Biodegradable Matrices from Regenerated Silk of Bombix mori

ISSN 16076729, Doklady Biochemistry and Biophysics, 2010, Vol. 433, pp. 201–204. © Pleiades Publishing, Ltd., 2010. Original Russian Text © I.I. Agapov, M.M. Moisenovich, T.V. Vasilyeva, O.L. Pustovalova, A.S. Kon’kov, A.Yu. Arkhipova, O.S. Sokolova, V.G. Bogush, V.I. Sevastianov, V.G. Debabov, M.P. Kirpichnikov, 2010, published in Doklady Akademii Nauk, 2010, Vol. 433, No. 5, pp. 699–702.

BIOCHEMISTRY, BIOPHYSICS AND MOLECULAR BIOLOGY

Biodegradable Matrices from Regenerated Silk of Bombix mori I. I. Agapova, M. M. Moisenovichb, T. V. Vasilyevab, O. L. Pustovalovab, A. S. Kon’kovb, A. Yu. Arkhipovab, O. S. Sokolovab, V. G. Bogushc, V. I. Sevastianova, Corresponding Member of the RAS V. G. Debabovc, and Academician M. P. Kirpichnikovb Received April 27, 2010

DOI: 10.1134/S1607672910040149

The structure and biological properties of devices obtained from regenerated fibroin of Bombix mori silk in the form of films, threedimensional matrices, and tubes intended for regenerative medicine were studied. It is shown that regenerated fibroin forms a substrate that maintains adhesion and proliferation of eukary otic cells by forming a structure that enables homoge neous distribution of proliferating cells both on the cell surface and in deep matrix layers. The obtained devices are characterized by a high strength and elas ticity. Matrices implanted into experimental animals undergo diodegradation and neovascularization with time. The properties of devices obtained from regener ated fibroin allow them to be considered as a base in designing artificial biological analogues of tissue struc tures. The key problem of modern medicine is the devel opment of technologies for replacement of damaged or lost organs and tissues with artificial biological ana logues constructed in vitro, which are also called tis sueengineering constructions. One of the stages of creating bioartificial organs is the formation of a frame from biocompatible materials mimicking the healthy tissue structure. Such threedimensional frames are called matrices. Artificial matrices determine the mechanical prop erties and shape of implants and form the substrate for cell adhesion. The latter determines the topology of focal adhesive complexes, thereby initiating a cascade of intracellular signals induced by mechanical tension of the cell membrane [4]. Cell adhesion leads to clus terization of cell receptors and, as a result, to activa tion of expression of genes involved in proliferation

a

Shumakov Institute of Transplantology and Artificial Organs, Federal Agency for HighTech Medical Services, ul. Shchukinskaya 1, Moscow, 113182 Russia b Faculty of Biology, Moscow State University, Moscow, 119991 Russia c Institute of Genetics and Selection of Industrial Microorganisms, Pervyi Dorozhnyi proezd, Moscow, 113545 Russia

and differentiation [5]. The interaction with substrate determines polarization of cells, changes in their mor phology, spatial orientation of cytoskeletal compo nents and cellular organelles, and organization of intracellular transport [4]. In addition, substrate func tions as a depot for soluble growth factors [6]; many substrates contain signal sequences belonging to the class of insoluble biologically active ligands, matrik ines [10, 11]. Substrate regulatory factors influence the repertoire of extracellular matrix components synthe sized by cells, which are, in turn, sources of signals that determine the functional state of the cell [9]. Thus, substrate is actively involved in instructing the cell morphotype, phenotype, and cell–cell interactions. Correct selection of material to be used as a shape forming element and cellular substrate in creating bio artificial organs is the prerequisite for successful replacement therapy. The group of materials used for obtaining matrices for replacement therapy includes ceramic composites, alginates, collagen, gelatin, chi tosan, hyaluronic acid, and polyesters of bacterial ori gin [12]. Recently, silk fibroin from the silkworm Bom bix mori cocoons was proposed as material to be used in tissue engineering [1]. Fibroin has a regular struc ture saturated with negatively charged amino acids and is capable of rapid phase transition into insoluble state from aqueous solutions in the presence of alco hols and other factors [3]. This is an ideal material for production of strong yet flexible structures. In addi tion, silk is thermally stable, and devices obtained from it can be sterilized by heating at temperatures up to 150°C [7]. In this study, we describe the characteristics of devices obtained from silk fibroin, which were devel oped by us and which can be used as a base for histo typic analogues of skin, blood vessels, and bone tissue. Fibroin was isolated from silkworm cocoons kindly provided by V. V. Bogoslovskii, Director of the Repub lican Sericulture Research Station, Russian Academy of Agricultural Sciences (Zheleznovodsk, Stavropol oblast). Sericin and other admixtures present in cocoons were eliminated by boiling for 1 h in 0.03 M NaHCO3 with subsequent washing and drying. Puri

201

202

AGAPOV et al.

