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Development of biodegradable porous scaffolds for tissue engineering
Materials Science and Engineering C 17 Ž2001. 63–69 www.elsevier.comrlocatermsec
Development of biodegradable porous scaffolds for tissue engineering Guoping Chen a,) , Takashi Ushida a,b, Tetsuya Tateishi a,b a
b
Tissue Engineering Research Center, National Institute of AdÕanced Industrial Science and Technology, Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan Biomedical Engineering Laboratory, Graduate School of Engineering, The UniÕersity of Tokyo, Tokyo 113-8656, Japan
Abstract Three-dimensional biodegradable porous scaffolds play an important role in tissue engineering. A new method of preparing porous scaffolds composed of synthetic biodegradable polymers was developed by combining porogen leaching and freeze-drying techniques using preprepared ice particulates as the porogen material. The pore structures of the polymer sponges could be manipulated by controlling processing variables such as the size and weight fraction of the ice particulates and the polymer concentration. The synthetic polymer sponges were further hybridized with collagen microsponges to prepare biodegradable hybrid porous sponges of synthetic polymer and collagen. The collagen microsponges were formed in the pores of synthetic polymer sponges. The hybrid sponges exhibited the advantages of both the synthetic polymers and collagen. Hybrid sponges of synthetic polymer, collagen, and inorganic hydroxyapatite were developed by depositing hydroxyapatite particulates on the surfaces of the collagen microsponges in the synthetic polymer–collagen sponges. The use of synthetic polymer sponge as a mechanical skeleton facilitated the formation of these hybrid sponges into desired shapes, contributed good mechanical strength and handling, while the collagen and hydroxyapatite facilitated cell seeding and promoted cell interaction. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Biodegradable polymer; Hybrid sponge; Porous; Collagen; Tissue engineering
1. Introduction The therapies of damaged or lost tissues or organs include tissue or organ transplantation, surgical reconstruction, drug therapy, synthetic prostheses, and medical devices. Tissue or organ transplantation is restricted by an insufficient number of donors. Although the other therapies are not limited by supply, they also have problems. For example, synthetic prostheses and medical devices are not able to replace all the functions of a damaged or lost organ or tissue. The efforts to address these problems and their limitations have elicited the development of new biomaterials and alternative therapies. Tissue engineering has emerged as one such promising alternative approach in treating patients suffering from these problems w1,2x. Tissue engineering involves the expansion of cells from a small biopsy, followed by the culturing of the cells in temporary three-dimensional scaffolds to form the new organ or tissue. By using the
) Corresponding author. Tel.: q81-298-61-3052; fax: q81-298-612565. E-mail address: [email protected] ŽG. Chen..
patient’s own cells, this approach has the advantages of autografts, but without the problems associated with adequate supply. With this approach, porous three-dimensional temporary scaffolds play an important role in manipulating cell functions. Isolated and expanded cells adhere to the temporary scaffold in all three dimensions, proliferate, and secrete their own extracellular matrices ŽECM., replacing the biodegrading scaffold. Therefore, in addition to permitting cell adhesion, promoting cell growth, and allowing retention of differentiated cell functions, the scaffold should be biocompatible, biodegradable, highly porous with a large surface to volume ratio, mechanically strong, and capable of being formed into desired shapes. The biomaterials used to construct these porous scaffolds include synthetic biodegradable polymers such as polyŽglycolic acid. ŽPGA., polyŽlactic acid. ŽPLA., and their copolymer polyŽDLlactic-co-glycolic acid. ŽPLGA., and naturally derived polymers such as collagen and inorganic hydroxyapatite w3–5x. To prepare three-dimensional biodegradable porous scaffolds, we developed a new method that incorporates the use of ice particulates as the porogen material. To develop new kinds of hybrid porous scaffolds for tissue engineering, we also hybridized synthetic biodegradable polymers with collagen and hydroxyapatite.
0928-4931r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 Ž 0 1 . 0 0 3 3 8 - 1
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2. Preparation of porous sponges of synthetic biodegradable polymers Porous three-dimensional scaffolds fabricated from synthetic biodegradable polymers such as PGA, PLA, and PLGA have been widely used in the tissue engineering of cartilage, bone, skin, ligament, etc. w6–8x. Several methods have been developed to prepare these kinds of porous
three-dimensional biodegradable scaffolds, including phase separation w9x, emulsion freeze drying w10x, gas foaming w11x, fiber bonding w12x and porogen leaching w13x. With phase separation, the pore structures of the scaffolds cannot be easily controlled; also, it is difficult to obtain large pores. Both the emulsion freeze-drying technique and an expansion technique using high-pressure CO 2 gas often result in a closed cellular structure within the scaffold.
Fig. 1. Preparation scheme of ice microparticulates Ža. and polymer sponges Žb..
