Novel biomaterials immobilized with biosignal molecules

MATERIALS SCIENCE & ENGINEERING ELSEVIER

Materials Science and Engineering C2 (1994) 67-72

t2

Novel biomaterials immobilized with biosignal molecules * Yoshihiro Ito, Ji Zheng, Shu Qin Liu *, Yukio Imanishi Divison of Material Chemistry, FacuBy of Engineering, Kyoto University, Kyoto 606-01, Japan

Abstract Bioinertness, the reducing interactions of the materials with biocomponents, has been considered to be the most important factor in the development of biomaterials. However, we recently proposed and realized a new strategy to design a bioactive material, on which biosignal molecules such as cell-growth-factor proteins and cell-adhesion-factor proteins were immobilized. T h e immobilized biosignal molecules were not only bioactive without being incorporated into the cell, but also more active than free (soluble) ones. In this review, some evidence to demonstrate that the effect of immobilized biosignal is based on biospecific interactions with cellular receptors is shown. In addtion, the explanation for the strong effect of the biosignalling, some efforts to further enhance the effect, and various applications of the biosignal molecules-immobilized biomaterials are described. Finally the further outlook of biomaterial research using this type of material is mentioned.

Keywords: Biomaterials; Immobilization; Growth factor; Cell culture; Hybrid organ; Biosignal proteins

1. Introduction

Considering biocompatibility of artificial organ materials, an emphasis was placed on the design and synthesis of bioinert materials which neither interact strongly with the living components nor induce responses to external materials. However, recently we found and developed a new concept to construct bioactive materials which stimulate cell functions. Cell functions are generally regulated by communications between the cell and biosignals such as those from other cells, the extracellular matrix (ECM), and biosignal molecules as shown in Fig. 1. Cadherin family is involved in cell-cell communications. Collagen is a typical ECM protein. These compounds control cell function heterogenously in the insoluble state. On the other hand, the third communication by hormones is conducted homogenously in the soluble state. The biomaterial which we developed was realized by immobilization or by insolubilization of biosignals on matrices. The soluble biosignals are classified into two types. One is that of low molecular weight, such as steroid hormones. This type of biosignal permeates the cell membrane and directly interacts with nucellus. On the * Paper presented at the Bionic Design Workshop '94, 22-23 February, 1994, Tsukuba, Japan. * Present address: Department of Chemistry, South China Normal University, Guang Dong, People's Republic of China.

0928-4931/94/$07.00 © 1994 Elsevier Science S.A. All rights reserved SSDI 0928-4931 (94)00045-T

Cell C o m m u n i c a t i o n s

Insoluble stat

e,-,x / ~ / N /N/'x ExtracellularMatriees

Insoluble stat~

Hormones Growth Factors

Soluble state]

Fig. 1. Cell communications among cell-cell, cell-extracellular matrices, and cell-hormones.

other hand, high molecular weight-biosignals such as polypeptide growth factors, interact with their receptors instead of permeating the cell membrane. These are by the receptors on the target cell surface and the complexes are internalized into the cell to be dissociated and decomposed in lysosome, and that a part of receptor

68

Y. Ito et al. / Materials Science and Engineering C2 (1994) 67-72

or

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2. Effect of immobilized biosignals

As growth factor proteins insulin, epidermal growth factor (EGF), and transferrin, which are usually contained in the commercial serum-free cell culture medium, were immobilized on various matrices. These included surface-hydrolyzed-poly(methylmethacrylate) [5-8,10,11,13,15] surface-hydrolyzed-poly(ethylene terephthalate) [5,6] polyurethane containing amino groups incorporated by glow-discharge in the presence of ammonia [12] poly(hydroxyethyl methacrylate)-copoly(ethyl methacrylate) of various compositions [14] surface-treated polyacrylamide [9] poly(acrylic acid)grafted polystyrene [16] and silane-coupled glass [9]. Various immobilization reagents, such water-soluble carbodiimide [5-11,13,15,16] cyanogen bromide [14] glutaraldehyde [9] and dimethyl suberimide [12] were used as illustrated in Fig. 3. Mouse fibroblast cells STO

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Fig. 2. Schematic illustration of interaction of biosignal molecules with cells. Low-molecular-weight molecules (e.g. steroid hormones) permeate the cell membrane to interact with nucleus. On the other hand, high-molecular-weight molecules interact with the receptors.

molecules liberated is transported back to the cell surface as shown in Fig. 2. However, it has not been made clear whether the process of endocytosis is essential for the action of peptidic growth factors and hormones. If the immobilized biosignal molecules biospecifically interacted with the receptor, they were expected to inhibit the endocytosis of the biosignal-receptor complex. We chose cell growth-factor proteins as the biosignal molecules [1-15]. The immobilized growth factor proteins realized complete protein-free medium cell culture. In this article, first the effect of immobilized biosignal molecules is described, secondly the evidences that the effect is based on the biospecific interactions are mentioned. Finally for the applications for in vivo and in vitro uses some efforts to enhance the effect of immobilized growth factor proteins are written, including future outlooks.

