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Fast and efficient recovery of injured bone tissue is the key task of modern orthopedics. One of the main approaches is the creation of bioartificial analogs of.

ISSN 16076729, Doklady Biochemistry and Biophysics, 2011, Vol. 440, pp. 228–230. © Pleiades Publishing, Ltd., 2011. Original Russian Text © I.I. Agapov, M.M. Moisenovich, T.V. Druzhinina, Ya.A. Kamenchuk, K.V. Trofimov, T.V. Vasilyeva, A.S. Konkov, A.Yu. Arhipova, O.S. Sokolova, V.V. Guzeev, M.P. Kirpichnikov, 2011, published in Doklady Akademii Nauk, 2011, Vol. 440, No. 6, pp. 830–833.

BIOCHEMISTRY, BIOPHYSICS AND MOLECULAR BIOLOGY

Biocomposite Scaffolds Containing Regenerated Silk Fibroin and Nanohydroxyapatite for Bone Tissue Regeneration I. I. Agapova, M. M. Moisenovichb, T. V. Druzhininac, Ya. A. Kamenchukc, K. V. Trofimovc, T. V. Vasilyevab, A. S. Konkovb, A. Yu. Arhipovab, O. S. Sokolovab, V. V. Guzeevc, and Academician M. P. Kirpichnikovb Received July 4, 2011

DOI: 10.1134/S1607672911050103

Fast and efficient recovery of injured bone tissue is the key task of modern orthopedics. One of the main approaches is the creation of bioartificial analogs of bone tissue. The main component of bone is minerals in the form of hydroxyapatite (HA) nanocrystals, which are tightly associated with collagen fibers. Con structs made from regenerated silk can replace the nat ural collagen, because they are an excellent substrate for cell growth and proliferation and exhibit a high mechanical strength [1, 2]. When implanted, these constructs are not rejected by the immune system and are replaced by the surrounding tissue with the involvement of immunocompetent cells. We have earlier obtained threedimensional scaf folds of regenerated silk fibroin [3] and recombinant spidroin [4], whose surface was a good substrate for adhesion and proliferation of mammalian cells. In vitro experiments showed that the threedimensional porous structure of scaffolds provides free permeation of cells from the surface to deeper layers [5]. When the scaffolds were implanted to experimental animals, their material was replaced with the recipient tissue. Within the implanted scaffolds, a large number of blood vessels and nerve fibers were found, indicating that active processes of angiogenesis and neurogenesis accompanying biodegradation of implants took place [6]. In this study, we investigated the properties of scaf folds made of regenerated silk fibroin after mineraliza tion with HA and assessed the possibility of using them for bone tissue regeneration.

a

Shumakov Institute of Transplantology and Artificial Organs, Federal Agency for HighTech Medical Services, ul. Shchukinskaya 1, Moscow, 113182 Russia b Biological Faculty, Moscow State University, Moscow, 119992 Russia c Hospital no. 81 of Federal Biomedical Agency of Russia, ul. Mira 4, Seversk, Tomsk oblast, 636035 Russia

Fibroin was isolated from silkworm cocoons kindly provided by Dr. V.V. Bogoslovskii, Director of State Sci entific Institution “Republican Research Station of Sericulture, Russian Academy of Agricultural Sci ences” (Zheleznovodsk, Stavropol krai). To remove sericin and other admixtures, finely minced cocoons were boiled for 1 h in 0.03 M NaHCO3, then washed with water, and dried. Material for bone regeneration was obtained in the form of threedimensional scaffolds using the leaching method, as described earlier [6]. Scaffolds were formed as cylinders 5 mm in length and 2 mm in diameter. Composite threedimensional scaf folds contained 10% nanohydroxyapatite (nanoGA), which was obtained by the original procedure from ani mal bone tissue [7]. According to scanning electron microscopy data, the scaffolds had a porous structure with a pore diam eter of 250 µm (Fig. 1). Large pores were connected with one another through channels and holes. Both types of scaffolds were permeable to ink particles, which easily penetrated from the surface into deeper layers. Thus, the mineralization of scaffolds does not influence the size and connection of pores. The incor poration of HA into the scaffolds increased the pore surface relief. Interaction of mineralized and nonmin eralized substrates with mammalian cells was studied earlier [3]. It was shown that the incorporation into scaffolds of nanoGA noticeably stimulated adhesion and accelerated proliferation of cells cultured on scaf folds. The microstructure of both scaffolds determined migration of cells from the surface into deeper layers, where the cells are provided with gas and metabolic product exchange [3]. Regeneration of bone tissue was studied in Wistar rats weighing 190–220 g. In experiments, we took into account all the requirements of the international qual ity system standard GLP (Good Laboratory Practice). Combined anesthesia was ensured by intramuscular injection into the femoral muscle of 0.05 mg zolytil and 0.01 mg xylovet (proceeding from the average

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BIOCOMPOSITE SCAFFOLDS CONTAINING REGENERATED SILK FIBROIN

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1 (а)

100 µm

Fig. 2. Formation of bone tissue within scaffold within one week after implantation. (1) The surface of the scaffold is partially covered with osteogenic cells; (2) intact mature bone. Histomorphological studies were performed by stan dard methods. Bone samples were fixed in Bouin fluid and then decalcified in 25% Trilon B. Tissue fragments were embedded in paraffin and serial sections were prepared by the conventional methods. Slices 5–6 µm thick were cut with a HM 355S rotary microtome (Microm) and stained with hematoxylineosin.