G. Chen et al.r Materials Science and Engineering C 17 (2001) 63–69
Scaffolds constructed by the fiber bonding technique lack structural stability. Compared to the above methods, the porogen leaching method provides easy control of the pore structure and has been well established. This method involves the casting of a mixture of polymer solution and porogen in a mold, drying the mixture, followed by a leaching out of the porogen with water to generate the pores. Usually, watersoluble particulates such as salts and carbohydrates are used as the porogen materials. The pore structures can easily be manipulated by controlling the property and fraction of the porogen, and the process is reproducible. We developed a new method to prepare porous scaffolds of synthetic biodegradable polymers by combining porogen leaching and freeze-drying techniques using preprepared ice particulates as the porogen material w14x. The preparative scheme is shown in Fig. 1. Ice particulates were formed by injecting cold deionized water into liquid
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nitrogen through a capillary. The formed ice particulates were almost spherical. Their diameters could be controlled by the spraying speed and travel distance. Polymer solutions in chloroform were precooled to y20 8C, then mixed with the ice particulates. The dispersion was then vortexed and poured into an aluminum mold. This process was conducted in a cool room Ž4 8C.. The dispersion was frozen by placing the mold in liquid nitrogen, freeze-dried for 48 h under liquid nitrogen freezing, and for another 48 h at room temperature, to completely remove the solvent to form the synthetic polymer sponges. The structures of the synthetic polymer sponges were observed by scanning electron microscopy ŽSEM.. The SEM photomicrographs of the cross-sections of polyŽLlactic acid. ŽPLLA. sponges prepared by this method are shown in Fig. 2. The sponges were highly porous with evenly distributed and interconnected pore structures. The pore shapes were almost the same as those of the ice particulates. The degree of interconnection within the sponges increased as the weight fraction of the ice particulates increased. The polymer concentration also had some effect on the pore wall structure, i.e. lower polymer concentration resulted in more porous pore wall structures. The porosity and surface arearweight ratio increased with the increase of the weight fraction of the ice particulates. Therefore, the pore structure of the porous temporary scaffolds could be manipulated by varying the shape, weight fraction, size of the ice particulates, and the polymer concentration. The use of ice particulates as the porogen material enabled replacement of the conventional method of porogen leaching by repeated washing with water with freezedrying. The porogen removal thus became easier and more complete.
3. Hybridization of synthetic biodegradable polymers with collagen
Fig. 2. SEM photomicrographs of cross-sections of PLLA sponges prepared with the weight fractions of ice particulates: 80% Ža. and 90% Žb..
The synthetic biodegradable polymers used for tissue engineering are easily formed into desired shapes with good mechanical strength. Their periods of degradation can also be manipulated by controlling the crystallinity, molecular weight, and copolymer ratio of lactic acid to glycolic acid. Despite these advantages, the scaffolds derived from synthetic polymers lack cell-recognition signals, and their hydrophobic property hinders smooth cell seeding. In contrast, naturally derived collagen has the potential advantages of specific cell interactions and hydrophilicity, but scaffolds constructed entirely of collagen have poor mechanical strength. Therefore, these two kinds of biodegradable polymers were hybridized, as shown in Fig. 3, to combine their advantages w15–18x. The sponges of synthetic polymers were immersed in a collagen solution under a vacuum so that the sponge pores
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Fig. 3. Schematic illustration of hybridization of synthetic biodegradable polymers with collagen.
filled with collagen solution. The synthetic polymer sponges containing the collagen solution were then frozen and freeze-dried to allow the formation of collagen microsponges in the sponge pores. The collagen microsponges were further cross-linked by treatment with glutaraldehyde. After being washed with deionized water and freeze-dried, the hybrid sponges of synthetic polymers and collagen were formed. The hybrid structure of the PLGA–collagen hybrid sponge is shown in Fig. 4a. Collagen microsponges with interconnected pore structures were formed in the pores of the PLGA sponge. SEM–electron probe microanalysis of elemental nitrogen indicates that microsponges of collagen were formed in the pores of the PLGA sponge and that the pore surfaces were coated with collagen. Collagen microsponges in the hybrid sponge were cross-linked by glutaraldehyde. There are two methods of cross-linking, which are generally used to cross-link collagen: physical cross-linking and chemical cross-linking w19,20x. Physical cross-linking includes dehydrothermal treatment and ultraviolet irradiation, while the chemical method uses chemical cross-linking agents such as glutaraldehyde and hexamethylene diisocyanate. Because the synthetic biodegradable polymer sponges could not withstand the dehydrothermal treatment and ultraviolet irradiation, the chemical method was used to cross-link the collagen microsponges formed in their pores. In addition, because the collagen sponge would be dissolved in aqueous solution without pretreatment with physical cross-linking, a method of cross-linking with gaseous glutaraldehyde was selected. Such gaseous glutaraldehyde treatment had already been proven effective for collagen cross-linking w21x. The wettability of a polymer scaffold is considered very important for homogeneous and sufficient cell seeding in three dimensions. Because biodegradable synthetic poly-
Fig. 4. SEM photomicrographs of cross-sections of PLGA–collagen hybrid sponge Ža. and L929 cells cultured in the hybrid sponge for 1 day Žb..
G. Chen et al.r Materials Science and Engineering C 17 (2001) 63–69
mers are relatively hydrophobic, it is difficult to deliver a cell suspension in a manner that uniformly distributes transplanted cells throughout their porous scaffolds w6,22x. Numerous techniques have been used to evenly seed cells in PLA- and PLGA-based porous scaffolds, such as prewetting the scaffold with ethanol solution and then replacing it with distilled water and cell suspension, introducing hydrophilic polyŽethyl glycol. into the hydrophobic PLA and PLGA network, or hydrolysis w23–25x. Hybridization with collagen improved the wettability of synthetic sponges with water, which facilitated cell seeding. The hybrid sponges of synthetic polymers and collagen possessed almost the same high degree of mechanical strength as those of synthetic polymers, much higher than that of collagen sponges alone. Mouse fibroblast L929 cells ŽFig. 4b. and bovine articular chondrocytes adhered well to the collagen microsponges of the hybrid sponge. More cells adhered to the hybrid sponges than to the sponges made of synthetic polymers. The cells that adhered to the hybrid sponges proliferated and secreted their respective extracellular matrices with culture time. The hybrid sponges showed a good degree of cell interaction and biocompatibility. The sponges are prime candidates for use as porous scaffolds for the tissue engineering of cartilage, bone, liver, blood vessel, esophagus, etc.