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Fig. 3. The growth factors immobilized upon various materials investigated to date. (a) Insulin or transferrin was immobilized upon surface-hydrolyzed poly(methyl methacrylate) or poly(ethylene terephthalate) membrane by means of water soluble carbodiimide. (b) Insulin was immobilized upon polyurethane tube or membrane containing incorporated amino groups by means of water soluble carbodiimide or dimethyl suberimidate. (c) Insulin was immobilized upon NaOCl-treated polyacrylamide beads by means of dimethyt suberimidate. (d) Insulin was immobilized upon silane-coupled glass beads by means of dimethyl suberimidate or glutaraldehyde. (e) Insulin was immobilized upon poly(hydroxyethyl methacrylate-coethyl methacrylate) membrane using CNBr. (f) Insulin was immobilized upon poly(acrylic acid)-grafted polystyrene dish.

[5-11,13-16] mouse fibroic sarcoma cells [9] mouse hybridomas [9] bovine endothelial cells [12] and CHO cells [17] were cultured on these materials. Fig. 4 shows that growth of CHO on insulin-immobilized poly(methyl methacrylate) film was accelerated [17]. In addition, the growth rate on the insulin-immobilized film was higher than that in the presence of free insulin. Such acceleration was observed on other materials, although the degree depended on the immobilization methods and the matrices. 3. Biospecific interaction between cells and immobilized biosignals

That the acceleration of cell growth is due to a biospecific interaction between the immobilized proteins and their receptor, is indicated by the following evidences. (1) Immobilized proteins other than cell growth proteins did not accelerate cell growth [7]. Fig. 5 shows

Y. lto et aL / Materials Science and Engineering C2 (1994) 67-72

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Fig. 4. T h e relative growth rate of m o u s e fibroblast cell STO on insulin-immobilized poly(methyl methacrylate) m e m b r a n e (O) or on surface-hydrolyzed poly(methyl methacrylate) m e m b r a n e in the presence of free insulin (@). T h e relative cell growth rate was calculated by counting cells on surface-hydrolyzed poly(methyl methacrylate) in the absence of insulin after 48 h in culture.

Immobilized

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Relative Cell Adhesion Fig. 5. Classification of immobilized proteins according to effect on the adhesion and growth of m o u s e fibroblast STO cell. A contains albumin and y-globulin, B contains fibronectin and collage and C contains insulin•

that the immobilized albumin and y-globulin did not affect either cell adhesion and growth, that immobilized collagen and fibronectin enhanced cell adhesion not growth, and that the immobilized insulin, EGF, and transferrin accelerated cell growth not adhesion. (2) Anti-insulin antibody inhibited cell growth on the insulin-immobilized material as shown in Fig. 6 [8]. With increasing coverage of immobilized insulin with the antibody, the cell growth rate decreased. Anti-albumin antibody partially inhibited cell growth by nonspecific adsorption onto the material• The anti-insulin antibody was considered to inhibit the biospecific interaction between the immobilized insulin and the insulin receptor. (3) The immobilized growth factor protein, insulin, accelerated only anchorage-dependent cells, not those that are anchorage-independent, such as hy-

Fig. 7. T h e effect of free and immobilized insulin onto the growth of different type of m a m m a l i a n cells. T h e anchorage-dependent cells were m o u s e fibroblast STO and m o u s e sarcoma HSDM~C1, and the anchorage independent ceils m o u s e hybridoma SJK 132-20 and R D P 45/20.

