U, Federlin K. Transplantation of microencapsulated islets in rats: evidence for induction of fibrotic overgrowth by islet alloantigens released from microcapsules. Transplant Proc 1994;26:784–6. [27] King A, Sandler S, Andersson A. The effect of host factors and capsule composition on the cellular overgrowth on implanted alginate capsules. J Biomed Mater Res 2001;57:374–83. [28] Strand BL, Ryan TL, In’t Veld P, Kulseng B, Rokstad AM, Skjak-Brek G, Espevik T. Poly L-Lysine induces fibrosis on alginate microcapsules via the induction of cytokines. Cell Transplant 2001;10:263–75.
[29] Iwata H, Amemiya H, Matsuda T, Takano H, Hayashi R, Akutsu T. Evaluation of microencapsulated islets in agarose gel as bioartificial pancreas by studies of hormone secretion in culture and by xenotransplantation. Diabetes 1989;38(Suppl 1): 224–5. [30] Matthew HW, Salley SO, Peterson WD, Klein MD. Complex coacervate microcapsules for mammalian cell culture and artificial organ development. Biotechnol Prog 1993;9:510–9. [31] Sawhney AS, Hubbell JA. Polyethylene oxide-graft-poly L-lysine) copolymers to enhance the biocompatibility of poly L-lysinealginate microcapsule membranes. Biomaterials 1992;13:863–70. [32] Prevost P, Flori S, Collier C, Muscat E, Rolland E. Application of AN69 hydrogel to islet encapsulation. Evaluation in streptozotocin-induced diabetic rat model. Ann NY Acad Sci 1997;831: 344–9. [33] Lanza RP, Kuhtreiber WM, Ecker D, Staruk JE, Marsh J, Chick WL. A simple method for transplanting discordant islets into rats using alginate gel spheres. Transplantation 1995;59: 1486–7. [34] Yang H, O’Hali W, Kearns H, Wright Jr JR. Long-term function of fish islet xenografts in mice by alginate encapsulation. Transplantation 1997;64:28–32. [35] Powers AC, Brissova M, Lacik I, Anilkumar AV, Shahrokhi K, Wang TG. Permeability assessment of capsules for islet transplantation. Ann NY Acad Sci 1997;831:208–16.