4. Hybridization of synthetic polymers, collagen, and hydroxyapatite Because hydroxyapatite has a composition and a structure very similar to that of natural bone mineral, it has frequently been used for implants, coatings, and scaffolds in bone tissue engineering w26–30x. In natural bone, hy-
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droxyapatite is deposited in a regular manner on the collagen matrix w31x. Additionally, it has been reported that the osteoconductivity of hydroxyapatite can be further improved by associating a protein matrix with the mineral structure w32x. To mimic the unique structure of collagen and hydroxyapatite in natural bone, we developed a hybrid sponge of synthetic polymer, collagen, and hydroxyapatite by depositing hydroxyapatite particulates on the collagen microsponge surfaces of the above hybrid sponge of synthetic polymer and collagen. The deposition of hydroxyapatite particulates was accomplished by alternate immersion of the PLGA–collagen sponge in CaCl 2 and Na 2 HPO4 aqueous solutions and centrifugation w33x. The PLGA–collagen sponge was first immersed in a CaCl 2 solution under a vacuum to fill the sponge spaces with the solution, then incubated in the solution at 37 8C for 12 h. After removal from the CaCl 2 solution, the sponge was centrifuged to remove the excess CaCl 2 solution from the sponge. The sponge was then immersed in a Na 2 HPO4 aqueous solution to introduce the solution into the sponge, after which it was then incubated in the solution at 37 8C for another 12 h. After removal from the Na 2 HPO4 solution, the sponge was again centrifuged, this time to remove the excess Na 2 HPO4 solution. This process of alternate immersion and centrifugation was defined as one cycle. Hydroxyapatite particulates were deposited on the PLGA–collagen sponge by repeating the cycles. With the mechanically strong PLGA–collagen sponge as a template, the PLGA–collagen–hydroxyapatite hybrid sponge was able to maintain its original shape. Centrifugation after each immersion removed the excess aqueous solution remaining on the surfaces of the collagen microsponges, avoiding over-precipitation of the hydroxyapatite onto the collagen surfaces. Fig. 5 is an SEM
Fig. 5. SEM photomicrograph of cross-section of PLGA–collagen–hydroxyapatite hybrid sponge after four cycles of alternate immersion.
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photomicrograph of PLGA–collagen–hydroxyapatite hybrid sponge after four cycles of alternate immersion and centrifugation. Hydroxyapatite particulates were formed on the surfaces of the collagen microsponges of the PLGA– collagen sponge. After one immersion cycle, the deposited particulates were scarce and small. They became denser and grew larger as the number of alternate immersion cycles increased. The surfaces of the collagen microsponges were completely covered with the flake-like hydroxyapatite particulates after three cycles of alternate immersion. Energy-dispersive spectroscopy ŽEDS. analysis showed that the main elements of the hybrid sponge were carbon, oxygen, nitrogen, calcium, and phosphorus. The calciumto-phosphorus molar ratios in the hybrid sponge increased with the number of immersion cycles and became almost the same as that of hydroxyapatite after four cycles. X-ray diffraction ŽXRD. showed that the characteristic absorption peaks of hydroxyapatite were very weak in the initial immersion and became more evident after the second and third immersion cycles. After four alternate immersion cycles, the XRD spectra became almost the same as that of commercially available hydroxyapatite. These results suggest that the deposited hydroxyapatite particulates increased, grew larger, and their level of crystallinity grew as the number of immersion cycles increased. The deposited crystals became hydroxyapatite-like after four cycles of alternate immersion. The use of synthetic polymer sponge as a mechanical skeleton facilitated the formation of the hybrid sponges into desired shapes and gave the hybrid sponges good mechanical properties. Because collagen and hydroxyapatite are the two primary components of extracellular bone matrix, they should provide the hybrid sponge with a good degree of cell interaction and osteoconductivity. Such synthetic polymer– collagen – hydroxyapatite hybrid sponges would prove useful as three-dimensional porous scaffolds for bone tissue engineering.
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The authors acknowledge support from the New Energy and Industrial Technology Development Organization of Japan.
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References
w22x
w1x P. Langer, J.P. Vacanti, Tissue engineering. Science 260 Ž1993. 920–926. w2x R.M. Nerem, A. Sambanis, Tissue engineering: from biology to biological substitutes. Tissue Eng. 1 Ž1995. 3–13. w3x S.J. Peter, M.J. Miller, A.W. Yasko, M.J. Yaszemski, A.G. Mikos, Polymer concepts in tissue engineering. J. Biomed. Mater. Res. 43 Ž1998. 422–427. w4x L.E. Freed, G. Vunjak-Novakovic, R.J. Biron, D.B. Eagles, D.C.