(b)

100 µm

Fig. 1. Porous structure of (a) nonmineralized and (b) mineralized scaffold obtained from regenerated fibroin. Mineralization causes changes in the surface relief of the scaffold obtained from regenerated silk.

weight of animals of ~200 g). After shaving the surgical field in the projection of the left femur and treating skin with 70% ethanol, an incision 25 mm long was made in parallel to the femur axis. Then, the femoral muscle fascias were dissected and muscle tissue was pulled apart by the blunt method to expose the outer surface of the femur. In the diaphysis of the femur, a through hole 2 mm in diameter was made. In the experimental groups of animals, the defect was filled with scaffold fragments corresponding to the shape of damages. Muscle tissue over the implantation site was sutured, after which skin sutures were placed on the wound. The hole in the femur in animals of the first and second groups was filled with the nonmineralized scaffold and with the HAcontaining scaffold, respec tively. In the control group, the defect was not filled. DOKLADY BIOCHEMISTRY AND BIOPHYSICS

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The level of bone regeneration was evaluated by Xray studies on a Somatom AR.HP computer tomography (Siemens). The results of Xray studies are presented in the table. Starting from the second week, a higher index of bone tissue regeneration (BTR) was observed in rats of experimental groups compared with the con trol group. The most pronounced advantage of the HAcontaining scaffold compared with the nonmin eralized scaffold was detected four weeks after opera tion (table). Thus, our data indicate that the HAcon taining scaffolds ensure higher BTR indices than the nonmineralized scaffolds. These data were confirmed by histological studies (Fig. 2). Bone tissue began to form in the mineralized scaffold within one week after implantation. In such scaffolds, single bone beams were present in deeper layers. In the nonmineralized scaffolds, the results of osteogenic activity were observed one week after implantation only at the periphery. The bulk of the nonmineralized implant at this stage remained intact. At the same time, no signs of osteogenesis were observed in the injured area in the 2011

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AGAPOV et al.

Dynamics of changes in average values of bone tissue regen eration index Bone tissue regeneration index Study group

week 1 after operation

week 2 after operation

week 4 after operation

Control Fibroin Fibroin + HA

28.2 ± 0.3 28.8 ± 0.06 29.2 ± 0.1

28.8 ± 0.2 30 ± 0.3 44.3 ± 0.2

30.7 ± 0.3 43.5 ± 0.4 46.8 ± 0.3

Note: Scanning range was determined by tomogram and by visual inspection. Maximum perforation of the cortical layer of each femur was indicated on scans. Reconstruction was performed separately for each bone to obtain density indices for the cortical layer and in the defect area on the Hounsfield scale. Axial sections were made at a voltage of 110 kV and current strength of 50 mA. Scan thickness was 3 mm; tomography step was 1 to 2 mm, the algorithm was extremity. Data are represented as HU ± SD.

control animals, and the area of the defect was par tially filled with the bone marrow. Four weeks after implantation, only single bone beams were identified in deep layers of the nonmin

1 1

1

eralized scaffold (Fig. 3). The size and number of bone trabeculae formed in the center of the HAcontaining scaffold significantly exceeded those in the scaffold obtained from fibroin without HA. In the mineralized scaffolds, newly formed bone tissue was detected in the place of the scaffold; osteogenic cells, osteoblasts, and osteoclasts were present at the edges of scaffolds. Thus, the results of this study show that the porous scaffolds obtained from regenerated fibroin can be used for bone tissue regeneration and that mineraliza tion of scaffolds with nanoHA improves their regener ative properties. ACKNOWLEDGMENTS The work was supported by the Ministry of Edu cation and Science of the Russian Federation within the Federal Target Program “Research and scientificpedagogical personnel of innovation in Russia 2009–2013 years” on the State contract number P 407 from 12.05.2010 (P 816 of 24.05.2010, the, P 2460 from 19.11.2009, the, no. 14.740.11.0461 01.10.2010), under the Federal Target Program “Research and development on pri ority directions of scientifictechnological complex of Russia for 2007–2012” by the State contract no. 16.512. 11.2177 from 01.03.2011, partially by means of the Russian Foundation for Basic Research (grant 090200173).

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REFERENCES 2

1. Kon’kov, A.S., Pustovalova, O.L., and Agapov, I.I., Biotekhnol., 2010, no. 1, pp. 9–16. 2. Omenetto, F.G. and Kaplan, D.L., Science, 2010, vol. 329, no. 5991, pp. 528–531. 3. Agapov, I.I., Moisenovich, M.M., Vasil’eva, T.V., et al., Dokl. Biochem. Biophys., 2010, vol. 433, no. 5, pp. 201– 204 [Dokl. Akad. Nauk, 2010, vol. 433, no. 5, pp. 699– 702]. 1

4. 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].

Fig. 3. Bone beams formed de novo within scaffold 4 weeks after implantation. Scaffold (1), partially covered with osteogenic cells, is in contact with newly formed bone beam (2); contact points are indicated with arrows. Part of bone tissue develops within the scaffold. The cavities between scaffold fragments and bone beams are filled with bone marrow cells (3). The multinucleated cell in the inset is an osteoclasts contacting with the scaffold material. Nuclei of osteogenic cells can be seen nearby on the scaf fold surface.

5. Pustovalova, O.L., Agapov, I.I., Moisenovich, M.M., et al., Vestn. Transplantol. Iskusstv. Org., 2009, vol. 11, no. 2, pp. 54–59. 6. Moisenovich, M.M., Pustovalova, O.L., Arhipova, A.Yu., et al., J. Biomed. Mat. Res. Pt. A, 2011, vol. 96, no. 1, pp. 125–131. 7. Kamenchuk, Ya.A., Zelichenko, E.A., Druzhinina, T.V., et al., Biotekhnologiya, 2010, no. 5, pp. 89–96.

DOKLADY BIOCHEMISTRY AND BIOPHYSICS

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2011

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