bridoma cells as shown in Fig. 7 [9]. The immobilized insulin remarkably accelerated the growth of STO and sarcoma cells, compared with free and adsorbed insulin. On the other hand, the growth rate of mouse hybridoma cells in the presence of immobilized insulin was less than that in the presence of free or adsorbed insulin. Because these anchorage-independent cells did not adhere to matrices, the interaction of cells with immobilized insulin was considered to be less than that with free or adsorbed insulin. (4) It is known that after complex formation of free insulin with insulin receptor, the insulin receptor is autophosphorylated and activated insulin receptor substrate-1 (IRS-1) and further phosphoinositide3 (PI-3) kinase. The immobilized insulin also phosphorylated insulin receptor and IRS-1, and activated PI-3 kinase [17]. The proteins activated by immobilized insulin was illustrated in Fig. 8. Recently integrin which is a receptor for extracellular matrix proteins, or protein neighboring proteins are phosphorylated by interaction with the ligands [18]. This phenomenon also indicates the receptor phosphorylation without internalization of complex• It is

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Y. lto et al. / Materials Science and Engineering C2 (1994) 67-72

with the receptor, rendered more accesible by an alteration of the cell surface geometry.

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concluded that ligand internalization is not necessary for biosignalling.

To increase the growth acceleration, three approaches were employed. (1) As described above, the crosslinking of the biosignal-receptor complex is considered to be very important. Therefore, a spacer chain consisting of polyethylene glycol was incorporated to enhance the flexibility of the immobilized protein as shown in Fig. 9(a) [11]. STO cell growth was accelerated by this approach to some degree. (2) Naturally there are a variety of biosignal molecules for cell communication. They form a complex network by cross communication to maintain cellular homeostasis. Considering this phenomenon, coim(a)

4. Explanation for the strong biosignalling effect of immobilized proteins The reasons why the immobilized growth factor accelerated cell growth more than when free or adsorbed are considered to be as follows. (1) The immobilized insulin provides a high local concentration of insulin, which should induce multivalent simultaneous stimulation, leading to enhanced complex formation with the receptors and also to promoted crosslinking of the complex. It is known that crosslinking of the complex is very important for signal transduction. The immobilized or polymerized biosignals should provide an advantage in accelerating this process. (2) As mentioned above, after growth factor proteins bind with their receptors, the complex is internalized into the cell to be dissociated and decomposed in lysosomes. This process is called down-regulation and for inhibition of over-stimulation by ligands. However, the immobilized biosignal proteins are considered to inhibit this down-regulation. In fact the receptor number on the surface of cell interacted with free insulin was less than that with immobilized o n e [8]. In addition although phosphrylation by free insulin decreased after 30 min contact, that by immobilized insulin increased with time over 12 h contact [17]. (3) Unknown nonbiospecific interactions must be taken into consideration. For example, nonspecific interaction may facilitate interaction of the proteins

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Matrix Fig. 9. Several attempts to enhance the effect of immobilized biosignal proteins. (a) Incorporation of a spacer chain induced aggregation of biosignal-receptor complex. (b) Coimmobilization of various biosignal proteins induced a synergetic effect by heterogeneous interaction with receptors. (c) Coimmobilization of cationic chemicals to enhance the cell adheison to increase the opportunity of interaction between immobilized protein and the receptor.

Y. Ito et al. / Materials Science and Engineering C2 (1994) 67-72

(3)

mobilization of different biosignal molecules should result in an interaction between the receptors as shown in Fig. 9(b). We coimmobilized cell-adhesionfactor proteins, collagen [12] and fibronectin [11] or the core peptide, Arg-Gly-Asp-Ser (RGDS) [2] with insulin. On these materials, both the cell adhesion and growth were remarkably enhanced. Because the growth acceleration was drastically enhanced by coimmobilization of growth factor protein and adhesion protein, to enhance cell adhesion cationic chemicals such as polyallylamine and polylysine were coimmobilized with insulin as shown in Fig. 9(c) [15]. The coimmobilized material also markedly enhanced both the cell adhesion and growth.

71

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6. Applications of the biosignai-immobilized materials The biosignal-immobilized materials were applied to in vivo and in vitro uses. The former was for an artificial organ, the latter, for cell culture engineering. (1) Hybridization of synthetic materials and organs is considered to be a promising source of high performance artificial organs. A representative hybrid artificial organ is an artificial blood vessel. The vessel is made of a plastic tube coated with blood endothelial cells. Im-

Endothelialization

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Time required to cover the interior of polymer tube (d)

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Collagen-immobilized

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>270

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Fig. 10. Bovine endothelial cells covered a polyurethane tube more rapidly and the endotheliaization was stable for a long time when insulin and collagen were coimmobilized.