Fig. 1. Appearance of threedimensional devices from regenerated fibroin.

fied fibroin was dissolved at 60°C in formic acid con taining 10% LiCl. The devices of interest were obtained from solutions with protein concentration 250 mg per ml solvent. Films from regenerated fibroin (RF) intended for subsequent use as a possible base for designing bioartificial skin were prepared by casting [2]. The base for regeneration of bone tissue consisted of threedimensional matrices prepared by leaching [13, 15]. Dry NaCl particles (200–400 µm) were used as a poreforming agent. The concentration of parti cles of the poreforming agent was selected so that the pores of formed matrix were connected whereas the matrix as such contained no isolated cavities. Matrices were obtained in the form of discs 2–10 mm thick and 10 mm in diameter. In addition, we prepared twolayer tubes as proto types of artificial vessels. The first layer (0.5 mm) was formed on the surface of a polyvinylchloride bar by leaching. The poreforming NaCl particles were 50 µm in size. The second layer (0.5 mm) was created by tenfold layering and drying of RF solution on the surface of the formed device. Thus obtained devices were characterized by high durability and elasticity. The mean tensile strength of matrices obtained from RF was 35 ± 2 N/cm2 at an extensibility of 242 ± 18%. The crosslinking of matri ces with bifunctional regents, such as glutaraldehyde, which reacts with free amino groups and forms addi tional covalent intermolecular bonds, increased the strength to 65 ± 5 N/cm2 having no effect on the elas ticity of material: the extensibility of crosslinked material was 237 ± 23%.

The structure of threedimensional matrices obtained from fibroin represents a system of pores connected by channels and holes (Fig. 1). The size of pores coincided with the size of poreforming parti cles, which was confirmed by scanning electron and confocal microscopy. The fact that pores in matrices were unclosed was confirmed not only microscopi cally but also checked in the ink particle distribution test. In general, the structure of matrices obtained from RF corresponds to the structure of devices described earlier, which were obtained from recombi nant spidroin 1. A substantial distinction between them is that the walls of pores formed by fibroin con tained no microreticular structure with 1–10 µm cells, which is characteristic of the walls of pores in matrices formed by spidroin [13, 15]. Regenerated fibroin proved to be a perfect substrate for cell adhesion and proliferation: the number of cells adhered to RF films was twice as great as the number of cells adhered to culture plastic (table). After four days of culturing, the cell density on the surface of RF films became maximum and was 2.5 times higher than that reached as a result of culturing on plastic. After 14 days of culturing, 100% cell death as a result of apo ptosis induced by contact inhibition was detected on flat surfaces (table). The use of a Zeiss Axiovert 200M LSM 510 confo cal microscope (Carl Zeiss, Germany) equipped with lenses with a large working segment made it possible to detect cell nuclei at depths up to 600 µm from the matrix surface (Fig. 2). The software for processing images (LSM Version 1.4.2, Carl Zeiss, Germany and Imaris 6.1.5, Bitplane AG, Switzerland) allowed us to quantitatively assess the time course of changes in the cell surface, determine the number of cells in different optical layers and the number of cell nuclei per matrix unit volume, as well as estimate the dynamics of the number of cells (table). The results of studies showed that 3T3 fibroblasts applied on the surface of matrix actively proliferated and migrated into deep layers, retaining the morphological features characteristic of fibroblasts (Fig. 2b). The presence of cells in deep lay ers indicates that the material of devices and their structure, including the unclosed type of pores, facili tate cell migration into the inner matrix layers and cre ate homogeneous conditions, ensuring exchange of gases and metabolic products.

Time course of changes in the number of cells cultured in devices obtained from regenerated fibroin Cultivation time, days

Plastic, cell number/mm2

Film, cell number/mm2

Matrix, cell number/mm3

Matrix with nanoFA, cell number/mm3

0 1 4 14

16 ± 2 22 ± 2 97 ± 8 Cell death

32 ± 3 38 ± 2 278 ± 25 Cell death

ND 18 ± 2 172 ± 2 4105 ± 320

ND 73 ± 7 697 ± 85 4923 ± 412

Note: ND, not detected. DOKLADY BIOCHEMISTRY AND BIOPHYSICS

Vol. 433

2010

BIODEGRADABLE MATRICES FROM REGENERATED SILK

(a)

100 µm

203

20 µm

(b)

Fig. 2. 3T3 Fibroblasts in inner matrix layers. The cells were applied on the matrix surface and cultured for 14 days. (a) Optical section obtained with a Zeiss Axiovert 200M LSM 510 microscope at a depth of 600 µm from the matrix surface. Fibroblast nuclei were revealed by staining with the Sytox Creen nucleic acid stain (Invitrogen, United States). (b) Matrix in the form of a 2mm disc cut in the middle transversely to the surface, analyzed under a Camscan S2 scanning electron microscope (Cambridge Instru ments, Great Britain). A cell monolayer on the outer side of the pore is shown.

