Autologous chondrocyte implantation is an effective treatment for damaged articular cartilage. However, this method involves surgical procedures that may cause further cartilage degeneration, and in vitro expansion of chondrocytes can result in dedifferentiation. Adipose-derived stem cells (ADSCs) may be an alternative autologous cell source for cartilage regeneration. In this study, we developed an effective method for large-scale in vitro chondrogenic differentiation, which is the procedure that would be required for clinical applications, and the subsequent in vivo cartilage formation of human ADSCs (hADSCs). The spheroid formation and chondrogenic differentiation of hADSCs were induced on a large scale by culturing hADSCs in three-dimensional suspension bioreactors (spinner flasks). In vitro chondrogenic differentiation of hADSCs was enhanced by a spheroid culture compared with a monolayer culture. The enhanced chondrogenesis was probably attributable to hypoxia-related cascades and enhanced cell-cell interactions in hADSC spheroids. On hADSCs loading in fibrin gel and transplantation into subcutaneous space of athymic mice for 4 weeks, the in vivo cartilage formation was enhanced by the transplantation of spheroid-cultured hADSCs compared with that of monolayer-cultured hADSCs. This study shows that the spheroid culture may be an effective method for large-scale in vitro chondrogenic differentiation of hADSCs and subsequent in vivo cartilage formation.

The therapeutic efficacy of drugs often depends on the drug delivery carrier. For efficient delivery of therapeutic proteins, delivery carriers should enable the loading of large doses, sustained release, and retention of the bioactivity of the therapeutic proteins. Here, it is demonstrated that graphene oxide (GO) is an efficient carrier for delivery of therapeutic proteins. Titanium (Ti) substrates are coated with GO through layer-by-layer assembly of positively (GO-NH₃⁺) and negatively (GO-COO⁻) charged GO sheets. Subsequently, a therapeutic protein (bone morphogenetic protein-2, BMP-2) is loaded on the GO-coated Ti substrate with the outermost coating layer of GO-COO⁻ (Ti/GO⁻). The GO coating on Ti substrate enables loading of large doses and the sustained release of BMP-2 with preservation of the structure and bioactivity of the drug. The extent of in vitro osteogenic differentiation of human bone marrow-derived mesenchymal stem cells is higher when they are cultured on Ti/GO- carrying BMP-2 than when they are cultured on Ti with BMP-2. Eight weeks after implantation in mouse models of calvarial defects, the Ti/GO-/BMP-2 implants show more robust new bone formation compared with Ti, Ti/GO-, or Ti/BMP-2 implants. Therefore, GO is an effective carrier for the controlled delivery of therapeutic proteins, such as BMP-2, which promotes osteointegration of orthopedic or dental Ti implants.

6455wileyonlinelibrary.com while graphene oxide (GO) induced differentiation of hMSCs to adipocytes [ 5 ] and myoblasts. [ 7 ] The previous studies indicate that G and GO have distinct properties when utilized as substrates for stem cell culture and differentiation. For example, GO showed higher protein adsorption compared with G. [ 5 ] Although they both enhanced cell adhesion and growth, proteins such as insulin interacted with G and GO in different manners. [ 5 ] GO formed an electrostatic interaction with insulin and preserved the protein structure, while G denatured the native structure. Because the insulin protein structure was preserved on GO, the adipogenic differentiation of hMSCs was signifi cantly enhanced when cultured on GO compared with G. [ 5 ] Although many studies have explored how various properties of G and GO differentiate adult stem cells, [5][6][7][8] there has been no report on the use of G or GO for chondrogenic differentiation. This study reports, for the fi rst time, the use of GO for the chondrogenic differentiation of stem cells.The chondrogenic differentiation of adult stem cells is conventionally achieved through the culture of cells in pellets. [ 9 ] Cell condensation into pellets mimics the chondrogenic progenitor cell derivation during embryogenesis through cell condensation. [ 10 ] However, because the major composition of the pellets is cells and the extracellular matrix (ECM) amount is negligible, the cell-ECM interaction that promotes chondrogenic differentiation [ 11 ] is absent. Addionally, the diffusional limit of approximately 150 -200 µm restricts the mass transportation of many molecules, including oxygen, into the pellets. [ 12 ] Such characteristics thus limit the size of the pellets because pellets larger than the diffusional limit display a necrotic core surrounded by a viable cell layer. To overcome such hurdles, we used GO sheets as both a cell-adhesion substrate and a chondrogenic inducer-delivery carrier for in vitro chondrogenic differentiation of human adipose-derived stem cells (hASCs) in pellets in this study ( Figure 1 ). GO sheets (size = 0.5-1 µm) were utilized to adsorb fi bronectin (FN, a cell-adhesion protein) and transforming growth factor-β3 (TGF-β3) (a chondrogenic inducer) and were then incorporated into hASC pellets. The adsorption of FN and TGF-β3 on GO sheets relies on the surface chemistry of GO. GO features both hydrophobic π domains Dual Roles of Graphene Oxide in Chondrogenic Differentiation of Adult Stem Cells: Cell-Adhesion Substrate and Growth Factor-Delivery CarrierHee Hun Yoon , Suk Ho Bhang , Taeho Kim , Taekyung Yu , Taeghwan Hyeon , and Byung-Soo Kim * Here, it is shown that graphene oxide (GO) can be utilized as both a celladhesion substrate and a growth factor protein-delivery carrier for the chondrogenic differentiation of adult stem cells. Conventionally, chondrogenic differentiation of stem cells is achieved by culturing cells in pellets and adding the protein transforming growth factor-β3 (TGF-β3), a chondrogenic factor, to ...