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Fig. 11. An insulin-immobilized microcarrier can be used repeatedly. After culture, the cells are removed, and the microcarrier can be used again.

mobilization of biosignal proteins was proven useful for endothelialization. As shown in Fig. 10, the rate of covering a polyurethane tube by endothelial cells was measured [12]. A tube coimmobilized with both insulin and collagen was rapidly covered with endothelial cells which were stable for more than a year. (2) Mammalian cell culture is an importent technology not only for the fundamental research but also for the industrial production of a large quantity of biologicallysignificant materials. Serum or serum substituents have to be used to support cell growth in addition to nutrients. However, the use of serum is undesirable because of its high cost and the complications introduced by the serum proteins for separation of various products. Accordingly, several serum-free media have been developed. Our concept of an immobilized biosignal provides a revolutionary culture technique. Our method realized complete protein-free medium cell culture and recycling as shown in Fig. 11 [9]. The immobilized biosignal has advantages because not only are immobilized enzymes useful as bioreactors, but they are also more potent than free biosignals.

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Y. Ito et al. / Materials Science and Engineering C2 (1994) 67-72

7. Future outlooks

References

We emphasized the usefulness of immobilization of biosignal molecules. In this article cell-growth-factor proteins were employed as the biosignal. However, the biosignal is not limited in such proteins. Other types of biosignal proteins including cell-differentiation-factor will be immobilized for design and construction of biofunctional materials. In addition to such material design we would like to propose cell line design for material in order to construct ideal cell culture engineering. As mentioned above, the immobilized insulin accelerated the growth of only anchorage-dependent cells, not anchorage-independent cells. This phenomenon was considered to be evidence for biospecific interaction of the immobilized insulin and the receptor. At the same time it indicates that the effect of biosignal-immobilized materials depends on the nature of cell line. Therefore in this case we suggest " design of cell line", for example, the transformation of anchorage-independent cells into those that are anchorage-dependent are suitable for materials. In other words, cells designed for the biomaterial, not biomaterial design for the cell. Various types of cell design including receptor design are possible for total design of cell culture system.

[1] Y. Ito, in Y. Imanishi (ed.), Synthesis of Biocomposite Materials, CRC Press, Boca Raton, 1992, p. 285. [2] Y. Ito and Y. Imanishi, in S. Okamura, T. Tsuruta, Y. Imanishi and J. Sunamoto (eds.), Fundamental Investigations on the Cre. ation of Biofunctional Materials, Kagaku-Dojin, Kyoto, 1991, p. 13. [3] Y. Ito and Y. Imanishi, STP Pharm. Sci., 3 (1993) 46. [4] Y. Ito and Y. Imanishi, Polymer News, 19 (1993) 198. [5] Y. Ito, S.Q. Liu and Y. Imanishi, Biomaterials, 12 (1991) 449. [6] S.Q. Liu, Y. Ito, and Y. Imanishi, Biomaterials, 13 (1992) 50. [7] Y. Ito, S.Q. Liu, M. Nakabayashi and Y. Imanishi, Biomaterials, 13 (1992) 789. [8] S.Q. Liu, Y. Ito and Y. Imanishi, J. Biophys. Biochem. Method, 25 (1992) 139. [9] Y. Ito, T. Uno, S.Q. Liu and Y. Imanishi, Biotech. Bioeng., 40 (1992) 1271. [10] S.Q. Liu, Y. Ito and Y. Imanishi, Enz. Microb. Tech., 15 (1993) 167. [11] Y. Ito, M. Inoue, S.Q. Liu and Y. Imanishi, J. Biomed. Mater, Res., 27 (1993) 901. [12] S.Q. Liu, Y. Ito and Y. Imanishi, Z Biomed. Mater. Res., 27 (1993) 909. [13] S.Q. Liu, Y. Ito and Y. Imanishi, Int. J. Biol. Macromol., 15 (1993) 221. [14] Y. Ito, S.Q. Liu, T. Orihara and Y. Imanishi, Z Bioact. Comp. Polym., 9 (1994) 170. [15] J. Zheng, Y. Ito and Y. Imauishi, Biomaterials, in press. [16] Y. Ito, G. Chen, J. Zheng and Y. Imanishi, manuscript in preparation. [17] Y. Ito, J. Zheng, Y. Imansihi, K. Yonezawa and M. Kasuga, manuscript in preparation. [18] R.O. Hynes, Cell, 69 (1992) 11.