w23x
w24x
w25x
Lesnoy, S.K. Barlow, R. Langer, Biodegradable polymer scaffolds for tissue engineering. BiorTechnology 12 Ž1994. 689–693. B.S. Kim, D.J. Mooney, Development of biocompatible synthetic extracellular matrices for tissue engineering. TIBTECH 16 Ž1998. 224–230. L.E. Freed, J.C. Marquis, A. Nohria, J. Emmanual, A.C. Mikos, R. Langer, Neocartilage formation in vitro and in vivo using cells cultured on synthetic biodegradable polymers. J. Biomed. Mater. Res. 27 Ž1993. 11–23. S.L. Ishaug, G.M. Crane, M.J. Miller, A.W. Yasko, M.J. Yaszemski, A.G. Mikos, Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds. J. Biomed. Mater. Res. 36 Ž1997. 17–28. M.G. Dunn, J.B. Liesch, M.L. Tiku, J.P. Zawadsky, Development of fibroblast-seeded ligament analogs for ACL reconstruction. J. Biomed. Mater. Res. 29 Ž1995. 1363–1371. Y.S. Nam, T.G. Park, Porous biodegradable polymeric scaffolds prepared by thermally induced phase separation. J. Biomed. Mater. Res. 47 Ž1999. 8–17. K. Whang, C.H. Thomas, K.E. Healy, A novel method to fabricate biodegradable scaffolds. Polymer 36 Ž1995. 837–842. D.J. Mooney, D.F. Baldwin, N.P. Suh, J.P. Vacanti, R. Langer, Novel approach to fabricate porous sponges of polyŽD,L-lactic-coglycolic acid. without the use of organic solvents. Biomaterials 17 Ž1996. 1417–1422. A.G. Mikos, Y. Bao, L.G. Cima, D.E. Ingber, J.P. Vacanti, R. Langer, Preparation of polyŽglycolic acid. bonded fiber structures for cell attachment and transplantation. J. Biomed. Mater. Res. 27 Ž1993. 183–189. A.G. Mikos, A.J. Thorsen, L.A. Czerwonka, Y. Bao, R. Langer, D.N. Winslow, J.P. Vacanti, Preparation and characterization of polyŽL-lactic acid. foams. Polymer 35 Ž1994. 1069–1077. G. Chen, T. Ushida, T. Tatsuya, Preparation of polyŽL-lactic acid. and polyŽDL-lactic-co-glycolic acid. foams by use of ice microparticulates. Biomaterials 22 Ž2001. 2563–2567. G. Chen, T. Ushida, T. Tatsuya, Fabrication of PLGA–collagen hybrid sponge. Chem. Lett. Ž1999. 561–562. G. Chen, T. Ushida, T. Tatsuya, Hybrid biomaterials for tissue engineering: a preparative method of PLA or PLGA–collagen hybrid sponge. Adv. Mater. 12 Ž2000. 455–457. G. Chen, T. Ushida, T. Tatsuya, A biodegradable hybrid sponge nested with collagen microsponges. J. Biomed. Mater. Res. 51 Ž2000. 273–279. G. Chen, T. Ushida, T. Tatsuya, A hybrid network of synthetic polymer mesh and collagen sponge. Chem. Commun. Ž2000. 1505– 1506. K.S. Weadock, E.J. Miller, E.L. Keuffel, M.G. Dunn, Effect of physical crosslinking methods on collagen-fiber durability in proteolytic solutions. J. Biomed. Mater. Res. 32 Ž1996. 221–226. L.H.H. Olde Damink, P.J. Dijkstra, M.J.A. van Luyn, P.B. van Wachem, P. Nieuwenhuis, J. Feijen, Glutaraldehyde as a crosslinking agent for collagen-based biomaterials. J. Mater. Sci. 6 Ž1995. 460–472. N. Barbani, P. Giusti, L. Lazzeri, G. Polacco, G. Pizzirani, Bioartificial materials based on collagen: 1. Collagen cross-linking with gaseous glutaraldehyde. J. Biomater. Sci., Polym. Ed. 7 Ž1995. 461–469. H.L. Wald, G. Sarakinos, M.D. Lyman, A.G. Mikos, J.P. Vacanti, R. Langer, Cell seeding in porous transplantation devices. Biomaterials 14 Ž1993. 270–278. D.J. Mooney, S. Park, P.M. Kaufmann, K. Sano, K. McNamara, J.P. Vacanti, R. Langer, Biodegradable sponges for hepatocyte transplantation. J. Biomed. Mater. Res. 29 Ž1995. 959–965. A.G. Mikos, M.D. Lyman, L.E. Freed, R. Langer, Wetting of polyŽL-lactic acid. and polyŽDL-lactic-co-glycolic acid. forms for tissue culture. Biomaterials 15 Ž1994. 55–58. J. Gao, L. Niklason, R. Langer, Surface hydrolysis of polyŽglycolic
G. Chen et al.r Materials Science and Engineering C 17 (2001) 63–69
w26x
w27x
w28x
w29x
acid. meshes increases the seeding density of vascular smooth muscle cells. J. Biomed. Mater. Res. 42 Ž1998. 417–424. J.E. Devin, M.A. Attawia, C.T. Laurencin, Three-dimensional degradable porous polymer-ceramic matrices for use in bone repair. J. Biomater. Sci., Polym. Ed. 7 Ž1996. 661–669. R. Zhang, P.X. Ma, PolyŽalpha-hydroxyl acid.rhydroxyapatite porous composites for bone-tissue engineering: I. Preparation and morphology. J. Biomed. Mater. Res. 44 Ž1999. 446–455. W.L. Murphy, D.H. Kohn, D.J. Mooney, Growth of continuous bone-like mineral within porous polyŽlactide-co-glycolide. scaffolds in vitro. J. Biomed. Mater. Res. 50 Ž2000. 50–58. D. Bakos, M. Soldan, I. Hernandez-Fuentes, Hydroxyapatite–collagen–hyaluronic acid composite. Biomaterials 20 Ž1999. 191–195.
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w30x M.B. Yaylaoglu, C. Yildiz, F. Korkusuz, V. Hasirci, A novel osteochondral implant. Biomaterials 20 Ž1999. 1513–1520. w31x K. Soballe, Hydroxyapatite ceramic coating for bone implant fixation: mechanical and histological studies in dogs. Acta. Orthop. Scand., Suppl. 255 Ž1993. 1–58. w32x A. Rovira, R. Bareille, I. Lopez, F. Rouais, L. Bordenave, C. Rey, M. Rabaud, Preliminary report on a new composite material made of calcium phosphate, elastin peptides and collagens. J. Mater. Sci.: Mater. Med. 4 Ž1993. 372–380. w33x G. Chen, T. Ushida, T. Tateishi, PolyŽDL-lactic-co-glycolic acid. sponge hybridized with collagen microsponges and deposited apatite particulates. J. Biomed. Mater. Res. 57 Ž2001. 273–279.
