In vitro and in vivo biocompatibility studies of a recombinant analogue of spidroin 1 scaffolds M. M. Moisenovich, 1 O. L. Pustovalova, 1 A. Yu Arhipova, 1 T. V. Vasiljeva, 1 O. S. Sokolova, 1 V. G. Bogush, 2 V. G. Debabov, 2 V. I. Sevastianov, 3 M. P. Kirpichnikov, 1 I. I. Agapov 3 1 Faculty of Biology, Lomonosov Moscow State University, Leninskiye Gory 1, Building 12, 119991 Moscow, Russian Federation 2 Laboratory for Protein Engineering, The State Scientific Center of Russian Federation ‘‘The State Research Institute for Genetics and Selection of Industrial Microorganisms,’’ 1st Dorozhny 1, 117545 Moscow, Russian Federation 3 Research Center for Biomaterials, Shumakov Federal Research Center of Transplantology and Artificial Organs, Shchukinskaya 1, 123182 Moscow, Russian Federation Received 25 August 2010; accepted 1 September 2010 Published online 4 November 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.32968 Abstract: The goal of this study was to generate porous scaf- folds from the genetically engineered protein, an analogue of Nephila clavipes spidroin 1 (rS1/9) and to assess the prop- erties of new rS1/9 scaffolds essential for bioengineering. The salt leaching technique was used to make the rS1/9 scaf- folds of interconnected macroporous structure with sponta- neously formed micropores. The tensile strength of scaffolds was 18 6 5 N/cm 2 . Scaffolds were relatively stable in a phos- phate buffer but degraded in oxidizing environment after 11 weeks of incubation. Applicability of the recombinant spidroin 1 as a substrate for cell culture was demonstrated by successful 3T3 cells growth on the surface of rS1/9 films (270 6 20 cells/mm 2 vs. 97 6 8 cells/mm 2 on the glass sur- face, p < 0.01). The 3T3 fibroblasts readily proliferated within the rS1/9 scaffold (from initially plated 19 6 2 cells/mm 3 to 3800 6 304 cells/mm 3 after 2 weeks). By this time, cells were uniformly distributed between the surface and deeper layers (27% 6 8% and 33% 6 4%, respectively; p > 0.05), whereas the initial distribution was 58% 6 7% and 11% 6 8%, respec- tively; p < 0.05). The rS1/9 scaffolds implanted subcutane- ously into Balb/c mice were well tolerated. Over a 2-month period, the scaffolds promoted an ingrowth of de novo formed vascularized connective tissue elements and nerve fibers. Thus, scaffolds made of the novel recombinant spidroin 1 analogue are potentially applicable in tissue engi- neering. V C 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 96A: 125–131, 2011. Key Words: spidroin, biopolymer, biodegradable scaffold, biocompatibility INTRODUCTION New biomaterials as scaffolds for artificial tissues and organs are extensively investigated. Biocompatible and bio- degradable scaffolds can support cell attachment and prolif- eration, as well as three-dimensional (3D) orientation of tissues. 1–3 Natural silk derivatives have been widely used for these purposes. The most popular source of silk is the cocoon of the silkworm Bombix mori. 4 Recently, many other types of silk have attracted the attention of research groups worldwide. 5–8 The spider dragline silk of Nephila clavipes consists of two fibrillar proteins (spidroins 1 and 2). 9 Because the biomedical applications of natural spider silk are limited because of insufficient amount of material, the recombinant analogues of spidroins have been recently developed. 10,11 The genetically engineered protein, an ana- logue of N. clavipes spidroin 1 (rS1/9), possesses all typical properties of full-length spidroins, that is, the ability for structural transition to the beta-sheet structure, a spon- taneous formation of nanofibrils and microspheres in aque- ous solutions, and the formation of tough fibers after spinning. 12,13 The goal of this study was to prepare the scaffolds based on recombinant spidroin 1 and to investigate their structural and mechanical properties. It was shown that recombinant spidroin 1 scaffolds are suitable for eukaryotic cell culture as well as for in vivo evaluation of the tissue response to implants. MATERIALS AND METHODS Purification of recombinant protein The artificial gene S1/9 consists of nine chemically synthe- sized ‘‘monomers’’ 1f1. 12 They were amplified as part of the plasmid followed by transformation into yeast cells of Pichia pastoris. The expressed rS1/9 protein was purified from yeast cell extract by the method developed previously, 12 Correspondence to: M. M. Moisenovich; e-mail: mmoisenovich@mail.ru Contract grant sponsor: Russian Foundation for Basic Research; contract grant number: 09-02-00173-a Contract grant sponsor: Federal Program ‘‘Research and Education Staff for Innovative Russia, 2009–2013’’ of the Ministry of Education and Science of Russian Federation; contract grant numbers: P2460, P2087, P816, P407 V C 2010 WILEY PERIODICALS, INC. 125