Nanoclays mediate stem cell differentiation and mineralized ECM formation on biopolymer scaffolds Avinash H. Ambre, Dinesh R. Katti, Kalpana S. Katti Department of Civil Engineering, North Dakota State University, Fargo, North Dakota 58105 Received 23 July 2012; revised 10 October 2012; accepted 27 November 2012 Published online 15 February 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.34561 Abstract: In this work, novel modified nanoclays were used to mineralize hydroxyapatite (HAP) mimicking biomineraliza- tion in bone. This in situ HAPclay was further incorporated into chitosan/polygalacturonic acid (Chi/PgA) scaffolds and films for bone tissue engineering. Differences in microstruc- ture of the scaffolds were observed depending on the changes in processing of in situ HAPclay with ChiPgA biopol- ymer system. Response of human mesenchymal stem cells (hMSCs) on these scaffolds and films was studied using imaging and assays. SEM micrographs indicate that hMSCs were able to adhere to ChiPgA/in situ HAPclay scaffolds and phase contrast images indicated formation of mineralized nodules on ChiPgA/in situ HAPclay films in absence of osteo- genic supplements used for differentiation of hMSCs. The formation of mineralized nodules by hMSCs was confirmed by positive staining of the nodules by Alizarin Red S dye. Via- bility and differentiation assays showed that ChiPgA/in situ HAPclay scaffolds were favorable for viability and differentia- tion of hMSCs. Unique two-stage cell seeding experiments were performed as a strategy to enhance tissue formation by hMSCs on ChiPgA/in situ HAPclay composite films. This work showed that biomaterials based on ChiPgA/in situ HAPclay composites can be used for bone tissue engineering applica- tions and in situ nanoclay-HAP system mediates osteoinduc- tive and osteoconductive response from hMSCs. V C 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A: 2644–2660, 2013. Key Words: human mesenchymal stem cells, chitosan, poly- galacturonic acid, hydroxyapatite, nanocomposite, scaffold How to cite this article: Ambre AH, Katti DR, Katti KS. 2013. Nanoclays mediate stem cell differentiation and mineralized ECM formation on biopolymer scaffolds. J Biomed Mater Res Part A 2013:101A:2644–2660. INTRODUCTION Tissue engineering introduced by Langer and Vacanti 1 is one of the most promising approaches in regenerative medi- cine to rectify tissue defects arising from pathological condi- tions and injuries sustained. In addition, tissue engineering strategies present relatively less invasive routes and capabil- ity to cure tissue defects by natural processes, compared with conventional treatment methods. Scaffolds, cells, and growth factors used for supplementing cell culture media are vital components that can be modulated for successful tissue regeneration in tissue engineering. Scaffolds can be designed to provide the required biophysical and biochemi- cal signals to the cells for tissue formation in a three dimen- sional environment similar to the in vivo environment. The limited availability of autologous tissue biopsies restricts the number of cells available for regeneration in case of large tissue defects. Also, cells obtained from such tissue biopsies exhibit limited proliferation and tissue formation abilities 2 in addition to improper phenotype expression for certain cell types. 3 Cells for tissue engineering need to be obtainable through existing cell isolation techniques with low donor site morbidity, show ‘‘immunological compat- ibility’’ and maintain a stable phenotype while being recep- tive to signals from the surrounding environment. 3–5 Mesen- chymal stem cells (MSCs) can proliferate in an undifferentiated state and also maintain their capability to differentiate into different cell lineages. The differentiation of MSCs is non-spontaneous unlike the embryonic stem cells and can be controlled by using appropriate stimuli (chemi- cal, mechanical, and electrical). MSCs also have also been reported for their ‘‘immunosuppressive’’ effects 6–8 although research related to this property of MSCs continues to evolve. These cells exert suppressive effects on cells (e.g., T, B, and natural killer cells) involved in the immune system. 6,9 This makes possible the use of allogenic MSCs for regenerat- ing a defective tissue in a host using tissue engineering principles. Although bone marrow has been the major source of MSCs, these cells are also found in different tis- sues. Thus, the important attributes of MSCs and their potential to be a major cell source for tissue engineering has stimulated research focused on the behavior of MSCs seeded on engineered scaffolds. Materials synthesis and materials processing for devel- oping scaffolds that satisfy tissue engineering requirements (biocompatibility, biodegradability, porosity, pore size, and adequate mechanical properties) have been the focus of Correspondence to: Dr. K. S. Katti; e-mail: Kalpana.katti@ndsu.edu Contract grant sponsors: National Science Foundation IMR and MRI; ND EPSCoR 2644 V C 2013 WILEY PERIODICALS, INC.