The effect of decellularized bone/bone marrow produced by high-hydrostatic pressurization on the osteogenic differentiation of mesenchymal stem cells Yoshihide Hashimoto a, b , Seiichi Funamoto a, c , Tsuyoshi Kimura a, d , Kwangwoo Nam a, d , Toshiya Fujisato e , Akio Kishida a, d, * a Division of Biofunctional Molecules, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan b Japan Society for the Promotion of Science, 8 Ichibancho, Chiyoda-ku, Tokyo 102-8472, Japan c Department of Thoracic and Cardiovascular Surgery, Sapporo Medical University,17 Minami 1 Jyou-Nishi, Chuo-ku, Sapporo, Hokkaido 060-8556, Japan d Japan Science and Technology Agency, CREST, 5 Sanbancho, Chiyoda-kui, Tokyo 102-0075, Japan e Faculty of Engineering, Osaka Institute of Technology, 5-16-1 Omiya, Asahi-ku, Osaka, 535-8585, Japan article info Article history: Received 18 May 2011 Accepted 4 June 2011 Available online 2 July 2011 Keywords: Decellularization Extracellular matrix Bone marrow Scaffold Hematopoiesis abstract Decellularized bone/bone marrow was prepared to provide a microenvironment mimicking that of the bone marrow for three-dimensional culture in vitro. Bone/bone marrows were hydrostatically pressed at 980 MPa at 30  C for 10 min to dismantle the cells. Then, they were washed with EGM-2 and further treated in an 80% EtOH to remove the cell debris and lipid, respectively. After being rinsed and shaken with PBS again, treated bone/bone marrows were stained with hematoxylin and eosin (H-E) to assess the efficacy of decellularization. Cells were determined to have been completely removed through H-E staining of their sections and DNA quantification. Rat mesenchymal stem cells (rMSCs) were seeded on the decellularized bone/bone marrows and cultured for 21 days. The adhesion of rMSCs on or into decellularized bone/bone marrows was confirmed and proliferated over time in culture. The osteogenic differentiation effect of decellularized bone/bone marrows on rMSCs in the presence or absence of dexamethasone was investigated. Decellularized bone/bone marrows without dexamethasone signifi- cantly increased alkaline phosphatase (ALP) activity, indicating promoted osteogenic differentiation of rMSCs. In an animal study, when decellularized bone/bone marrows were implanted into the rat subcutaneous, no immune reaction occurred and clusters of the hematopoietic cells could be observed, suggesting the decellularized bone/bone marrows can provide a microenvironment in vivo. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Appropriate three-dimensional (3D) scaffolds must be devel- oped to provide a cellular microenvironment for tissue engineering and cell culture applications [1,2]. This is because nearly all cells reside in an extracellular matrix (ECM) consisting of a complex 3D fibrous network and ECMs regulate many cellular behaviors, including migration, proliferation, and differentiation [3,4]. Specifically, the microenvironments of stem cells are inherently 3D, and their biochemistry, topology, and elasticity strongly affect the differentiation process [5e7]. However, conventional two- dimensional (2D) cultures are considerably limited in mimicking these 3D environments due to the lack of structural architecture and unique ECM of each cell type. To overcome 2D culture limitations, many researchers have been investigating 3D porous scaffolds with different chemicals and structural properties such as polymers, metals, ceramics, and combinations of two or three components [8e15]. These 3D scaf- folds have been utilized to grow or guide the differentiation of different stem cell types including mesenchymal [8,12e15], hematopoietic [16], and embryonic stem cells [17,18]. These 3D scaffolds may not, however, represent ideal environments for studying responses of cells since each type of cell is embedded in a considerably different 3D environment. Therefore, one of the difficulties in investigating a 3D cellular microenvironment is selecting the appropriate 3D scaffold. Our approach to developing 3D scaffolds for investigating the cellular microenvironments is to use decellularized tissue. Decel- lularized tissue has the same structure and composition as natural * Corresponding author. Division of Biofunctional Molecules, Institute of Bioma- terials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda- Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. Tel./fax: þ81 3 5280 8028. E-mail address: kishida.fm@tmd.ac.jp (A. Kishida). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.06.008 Biomaterials 32 (2011) 7060e7067