Development of biodegradable porous scaffolds for tissue engineering Guoping Chen a,) , Takashi Ushida a,b, Tetsuya Tateishi a,b a
b
Tissue Engineering Research Center, National Institute of AdÕanced Industrial Science and Technology, Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan Biomedical Engineering Laboratory, Graduate School of Engineering, The UniÕersity of Tokyo, Tokyo 113-8656, Japan
Abstract Three-dimensional biodegradable porous scaffolds play an important role in tissue engineering. A new method of preparing porous scaffolds composed of synthetic biodegradable polymers was developed by combining porogen leaching and freeze-drying techniques using preprepared ice particulates as the porogen material. The pore structures of the polymer sponges could be manipulated by controlling processing variables such as the size and weight fraction of the ice particulates and the polymer concentration. The synthetic polymer sponges were further hybridized with collagen microsponges to prepare biodegradable hybrid porous sponges of synthetic polymer and collagen. The collagen microsponges were formed in the pores of synthetic polymer sponges. The hybrid sponges exhibited the advantages of both the synthetic polymers and collagen. Hybrid sponges of synthetic polymer, collagen, and inorganic hydroxyapatite were developed by depositing hydroxyapatite particulates on the surfaces of the collagen microsponges in the synthetic polymer–collagen sponges. The use of synthetic polymer sponge as a mechanical skeleton facilitated the formation of these hybrid sponges into desired shapes, contributed good mechanical strength and handling, while the collagen and hydroxyapatite facilitated cell seeding and promoted cell interaction. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Biodegradable polymer; Hybrid sponge; Porous; Collagen; Tissue engineering
1. Introduction The therapies of damaged or lost tissues or organs include tissue or organ transplantation, surgical reconstruction, drug therapy, synthetic prostheses, and medical devices. Tissue or organ transplantation is restricted by an insufficient number of donors. Although the other therapies are not limited by supply, they also have problems. For example, synthetic prostheses and medical devices are not able to replace all the functions of a damaged or lost organ or tissue. The efforts to address these problems and their limitations have elicited the development of new biomaterials and alternative therapies. Tissue engineering has emerged as one such promising alternative approach in treating patients suffering from these problems w1,2x. Tissue engineering involves the expansion of cells from a small biopsy, followed by the culturing of the cells in temporary three-dimensional scaffolds to form the new organ or tissue. By using the
) Corresponding author. Tel.: q81-298-61-3052; fax: q81-298-612565. E-mail address: [email protected] ŽG. Chen..
patient’s own cells, this approach has the advantages of autografts, but without the problems associated with adequate supply. With this approach, porous three-dimensional temporary scaffolds play an important role in manipulating cell functions. Isolated and expanded cells adhere to the temporary scaffold in all three dimensions, proliferate, and secrete their own extracellular matrices ŽECM., replacing the biodegrading scaffold. Therefore, in addition to permitting cell adhesion, promoting cell growth, and allowing retention of differentiated cell functions, the scaffold should be biocompatible, biodegradable, highly porous with a large surface to volume ratio, mechanically strong, and capable of being formed into desired shapes. The biomaterials used to construct these porous scaffolds include synthetic biodegradable polymers such as polyŽglycolic acid. ŽPGA., polyŽlactic acid. ŽPLA., and their copolymer polyŽDLlactic-co-glycolic acid. ŽPLGA., and naturally derived polymers such as collagen and inorganic hydroxyapatite w3–5x. To prepare three-dimensional biodegradable porous scaffolds, we developed a new method that incorporates the use of ice particulates as the porogen material. To develop new kinds of hybrid porous scaffolds for tissue engineering, we also hybridized synthetic biodegradable polymers with collagen and hydroxyapatite.
0928-4931r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 Ž 0 1 . 0 0 3 3 8 - 1
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G. Chen et al.r Materials Science and Engineering C 17 (2001) 63–69
2. Preparation of porous sponges of synthetic biodegradable polymers Porous three-dimensional scaffolds fabricated from synthetic biodegradable polymers such as PGA, PLA, and PLGA have been widely used in the tissue engineering of cartilage, bone, skin, ligament, etc. w6–8x. Several methods have been developed to prepare these kinds of porous
three-dimensional biodegradable scaffolds, including phase separation w9x, emulsion freeze drying w10x, gas foaming w11x, fiber bonding w12x and porogen leaching w13x. With phase separation, the pore structures of the scaffolds cannot be easily controlled; also, it is difficult to obtain large pores. Both the emulsion freeze-drying technique and an expansion technique using high-pressure CO 2 gas often result in a closed cellular structure within the scaffold.
Fig. 1. Preparation scheme of ice microparticulates Ža. and polymer sponges Žb..
