Finally, dentin-related features were detected in the spheroids containing BMPs-loaded microparticles after 5 weeks, suggesting that these hDPSC-LSS spheroids might be useful for dentin tissue regeneration. Rabbit polyclonal to IL29 1. are available from the corresponding authors on reasonable request. Abstract Human dental pulp stem cells (hDPSCs) are the primary cells responsible for dentin regeneration. Typically, in order to allow for odontoblastic differentiation, hDPSCs are cultured over weeks with Mcl1-IN-11 differentiation-inducing factors in a typical monolayered culture. However, monolayered cultures have significant drawbacks including inconsistent differentiation efficiency, require a higher BMP concentration than should be necessary, and require periodic treatment with BMPs for weeks to see results. To solve these problems, we developed a 3D-cell spheroid culture system for odontoblastic differentiation using microparticles with leaf-stacked structure (LSS), which allow for the sustained release of BMPs and adequate supply of oxygen in cell spheroids. BMPs were continuously released and maintained an effective concentration over 37 days. hDPSCs in the spheroid maintained their viability for 5 weeks, and the odontoblastic differentiation efficiency was increased significantly compared to monolayered cells. Finally, dentin-related features were detected in the spheroids containing BMPs-loaded microparticles after 5 weeks, suggesting that these hDPSC-LSS spheroids might be useful for dentin tissue regeneration. 1. Introduction In a normal biological system, stem cells undergo intensive cell-cell contacts to develop tissues with three-dimensional (3D) structures. This is mediated by the microenvironment including the extracellular matrix surrounding cells, which are distinct physiological cues for cell differentiation and proliferation. Two-dimensional (2D) monolayer cultures inadequately reproduce Mcl1-IN-11 the native microenvironment of stem cells created by intrinsic and extrinsic cellular communications, resulting in an unnatural spatial distribution of signaling molecules, oxygen, and nutrients [1]. Moreover, the normal physiological behaviors of stem cells can be distorted Mcl1-IN-11 in monolayer cultures, leading to the loss of the multilineage potential and replicative ability [2, 3]. To mimic the physiological environment, various methods have been reported for culturing cells in a 3D structure, including scaffold-free cultures [4, 5], cultures grown on various scaffold materials [6C8], and cultures embedded in gel materials [9, 10]. 3D cell aggregates, known as spheroids, yield a multicellular mass that mimics the natural cell niche [11]. Scaffold-free 3D culture systems have advantages in preventing the inflammation and infection caused by scaffold material degradation [12] and disadvantages of poor guidance on cells required for cell proliferation and differentiation. Cell spheroids have been utilized in various tissue engineering [13C15] and stem cell applications [10, 16C19]. However, only recently have cell spheroids been investigated for dental tissue regeneration, leaving much data on the topic still to be discovered. Spheroid culturing of human periodontal ligament stem cells (hPDLSCs) significantly enhanced stemness and osteogenic potential compared with hPDLSCs cultured in a monolayer [11, 20]. In addition, osteo/odontoblastic gene expression and mineralization are upregulated in immortalized mouse dental Mcl1-IN-11 papilla cells cultured as 3D spheroids when compared with 2D monolayer-cultured cells [21]. In addition to the 3D aspects of the cell culture system, the sustained supply of cytokines or growth factors also plays an important role in mimicking the natural microenvironment and achieving effective differentiation. Biomaterial-based delivery systems in culture have been previously reported to form nanoparticles, microparticles, hydrogels, and tissue engineering scaffolds, improving the pharmacokinetics of retained factors and reducing toxicity [13C15, 22]. Mcl1-IN-11 Recently, 3D cell culture models, including cell spheroids, have been assessed in terms of drug delivery efficacy [5C7]. In previous studies, signaling pathways stimulated by Wnt proteins, transforming growth factor-(TGFvalue of less than 0.05 was considered statistically significant. 3. Results 3.1. Development of BMPs-Loaded Microparticles for Sustainable Delivery Microparticles with a leaf-stacked structure (LSS) were developed to provide an appropriate microenvironment with a continuous supply of factors and sufficient surface for cell adhesion [27]. Diameters of these particles were 25~53? 0.05; ?? 0.01; ??? 0.001; NS: not significant. (d) H&E staining of paraffin section of hDPSC-spheroids containing LSS particles. As well as covering on spheroid by cell layers, cells were detected inside of spheroid. (A) hDPSC spheroid with particles. (B) hDPSC spheroid with BMPs-loaded particles. 3.3. Enhancement of Odontoblastic Differentiation in hDPSC/LSS Spheroids Releasing BMP-2 and BMP-4 Odonto/osteoblastic differentiation of hDPSCs cultured in spheroids was evaluated based on the transcriptional expression of odonto/osteogenic markers. Overall, gene expression levels of representative markers were increased in spheroids even without BMP-2 or BMP-4 (Figure.