Research News Hybrid for Tissue Engineering: A Preparative Method for PLA or PLGA± Collagen Hybrid Sponges

By Guoping Chen,* Takashi Ushida, and Tetsuya Tateishi

Tissue engineering has emerged as a promising alterna- which hinders even seeding of sufficient cell mass in three tive approach in the treatment of malfunctioning or lost or- dimensions. In contrast, naturally derived polymers have gans.[1±3] In this approach, a temporary scaffold is needed the potential advantages of specific cell interactions and to serve as an adhesive substrate for the implanted cells easy seeding of cells because of their hydrophilicity. How- and a physical support to guide the formation of the new ever, scaffolds constructed from naturally derived polymers organs. Transplanted cells adhere to the scaffold, prolifer- are mechanically unstable, and do not easily contribute to ate, secrete their own extracellular matrices (ECM), and the creation of tissue structures with a specific predefined stimulate new tissue formation. During this process, the shape for transplantation. scaffold gradually degrades and is eventually eliminated. As a result, hybrid three-dimensional porous scaffolds of Therefore, in addition to facilitating cell adhesion, promot- synthetic and naturally derived biodegradable polymers ing cell growth, and allowing the retention of differentiated have been developed for tissue engineering. A hybrid bio- cell functions, the scaffold should be biocompatible, biode- material of collagen fibers embedded within a PLA matrix gradable, highly porous with a large surface/volume ratio, has been reported to strengthen collagen fibers for applica- mechanically strong, and malleable into desired shapes. tion to tendon or ligament reconstruction.[9] A method of The most commonly used three-dimensional porous scaf- coating PLLA sponge (PLLA = poly-(L-)) with folds are constructed from two classes of biomaterials. One collagen or embedding collagen gel within PLLA sponge class consists of synthetic biodegradable polymers such as has been used to improve the interaction between PLLA aliphatic , poly() (PGA), poly(lactic sponge and hepatocytes.[10] However, the surface area/vol- acid) (PLA), and their copolymer of poly-(DL-lactic-co-gly- ume ratios of these hybrid biomaterials remained un- colic acid) (PLGA). The other class consists of naturally changed. A novel kind of hybrid biodegradable porous derived polymers such as collagen.[4±8] PLA, PGA, and scaffold has been developed by our group by nesting col- PLGA are biocompatible and among the few synthetic lagen microsponge in the pores of poly(alpha ester) polymers approved by the Food and Drug Administration sponge.[11] This kind of hybrid not only retains for specific human clinical applications, such as surgical su- the advantages of both biodegradable synthetic poly(alpha tures and some implantable devices. They have the poten- ester)s and naturally derived collagen, also increased the tial advantages of being easily formed into scaffolds having surface area/volume ratio for transplanted cells. the same shape as the tissue to be replaced and, if designed At first, PLA or PLGA sponges were prepared by a par- with sufficient mechanical strength, can retain this struc- ticulate-leaching technique using sieved sodium chloride ture until the new tissue forms. Their rate of biodegrada- particulates[12] or pre-prepared ice particulates[13] as a poro- tion can also be tailored to match the rate of regeneration gen. After sodium chloride or pre-prepared ice particulates of the new tissue. The disadvantages of these synthetic were removed by washing with or freeze-drying, por- polymers are the lack of cell-recognition signals, which re- ous structures were formed. The PLA or PLGA sponge sults in insufficient cell adhesion, and hydrophobicity, showed the same pore size and morphology as the NaCl or ice particulates that were used. Their pore structure can be controlled by adjusting the particulate size or weight ratio ± [*] Dr. G. Chen, Prof. T. Ushida, Prof. T. Tateishi of NaCl or ice to the polymer. Subsequently, the PLA or 3D Tissue Engineering Group PLGA sponges were immersed in type I or type II acidic National Institute for Advanced Interdisciplinary Research collagen solutions (pH 3.2) under a vacuum so that the 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562 (Japan) sponge pores filled with the collagen solution. The collagen and Institute of Medical Research, Graduate School of Medicine solution-containing PLA or PLGA sponges were then fro- University of Tsukuba, Tsukuba, Ibaraki 305-8575 (Japan) zen at ±80 C and freeze-dried to allow the formation of

Adv. Mater. 2000, 12, No. 6 Ó WILEY-VCH Verlag GmbH, D-69469 Weinheim, 2000 0935-9648/00/0603-0455 $ 17.50+.50/0 455 CMYB

Research News

collagen microsponges in the sponge pores. Finally, the col- lagen microsponges were further cross-linked by treatment with glutaraldehyde vapor saturated with 25 % glutaralde- hyde aqueous solution at 37 C for 4 h; and non-reacting al- dehyde groups were blocked by treating with 0.1 M glycine aqueous solution. Figure 1 shows the pore structure of one of such kind of hybrid sponges. Microsponges of collagen with intercon- nected pore structures were nested in the pores of PLGA sponge. Its hybrid structure was further confirmed by detect- ing elemental nitrogen, which exists in collagen but not in PLGA copolymer, with scanning electron microscopy (SEM)-electron probe microanalysis (SEM-EPMA) (Fig. 2). Nitrogen was detected in the microsponges of collagen and the surfaces of the PLGA pores, but not in the cross-sections of the PLGA regions, indicating that microsponges of col- lagen were formed in the pores of the PLGA sponge and that the pore surfaces were also coated with collagen. C M Y B

Fig. 2. SEM (a) and SEM-EPMA (b) photomicrographs of a cross section of a PLGA±collagen hybrid sponge. The colors indicate elemental nitrogen with red representing the highest content.