G. Chen et al.r Materials Science and Engineering C 17 (2001) 63–69
Scaffolds constructed by the fiber bonding technique lack structural stability. Compared to the above methods, the porogen leaching method provides easy control of the pore structure and has been well established. This method involves the casting of a mixture of polymer solution and porogen in a mold, drying the mixture, followed by a leaching out of the porogen with water to generate the pores. Usually, watersoluble particulates such as salts and carbohydrates are used as the porogen materials. The pore structures can easily be manipulated by controlling the property and fraction of the porogen, and the process is reproducible. We developed a new method to prepare porous scaffolds of synthetic biodegradable polymers by combining porogen leaching and freeze-drying techniques using preprepared ice particulates as the porogen material w14x. The preparative scheme is shown in Fig. 1. Ice particulates were formed by injecting cold deionized water into liquid
65
nitrogen through a capillary. The formed ice particulates were almost spherical. Their diameters could be controlled by the spraying speed and travel distance. Polymer solutions in chloroform were precooled to y20 8C, then mixed with the ice particulates. The dispersion was then vortexed and poured into an aluminum mold. This process was conducted in a cool room Ž4 8C.. The dispersion was frozen by placing the mold in liquid nitrogen, freeze-dried for 48 h under liquid nitrogen freezing, and for another 48 h at room temperature, to completely remove the solvent to form the synthetic polymer sponges. The structures of the synthetic polymer sponges were observed by scanning electron microscopy ŽSEM.. The SEM photomicrographs of the cross-sections of polyŽLlactic acid. ŽPLLA. sponges prepared by this method are shown in Fig. 2. The sponges were highly porous with evenly distributed and interconnected pore structures. The pore shapes were almost the same as those of the ice particulates. The degree of interconnection within the sponges increased as the weight fraction of the ice particulates increased. The polymer concentration also had some effect on the pore wall structure, i.e. lower polymer concentration resulted in more porous pore wall structures. The porosity and surface arearweight ratio increased with the increase of the weight fraction of the ice particulates. Therefore, the pore structure of the porous temporary scaffolds could be manipulated by varying the shape, weight fraction, size of the ice particulates, and the polymer concentration. The use of ice particulates as the porogen material enabled replacement of the conventional method of porogen leaching by repeated washing with water with freezedrying. The porogen removal thus became easier and more complete.
3. Hybridization of synthetic biodegradable polymers with collagen
Fig. 2. SEM photomicrographs of cross-sections of PLLA sponges prepared with the weight fractions of ice particulates: 80% Ža. and 90% Žb..
The synthetic biodegradable polymers used for tissue engineering are easily formed into desired shapes with good mechanical strength. Their periods of degradation can also be manipulated by controlling the crystallinity, molecular weight, and copolymer ratio of lactic acid to glycolic acid. Despite these advantages, the scaffolds derived from synthetic polymers lack cell-recognition signals, and their hydrophobic property hinders smooth cell seeding. In contrast, naturally derived collagen has the potential advantages of specific cell interactions and hydrophilicity, but scaffolds constructed entirely of collagen have poor mechanical strength. Therefore, these two kinds of biodegradable polymers were hybridized, as shown in Fig. 3, to combine their advantages w15–18x. The sponges of synthetic polymers were immersed in a collagen solution under a vacuum so that the sponge pores
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Fig. 3. Schematic illustration of hybridization of synthetic biodegradable polymers with collagen.
filled with collagen solution. The synthetic polymer sponges containing the collagen solution were then frozen and freeze-dried to allow the formation of collagen microsponges in the sponge pores. The collagen microsponges were further cross-linked by treatment with glutaraldehyde. After being washed with deionized water and freeze-dried, the hybrid sponges of synthetic polymers and collagen were formed. The hybrid structure of the PLGA–collagen hybrid sponge is shown in Fig. 4a. Collagen microsponges with interconnected pore structures were formed in the pores of the PLGA sponge. SEM–electron probe microanalysis of elemental nitrogen indicates that microsponges of collagen were formed in the pores of the PLGA sponge and that the pore surfaces were coated with collagen. Collagen microsponges in the hybrid sponge were cross-linked by glutaraldehyde. There are two methods of cross-linking, which are generally used to cross-link collagen: physical cross-linking and chemical cross-linking w19,20x. Physical cross-linking includes dehydrothermal treatment and ultraviolet irradiation, while the chemical method uses chemical cross-linking agents such as glutaraldehyde and hexamethylene diisocyanate. Because the synthetic biodegradable polymer sponges could not withstand the dehydrothermal treatment and ultraviolet irradiation, the chemical method was used to cross-link the collagen microsponges formed in their pores. In addition, because the collagen sponge would be dissolved in aqueous solution without pretreatment with physical cross-linking, a method of cross-linking with gaseous glutaraldehyde was selected. Such gaseous glutaraldehyde treatment had already been proven effective for collagen cross-linking w21x. The wettability of a polymer scaffold is considered very important for homogeneous and sufficient cell seeding in three dimensions. Because biodegradable synthetic poly-
Fig. 4. SEM photomicrographs of cross-sections of PLGA–collagen hybrid sponge Ža. and L929 cells cultured in the hybrid sponge for 1 day Žb..
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mers are relatively hydrophobic, it is difficult to deliver a cell suspension in a manner that uniformly distributes transplanted cells throughout their porous scaffolds w6,22x. Numerous techniques have been used to evenly seed cells in PLA- and PLGA-based porous scaffolds, such as prewetting the scaffold with ethanol solution and then replacing it with distilled water and cell suspension, introducing hydrophilic polyŽethyl glycol. into the hydrophobic PLA and PLGA network, or hydrolysis w23–25x. Hybridization with collagen improved the wettability of synthetic sponges with water, which facilitated cell seeding. The hybrid sponges of synthetic polymers and collagen possessed almost the same high degree of mechanical strength as those of synthetic polymers, much higher than that of collagen sponges alone. Mouse fibroblast L929 cells ŽFig. 4b. and bovine articular chondrocytes adhered well to the collagen microsponges of the hybrid sponge. More cells adhered to the hybrid sponges than to the sponges made of synthetic polymers. The cells that adhered to the hybrid sponges proliferated and secreted their respective extracellular matrices with culture time. The hybrid sponges showed a good degree of cell interaction and biocompatibility. The sponges are prime candidates for use as porous scaffolds for the tissue engineering of cartilage, bone, liver, blood vessel, esophagus, etc.
