Figure 12, Greatly enhanced total calcium deposition on CAP modified nHA/Chitosan scaffold after 3 weeks of culture. Data are mean ±SEM; n=9. *p<0.01 when compare to all other substrates. **p<0.05 when compare to nontreated substrates. Enhanced Osteogenic Differentiation of Stem Cells in Novel Bioactive Cold Plasma Treated Nanostructured Bone Scaffolds Mian Wang 1 ; Xiaoqian Cheng 1 ; Benjamin Holmes 1 ; Wei Zhu 1, Michael Keidar 1 ; Lijie Grace Zhang 1,2* 1 Department of Mechanical and Aerospace Engineering, 2 Department of Medicine; The George Washington University, Washington, DC Introduction As an interdisciplinary field, regenerative medicine and tissue engineering aim to recreate living and functional tissues via the use of biomaterials, growth factors, and stem cells. To date, they hold huge potential for bone regeneration. Specifically, autologous human mesenchymal stem cells (MSCs) from bone marrow are readily accessible and harvested, i.e. from the iliac crest, and display several key biological characteristics. However, current stem cell-based bone regeneration still has many limitations such as low levels of stem cell engraftment and transplant survival, uncontrollable osteogenic differentiation and decreased production of extracellular matrix proteins, which have greatly inhibited the full clinical potential. Native stem cells reside in a nanostructured bioactive extracellular matrix (ECM) environment and are extremely sensitive to minute changes to their surroundings. Thus, the objective of this study is to create a biomimetic and bioactive cold atmospheric plasma (CAP) modified three-dimensional nanocomposite scaffold to address the aforementioned limitations and current challenges to further improve MSC osteogenic differentiation and bone regeneration in situ. Materials and Methods  Fabrication of chitosan/nHA scaffold Biomimetic nanocystalline hydroxyapatites (nHAs) were synthesized via a wet chemistry precipitation method plus a special hydrothermal treatment. 3D porous chitosan scaffolds with 10% hydrothermally treated nHA were fabricated via a simple lyophilization procedure.  CAP modification The prepared hydrated nHA/Chitosan scaffolds were placed in a 48-well plate. The CAP generator had a 1 mm diameter central powered electrode and a grounded outer electrode wrapped around a 4.5 mm diameter quartz tube. Different CAP treatment conditions (i.e., output voltage: 3.4, 3.6, and 3.8 kV) were investigated. The distance between the CAP jet and samples was 20 mm.  Scaffold Characterization Surface topographies of the untreated scaffold and CAP modified scaffolds were imaged via a scanning electron microscopy (SEM) and white light interferometry. Surface wettability of scaffolds was measured using a contact angle analyzer (DSA4, Krüss). Specific proteins absorptions were evaluated via ELISA assays.  MSC adhesion study and osteogenic differentiation study in vitro For adhesion study, MSCs were seeded at a density of 50,000 cells per scaffold on different CAP modified nHA/chitosan scaffolds and cultured for 4h. For differentiation study, MSCs were seeded at a density of 150,000 cells/cm 2 in 0, 3, and 5 minute 3.6 kV output voltage CAP treated nHA/chitosan scaffolds and were cultured in an osteogenic medium for 1, 2 and 3 weeks. Total protein, collagen synthesis and calcium deposition were quantified via biochemistry assay.  MSC growth morphology under a confocal microscope The MSCs were cultured in CAP treated scaffold for 3 days, then stained with DAPI and Rhodamine dyes. Cell growth morphology was imaged under a confocal microscope. Results Figure 2, Plasma appearance as function of output voltage. Figure 4, (a) Emission intensity as function of output voltage; and (b) O (777 nm)/N2+ (391 nm) ratio as a function of output voltage. Figure 14, Confocal microscopy images of MSC growth in different CAP modified bone scaffold. (A) and (a) control scaffolds; (B) and (b) 3 min CAP treated scaffolds; (C) and (c) 3 min CAP treated scaffolds. (a), (b) and (c) are cell only images. Conclusion In summary, biomimetic and bioactive nHA/chitosan scaffolds were fabricated via a lyophilization method and were surface modified via a CAP treatment technique. Significantly improved MSC adhesion, migration and osteogenic differentiation were observed in 3 and 5 min CAP modified nHA/chitosan scaffolds. A fibrous morphology, more open pores and hydrophilic surface can be formed on scaffolds after CAP treatment, which can create a more biomimetic microenvironment for specific protein absorption, cell attachment, infiltration and differentiation. Our results show the potential of CAP modified nHA/chitosan scaffolds for bone regeneration. In particular, as an emerging technique for medicine, the CAP treatment can be easily used for many other complex tissue scaffold modification. (b) (a) Figure 7, MSC cell adhesion on modified scaffold with different CAP conditions. Data are mean ±SEM; n = 9. Output voltages of Conditions 1, 2, 3 are 3.4, 3.6, and 3.8 kV, respectively. (A) Figure 3, Emission spectrum of helium plasma. Figure 11, Enhanced total protein synthesis on CAP modified nHA/Chitosan scaffold after 1 and 2 weeks of culture. Data are mean ±SEM; n=9. *p<0.01 when compare to all other substrates. Figure 8, Low and high magnification SEM images of unmodified nHA/chitosan scaffold (A, a1 and a2), and CAP modified nHA/chitosan scaffolds with different treatment time (B, b1 and b2 is 3 min; C, c1 and c2 is 5 min; D, d1 and d2 is 10 min) under the 3.6 kV output voltage. Figure 1, (a) Configuration of CAP, probe, and holder (b) Schematic diagram of probe and holder of emission intensity of output voltage. Figure 5. Transmission electron microscopy images of nHA with (A) low magnification and (B) high magnification. Figure 6, Increased surface hydrophilicity of nHA/chitosan after 3, 5 and 10min CAP treatments. Data are mean ± standard deviation, n=3. *p<0.05 when compared to all other samples; and **p<0.05 when compared to 5 min CAP treated sample. Figure 13, Total collagen synthesis on CAP modified nHA/chitosan scaffold after 1, 2 and 3 weeks of culture. Data are mean ±SEM; n=9. *p<0.01 when compared to controls after 3 weeks of culture; **p<0.01 when compared to all other substrates at week 1 and 2. Figure 10. Vitronectin and fibronectin absorptions on nHA/chitosam after 3 and 5 min CAP treatment by ELISA, n=3. *p<0.01 when compared to 0 min CAP treated group; and **p<0.1 and ***p<0.05 when compared to 0 min CAP treated group. Figure 9. 3D surface topography images of nHA/chitosan obtained using white light interferometry, without CAP treatment (A) and 3 min CAP treatment