We also prepared composite threedimensional matrices containing RF and 10% nanohydroxyapatite (nanoHA), which was kindly provided by Professor V.V. Guzeev (Interregional Orthopedic Center, Clini cal Hospital no. 81, Seversk, Russia). Nanohydroxya patite was obtained by the original method from ani mal bone tissue [14]. Matrix modification with nanoHA did not disturb the homogeneity of the struc

10 µm

ture; it markedly stimulated adhesion and accelerated proliferation of cells cultured on the devices (table). Thus obtained threedimensional matrices were used for subcutaneous implantation to BALB/c mice. Histological analysis of samples obtained after their extraction two months later showed that connective tissue is formed in the matrix in the form of cords con sisting of fibroblastlike cells and collagen fibers (Fig. 3). Note that the material of matrix underwent degradation, being gradually replaced with young fibroblasts revealed by morphological traits, one of which is loose nuclear chromatin (euchromatin and the presence of one or two nucleoli). Histological analysis of implants revealed no phenotypic changes in tissues that might indicate disturbances in physiology of cells contacting with the matrix. We also detected a high level of vascularization of tissues replacing matrix after implantation. Blood ves sels lined with flat endothelium were found as well as de novo formed vessels with “high” endothelium. The large number of de novo formed vessels is indicative of active angiogenesis and tissue formation taking place in the matrix two months after its implantation into laboratory animals. The presence of vessels of different type and diameter is characteristic of natural vascular architecture of tissue [8]. The replacement of matrix with vascularized tissue confirms the possibility of using the designed devices as a base in constructing bioartificial organs.

Fig. 3. Replacement of matrices with connective tissue (staining with hematoxylin/eosin). The matrix material was replaced with de novo formed tissue 2 months after implantation. Vessels of different diameter and inhomoge neous matrix degradation can be seen. Certain fragments of macropore walls retain the initial structure of the matrix. Arrows indicate the de novo formed blood vessels.

ACKNOWLEDGMENTS This study was supported in part by the Russian Foundation for Basic Research (project no. 0902 00173) and the Ministry of Education and Science of the Russian Federation within the Framework of the

DOKLADY BIOCHEMISTRY AND BIOPHYSICS

Vol. 433

2010

204

AGAPOV et al.

Federal SpecialPurpose Program “Scientific and Sci entific–Pedagogical Personnel of the Innovative Rus sia in 2009–2013” under State Contract no. P2460 from November 19, 2009, and State Contract no. P2087 from November 3, 2009. REFERENCES 1. Altman, G.H., Diaz, F., Jakuba, C., et al., Biomaterials, 2003, vol. 24, pp. 401–416. 2. Bogush, V.G., Sokolova, O.S., Davydova, L.I., et al., J. Neuroimmune Pharmacol., 2009, vol. 4, pp. 17–27. 3. Chen, X., Shao, Z., Marinkovic, N.S., et al., Biophys. Chem., 2001, vol. 89, pp. 25–34. 4. Shumakov, V.I. and Sevast’yanov, V.I., Zdravookhr. Med. Tekhn., 2003, no. 4, pp. 30–32. 5. Huang, S. and Ingber, D.E., Nat. Cell. Biol., 1999, vol. 1, pp. E131–E138. 6. Hynes, R.O., Science, 2009, vol. 326, pp. 1216–1219.

7. Martel, A., Burghammer, M., Davies, R.J., and Riekel, C., Biomacromolecules, 2007, vol. 8, pp. 3548– 3556. 8. Sieminski, A.L. and Gooch, K.J., Biomaterials, 2000, vol. 21, pp. 2232–2241. 9. Streuli, C.H. and Bissell, M.J., J. Cell Biol., 1990, vol. 110, pp. 1405–1415. 10. Swindle, C.S., Tran, K.T., Johnson, T.D., et al., J. Cell Biol., 2001, vol. 154, pp. 459–468. 11. Tran, K.T., Griffith, L., and Wells, A., Wound Repair Regen., 2004, vol. 12, pp. 262–268. 12. Sevastianov, V.I., Vasilets, V.N., and Agapov, I.I., Rare Metals, 2009, vol. 28, pp. 84–86. 13. Agapov, I.I., Pustovalova, O.L., Moisenovich, M.M., et al., Dokl. Biochem. Biophys., 2009, vol. 426, no. 1, pp. 127–130 [Dokl. Akad. Nauk, 2009, vol. 426, no. 1, pp. 115–118]. 14. Druzhinina, T.V., Khlusov, I.A., Karlov, A.V., and Ros tovtsev, A.V., Genii Ortopedii, 2007, vol. 4, pp. 83–88. 15. Pustovalova, O.L., Moisenovich, M.M., Eremin, P.S., et al., Ros. Immun. Zh., 2009, vol. 3, no. 12, pp. 139– 146.

DOKLADY BIOCHEMISTRY AND BIOPHYSICS

Vol. 433

2010