The effective concentration of collagen solution for the preparation of the hybrid sponges was in the range from 0.1 to 1.5 % (w/v). Formation of collagen microsponges in the pores of PLA or PLGA sponge was difficult if the con- centration of collagen solution was lower than 0.1 % (w/v). A collagen solution with a concentration higher than 1.5 % (w/v) could not infiltrate the pores of the PLA or PLGA sponge because of its high viscosity. Three-dimensional poly(alpha ester) sponges are rela- tively hydrophobic, making it difficult to deliver a cell sus- pension in a manner that uniformly distributes transplanted cells throughout the sponges.[14,15] Hybridization of poly(al- pha ester) sponges with collagen microsponges increased their wettability and facilitated cell seeding in the sponges. When a cell suspension was loaded onto the poly(alpha es- ter)±collagen hybrid sponges, cell suspension infiltrated the Fig. 1. SEM photomicrographs of a cross section of PLGA sponge prepared hybrid sponges, and transplanted cells adhered to the col- with NaCl particulates ranging in size from 355 to 425 mm (a), and of PLGA±collagen hybrid sponge prepared with 1.0 % type I collagen acidic lagen microsponges and the collagen-coated PLGA pore solution (b). surfaces in the hybrid sponges. The hydrophilicity, as well

456 Ó WILEY-VCH Verlag GmbH, D-69469 Weinheim, 2000 0935-9648/00/0603-0456 $ 17.50+.50/0 Adv. Mater. 2000, 12, No. 6 Research News

as the interconnected pore structure, resulted in a spatially erated a cartilagious matrix, and maintained their pheno- uniform cell distribution throughout the hybrid sponges. typical round morphology after culturing for 6 weeks in the The hybrid sponges showed good cell interactions.[16] Fig- PLGA-collagen hybrid sponge. The hybrid sponges also ure 3 shows mouse fibroblast L929 cells and bovine articu- showed higher mechanical strength than either poly(alpha lar chondrocytes adhered in a PLGA±collagen hybrid ester) sponge or collagen sponge. sponge. Bovine articular chondrocytes proliferated, regen- The hybridization method of nesting collagen micro- sponges in the pores of poly(alpha ester) sponges provides an effective method for preparing hybrid biomaterials for tissue engineering. Use of poly(alpha ester) sponge as a skeleton facilitates easy formation of the hybrid sponges into desired shapes that have a high degree of mechanical strength, while the collagen microsponges nested in the pores of poly(alpha ester) sponges allowed the cells to in- teract with the collagen surfaces, instead of direct interac- tion with the poly(alpha ester) surfaces. Nested collagen microsponges and coated collagen gave the hybrid sponges a good degree of cell interaction.

± [1] R. Langer, J. P. Vacanti, Science 1993, 260, 920. [2] R. M. Nerem, A. Sambanis, Tissue Eng. 1995, 1,3. [3] F. Oberpenning, J. Meng, J. J. Yoo, A. Atala, Nature Biotechnol. 1999, 17, 149. [4] S. J. Peter, M. J. Miller, A. W. Yasko, M. J. Yaszemski, A. G. Mikos, J. Biomed. Mater. Res. 1998, 43, 422. [5] R. C. Thomson, M. J. Yaszemski, J. M. Powers, A. G. Mikos, J. Bioma- ter. Sci., Polym. Ed. 1995, 7, 23. [6] L. E. Freed, G. Vunjak-Novakovic, R. J. Biron, D. B. Eagles, D. C. Lesnoy, S. K. Barlow, R. Langer, Biotechnology 1994, 12, 689. [7] A. G. Mikos, G. Sarakinos, S. M. Leite, J. P. Vacanti, R. Langer, Bio- materials 1993, 14, 323. [8] B. S. Kim, D. J. Mooney, TIBTECH 1998, 16, 224. [9] M. G. Dunn, L. D. Bellincampi, A. J. Tria, J. P. Zawadsky, J. Appl. Polym. Sci. 1997, 63, 1423. [10] P. M. Kaufmann, S. Heimrath, B. S. Kim, D. J. Mooney, Cell Trans- plant. 1997, 6, 463. [11] G. Chen, T. Ushida, T. Tateishi, Chem. Lett. 1999, 561. [12] A. G. Mikos, A. J. Thorsen, L. A. Czerwonka, Y. Bao, R. Langer, D. N. Winslow, J. P. Vacanti, Polymer 1994, 35, 1069. [13] G. Chen, T. Ushida, T. Tateishi, Trans. Mater. Res. Soc. Jpn., in press. [14] A. G. Mikos, M. D. Lyman, L. E. Freed, R. Langer, Biomaterials 1994, 15, 55. Fig. 3. SEM photomicrographs of L929 cells (a) and chondrocytes (b) cul- [15] J. Gao, L. Niklason, R. Langer, J. Biomed. Mater. Res. 1998, 42, 417. tured in a PLGA±collagen hybrid sponge for one day. [16] G. Chen, T. Ushida, T. Tateishi, J. Biomed. Mater. Res., in press.

______

Adv. Mater. 2000, 12, No. 6 Ó WILEY-VCH Verlag GmbH, D-69469 Weinheim, 2000 0935-9648/00/0603-0457 $ 17.50+.50/0 457