To date, expansion of bone-marrow-derived mesenchymal stem cells (MSCs) is typically carried out on twodimensional (2D) tissue culture plastic. Since this 2D substratum is very different from the physiological situation, MSCs gradually lose their unique multipotent properties during expansion. Recently, the role of the extracellular matrix (ECM) microenvironment (''niche'') in facilitating and regulating stem cell behavior in vivo has been elucidated. As a result, investigators have shifted their efforts toward developing threedimensional (3D) scaffolds capable of functioning like the native tissue ECM. In this study, we demonstrated that stromal-cell-derived ECM, formed within a collagen/hydroxyapatite (Col/HA) scaffold to mimic the bone marrow ''niche,'' promoted MSC proliferation and preserved their differentiation capacity. The ECM was synthesized by MSCs to reconstitute the tissue-specific 3D microenvironment in vitro. Following deposition of the ECM inside Col/HA scaffold, the construct was decellularized and reseeded with MSCs to study their behavior. The data showed that MSCs cultured on the ECM-Col/HA scaffolds grew significantly faster than the cells from the same batch cultured on the regular Col/HA scaffolds. In addition, MSCs cultured on the ECMCol/HA scaffolds retained their ''stemness'' and osteogenic differentiation capacity better than MSCs cultured on regular Col/HA scaffolds. When ECM-Col/HA scaffolds were implanted into immunocompromised mice, with or without loading MSCs, it was found that those scaffolds formed less bone as compared with regular Col/ HA scaffolds (i.e., without ECM), in both cases of with or without loading MSCs. The in vivo study further confirmed that the ECM-Col/HA scaffold was a suitable mimic of the bone marrow ''niche.'' This novel 3D stromal-cell-derived ECM system has the potential to be developed into a biomedical platform for regenerative medicine applications.

Salivary gland hypofunction often results from a number of causes, including the use of various medications, radiation for head and neck tumors, autoimmune diseases, diabetes, and aging. Since treatments for this condition are lacking and adult salivary glands have little regenerative capacity, there is a need for cell-based therapies to restore salivary gland function. Development of these treatment strategies requires the establishment of a system that is capable of replicating the salivary gland cell "niche" to support the proliferation and differentiation of salivary gland progenitor cells. In this study, a culture system using three-dimensional silk fibroin scaffolds (SFS) and primary salivary gland epithelial cells (pSGECs) from rat submandibular (SM) gland and parotid gland (PG) was established and characterized. pSGECs grown on SFS, but not tissue culture plastic (TCP), formed aggregates of cells with morphological features resembling secretory acini. High levels of amylase were released into the media by both cell types after extended periods in culture on SFS. Remarkably, cultures of PG-derived cells on SFS, but not SM cells, responded to isoproterenol, a β-adrenergic receptor agonist, with increased enzyme release. This behavior mimics that of the salivary glands in vivo. Decellularized extracellular matrix (ECM) formed by pSGECs in culture on SFS contained type IV collagen, a major component of the basement membrane. These results demonstrate that pSGECs grown on SFS, but not TCP, retain important functional and structural features of differentiated salivary glands and produce an ECM that mimics the native salivary gland cell niche. These results demonstrate that SFS has potential as a scaffold for creating the salivary gland cell niche in vitro and may provide an approach for inducing multipotent stem cells to provide therapeutically meaningful numbers of salivary gland progenitor cells for regenerating these tissues in patients.