In this study, we designed graphene oxide-functionalized polyethylene glycol diacrylate hydrogels to assign cell adhesion-dependent biofunctionality, which resulted in cell adhesion dependent osteogenic differentiation of encapsulated stem cells.

In this study, we demonstrate that graphene oxide (GO) can be used for the delivery of bone morphogenetic protein-2 (BMP-2) and substance P (SP), and that this delivery promotes bone formation on titanium (Ti) implants that are coated with GO. GO coating on Ti substrate enabled a sustained release of BMP-2. BMP-2 delivery using GO-coated Ti exhibited a higher alkaline phosphatase activity in bone-forming cells in vitro compared with bare Ti. SP, which is known to recruit mesenchymal stem cells (MSCs), was co-delivered using Ti or GO-coated Ti to further promote bone formation. SP induced the migration of MSCs in vitro. The dual delivery of BMP-2 and SP using GO-coated Ti showed the greatest new bone formation on Ti implanted in the mouse calvaria compared with other groups. This approach may be useful to improve osteointegration of Ti in dental or orthopedic implants.

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The culture surface can affect the in vitro differentiation of stem cells. In this study, we investigated whether modifying the culture surface with 3,4-dihydroxy-l-phenylalanine (DOPA), an element of mussel adhesion protein, could enhance the in vitro osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMMSCs). hBMMSCs cultured on DOPA-coated plates exhibited better cell adhesion and spreading compared with noncoated conventional tissue culture plates. The DOPA coated did not affect the apoptosis or viability of the cultured hBMMSCs. Also, hBMMSCs cultured on DOPA-coated plates exhibited a higher degree of osteogenic differentiation than did hBMMSCs cultured on noncoated plates, as evaluated with alkaline phosphate (ALP) activity, calcium content, and the mRNA expression of runt-related transcription factor 2, ALP, and osteocalcin. Further, hBMMSCs cultured on DOPA-coated plates demonstrated a higher capability of ectopic bone formation in vivo following implantation in the subcutaneous space of athymic mice compared with hBMMSCs cultured on noncoated plates, as evaluated with microcomputer topography analysis and histomorphometry. These results indicate that modifying the culture surface with DOPA can enhance the in vitro osteogenic differentiation of hBMMSCs.

In this article, we examined the feasibility of using 3,4-dihydroxy-L-phenylalanine (DOPA) as a cell adhesion molecule in serum-free cultures of anchorage-dependent mammalian cells. DOPA is a critical, functional element in mussel adhesive proteins and is known to bind strongly to various natural or synthetic materials. DOPA coating on culture plates was confirmed using X-ray photoelectron spectroscopy and energy-dispersive spectroscopy. Human dermal fibroblasts (HDFs) were cultured on DOPA-coated, fibronectin-coated, or no material-coated culture plates in serum-free medium. HDFs cultured on DOPA showed the highest cell adhesion ratio, spreading, and viability but the lowest apoptotic activity. Therefore, DOPA may be a useful cell-adhesion molecule for serum-free culture.