4. Hybridization of synthetic polymers, collagen, and hydroxyapatite Because hydroxyapatite has a composition and a structure very similar to that of natural bone mineral, it has frequently been used for implants, coatings, and scaffolds in bone tissue engineering w26–30x. In natural bone, hy-
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droxyapatite is deposited in a regular manner on the collagen matrix w31x. Additionally, it has been reported that the osteoconductivity of hydroxyapatite can be further improved by associating a protein matrix with the mineral structure w32x. To mimic the unique structure of collagen and hydroxyapatite in natural bone, we developed a hybrid sponge of synthetic polymer, collagen, and hydroxyapatite by depositing hydroxyapatite particulates on the collagen microsponge surfaces of the above hybrid sponge of synthetic polymer and collagen. The deposition of hydroxyapatite particulates was accomplished by alternate immersion of the PLGA–collagen sponge in CaCl 2 and Na 2 HPO4 aqueous solutions and centrifugation w33x. The PLGA–collagen sponge was first immersed in a CaCl 2 solution under a vacuum to fill the sponge spaces with the solution, then incubated in the solution at 37 8C for 12 h. After removal from the CaCl 2 solution, the sponge was centrifuged to remove the excess CaCl 2 solution from the sponge. The sponge was then immersed in a Na 2 HPO4 aqueous solution to introduce the solution into the sponge, after which it was then incubated in the solution at 37 8C for another 12 h. After removal from the Na 2 HPO4 solution, the sponge was again centrifuged, this time to remove the excess Na 2 HPO4 solution. This process of alternate immersion and centrifugation was defined as one cycle. Hydroxyapatite particulates were deposited on the PLGA–collagen sponge by repeating the cycles. With the mechanically strong PLGA–collagen sponge as a template, the PLGA–collagen–hydroxyapatite hybrid sponge was able to maintain its original shape. Centrifugation after each immersion removed the excess aqueous solution remaining on the surfaces of the collagen microsponges, avoiding over-precipitation of the hydroxyapatite onto the collagen surfaces. Fig. 5 is an SEM
Fig. 5. SEM photomicrograph of cross-section of PLGA–collagen–hydroxyapatite hybrid sponge after four cycles of alternate immersion.
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photomicrograph of PLGA–collagen–hydroxyapatite hybrid sponge after four cycles of alternate immersion and centrifugation. Hydroxyapatite particulates were formed on the surfaces of the collagen microsponges of the PLGA– collagen sponge. After one immersion cycle, the deposited particulates were scarce and small. They became denser and grew larger as the number of alternate immersion cycles increased. The surfaces of the collagen microsponges were completely covered with the flake-like hydroxyapatite particulates after three cycles of alternate immersion. Energy-dispersive spectroscopy ŽEDS. analysis showed that the main elements of the hybrid sponge were carbon, oxygen, nitrogen, calcium, and phosphorus. The calciumto-phosphorus molar ratios in the hybrid sponge increased with the number of immersion cycles and became almost the same as that of hydroxyapatite after four cycles. X-ray diffraction ŽXRD. showed that the characteristic absorption peaks of hydroxyapatite were very weak in the initial immersion and became more evident after the second and third immersion cycles. After four alternate immersion cycles, the XRD spectra became almost the same as that of commercially available hydroxyapatite. These results suggest that the deposited hydroxyapatite particulates increased, grew larger, and their level of crystallinity grew as the number of immersion cycles increased. The deposited crystals became hydroxyapatite-like after four cycles of alternate immersion. The use of synthetic polymer sponge as a mechanical skeleton facilitated the formation of the hybrid sponges into desired shapes and gave the hybrid sponges good mechanical properties. Because collagen and hydroxyapatite are the two primary components of extracellular bone matrix, they should provide the hybrid sponge with a good degree of cell interaction and osteoconductivity. Such synthetic polymer– collagen – hydroxyapatite hybrid sponges would prove useful as three-dimensional porous scaffolds for bone tissue engineering.
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The authors acknowledge support from the New Energy and Industrial Technology Development Organization of Japan.
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References
w22x
w1x P. Langer, J.P. Vacanti, Tissue engineering. Science 260 Ž1993. 920–926. w2x R.M. Nerem, A. Sambanis, Tissue engineering: from biology to biological substitutes. Tissue Eng. 1 Ž1995. 3–13. w3x S.J. Peter, M.J. Miller, A.W. Yasko, M.J. Yaszemski, A.G. Mikos, Polymer concepts in tissue engineering. J. Biomed. Mater. Res. 43 Ž1998. 422–427. w4x L.E. Freed, G. Vunjak-Novakovic, R.J. Biron, D.B. Eagles, D.C.
w23x
w24x
w25x
Lesnoy, S.K. Barlow, R. Langer, Biodegradable polymer scaffolds for tissue engineering. BiorTechnology 12 Ž1994. 689–693. B.S. Kim, D.J. Mooney, Development of biocompatible synthetic extracellular matrices for tissue engineering. TIBTECH 16 Ž1998. 224–230. L.E. Freed, J.C. Marquis, A. Nohria, J. Emmanual, A.C. Mikos, R. Langer, Neocartilage formation in vitro and in vivo using cells cultured on synthetic biodegradable polymers. J. Biomed. Mater. Res. 27 Ž1993. 11–23. S.L. Ishaug, G.M. Crane, M.J. Miller, A.W. Yasko, M.J. Yaszemski, A.G. Mikos, Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds. J. Biomed. Mater. Res. 36 Ž1997. 17–28. M.G. Dunn, J.B. Liesch, M.L. Tiku, J.P. Zawadsky, Development of fibroblast-seeded ligament analogs for ACL reconstruction. J. Biomed. Mater. Res. 29 Ž1995. 1363–1371. Y.S. Nam, T.G. Park, Porous biodegradable polymeric scaffolds prepared by thermally induced phase separation. J. Biomed. Mater. Res. 47 Ž1999. 8–17. K. Whang, C.H. Thomas, K.E. Healy, A novel method to fabricate biodegradable scaffolds. Polymer 36 Ž1995. 837–842. D.J. Mooney, D.F. Baldwin, N.P. Suh, J.P. Vacanti, R. Langer, Novel approach to fabricate porous sponges of polyŽD,L-lactic-coglycolic acid. without the use of organic solvents. Biomaterials 17 Ž1996. 1417–1422. A.G. Mikos, Y. Bao, L.G. Cima, D.E. Ingber, J.P. Vacanti, R. Langer, Preparation of polyŽglycolic acid. bonded fiber structures for cell attachment and transplantation. J. Biomed. Mater. Res. 27 Ž1993. 183–189. A.G. Mikos, A.J. Thorsen, L.A. Czerwonka, Y. Bao, R. Langer, D.N. Winslow, J.P. Vacanti, Preparation and characterization of polyŽL-lactic acid. foams. Polymer 35 Ž1994. 1069–1077. G. Chen, T. Ushida, T. Tatsuya, Preparation of polyŽL-lactic acid. and polyŽDL-lactic-co-glycolic acid. foams by use of ice microparticulates. Biomaterials 22 Ž2001. 2563–2567. G. Chen, T. Ushida, T. Tatsuya, Fabrication of PLGA–collagen hybrid sponge. Chem. Lett. Ž1999. 561–562. G. Chen, T. Ushida, T. Tatsuya, Hybrid biomaterials for tissue engineering: a preparative method of PLA or PLGA–collagen hybrid sponge. Adv. Mater. 12 Ž2000. 455–457. G. Chen, T. Ushida, T. Tatsuya, A biodegradable hybrid sponge nested with collagen microsponges. J. Biomed. Mater. Res. 51 Ž2000. 273–279. G. Chen, T. Ushida, T. Tatsuya, A hybrid network of synthetic polymer mesh and collagen sponge. Chem. Commun. Ž2000. 1505– 1506. K.S. Weadock, E.J. Miller, E.L. Keuffel, M.G. Dunn, Effect of physical crosslinking methods on collagen-fiber durability in proteolytic solutions. J. Biomed. Mater. Res. 32 Ž1996. 221–226. L.H.H. Olde Damink, P.J. Dijkstra, M.J.A. van Luyn, P.B. van Wachem, P. Nieuwenhuis, J. Feijen, Glutaraldehyde as a crosslinking agent for collagen-based biomaterials. J. Mater. Sci. 6 Ž1995. 460–472. N. Barbani, P. Giusti, L. Lazzeri, G. Polacco, G. Pizzirani, Bioartificial materials based on collagen: 1. Collagen cross-linking with gaseous glutaraldehyde. J. Biomater. Sci., Polym. Ed. 7 Ž1995. 461–469. H.L. Wald, G. Sarakinos, M.D. Lyman, A.G. Mikos, J.P. Vacanti, R. Langer, Cell seeding in porous transplantation devices. Biomaterials 14 Ž1993. 270–278. D.J. Mooney, S. Park, P.M. Kaufmann, K. Sano, K. McNamara, J.P. Vacanti, R. Langer, Biodegradable sponges for hepatocyte transplantation. J. Biomed. Mater. Res. 29 Ž1995. 959–965. A.G. Mikos, M.D. Lyman, L.E. Freed, R. Langer, Wetting of polyŽL-lactic acid. and polyŽDL-lactic-co-glycolic acid. forms for tissue culture. Biomaterials 15 Ž1994. 55–58. J. Gao, L. Niklason, R. Langer, Surface hydrolysis of polyŽglycolic
G. Chen et al.r Materials Science and Engineering C 17 (2001) 63–69
w26x
w27x
w28x
w29x
acid. meshes increases the seeding density of vascular smooth muscle cells. J. Biomed. Mater. Res. 42 Ž1998. 417–424. J.E. Devin, M.A. Attawia, C.T. Laurencin, Three-dimensional degradable porous polymer-ceramic matrices for use in bone repair. J. Biomater. Sci., Polym. Ed. 7 Ž1996. 661–669. R. Zhang, P.X. Ma, PolyŽalpha-hydroxyl acid.rhydroxyapatite porous composites for bone-tissue engineering: I. Preparation and morphology. J. Biomed. Mater. Res. 44 Ž1999. 446–455. W.L. Murphy, D.H. Kohn, D.J. Mooney, Growth of continuous bone-like mineral within porous polyŽlactide-co-glycolide. scaffolds in vitro. J. Biomed. Mater. Res. 50 Ž2000. 50–58. D. Bakos, M. Soldan, I. Hernandez-Fuentes, Hydroxyapatite–collagen–hyaluronic acid composite. Biomaterials 20 Ž1999. 191–195.
69
w30x M.B. Yaylaoglu, C. Yildiz, F. Korkusuz, V. Hasirci, A novel osteochondral implant. Biomaterials 20 Ž1999. 1513–1520. w31x K. Soballe, Hydroxyapatite ceramic coating for bone implant fixation: mechanical and histological studies in dogs. Acta. Orthop. Scand., Suppl. 255 Ž1993. 1–58. w32x A. Rovira, R. Bareille, I. Lopez, F. Rouais, L. Bordenave, C. Rey, M. Rabaud, Preliminary report on a new composite material made of calcium phosphate, elastin peptides and collagens. J. Mater. Sci.: Mater. Med. 4 Ž1993. 372–380. w33x G. Chen, T. Ushida, T. Tateishi, PolyŽDL-lactic-co-glycolic acid. sponge hybridized with collagen microsponges and deposited apatite particulates. J. Biomed. Mater. Res. 57 Ž2001. 273–279.