The ability of epithelial cells to organize into polarized, three-dimensional (3D) structures correlates closely with their normal or malignant status. In a versatile model of morphogenesis, we have shown in past studies that inhibiting a number of key signaling pathways in human breast cancer cells grown in laminin-rich ECM gels leads to ‘reversion&’ of the malignant phenotype. The resulting growth-arrested polarized structures resemble normal ‘acini&’. These studies have helped us understand how polarity of the normal structures in the mammary gland may be disrupted as breast cancer progresses. We now have used two additional models to study signaling integration in single mammary cells and also mammary organoids, where we have modeled mammary invasion into the stromal collagen during branching morphogenesis and discovered unexpected mechanisms by which MMPs (matrix metalloproteinases) signal and how tumor cells usurp these pathways to invade. Most recently we have succeeded in modeling where breast cancer cells go to become dormant and how and why they may ‘wake up&’. I will discuss data under review to provide additional proof of how single cells from adult breast can make physiologically correct structures (acini) of the mammary gland, and show that all signaling pathways including those of glucose metabolism, must integrate to maintain homeostasis. We show how biochemical and mechanical signaling from the ECM, the ECM receptors and MMPs interconnect with cells and their nuclei to build tissue architecture by reciprocal and reiterating loops to achieve tissue specificity.

Introduction: Glioblastoma (GBM) is the most common and aggressive form of primary brain cancer in adults. The GBM tumor niche consists of biochemical and mechanical cues that act in conjunction to fuel tumor growth. The gold standards for studying cancer biology are 2D monolayer culture, organotypic explant cultures, and animal models. Currently, there is a lack of 3D in vitro models that permit studying cancer cells in a more physiologically relevant and controllable manner. Clinical evidence has shown that GBM tumor development is coupled with a substantial increase in matrix stiffness, with ~26 kPa in GBM vs. ~1 kPa in normal brain tissues. While the important roles of niche cues on GBM progression have been well recognized, little is known about how matrix stiffness regulates GBM cell fates in 3D. Here we report the development of 3D biomimetic hydrogels to study the effect of matrix stiffness on brain cancer cell behavior.
Materials and Methods: Synthetic materials, such as poly(ethylene-glycol) (PEG), allow independent control of various hydrogel properties and present a blank slate for incorporating naturally-derived ECM ligands. To tune the hydrogel stiffness, the concentration of multi-arm PEG molecules was varied. To allow for cell-mediated degradation, MMP-cleavable sequences and nondegradable linear PEG molecules were used to crosslink the hydrogel. To mimic the biochemical properties of brain tissue and allow cell adhesion, hyaluronic acid (HA) and CRGDS peptides were also incorporated. A commercially available brain tumor cell line (U87) was used and cultured in hydrogels mimicking normal brain (1 kPa) or tumor (26 kPa) tissue stiffness. Hydrogels were characterized using mechanical testing and quantification of equilibrium swelling ratio over time. Cellular fates in 3D hydrogels were analyzed by monitoring cell proliferation, cell morphology, and gene expression.
Results and Discussion: Both soft and stiff hydrogels supported substantial cell proliferation of U87 cells in 3D, with formation of tumor spheroids throughout the hydrogels. Within 21 days of culture, cells proliferated 16-fold in hydrogels with brain-like stiffness (1 kPa) and 3-fold in hydrogels with tumor-like stiffness (26 kPa). In addition, increasing hydrogel stiffness limited cell spreading at early time points, but seemed to permit longer cell protrusion at later time points. Furthermore, changes in hydrogel stiffness resulted in differential gene expression for Ras protein HRAS, mechanotransduction proteins RhoA and ROCK1, and ECM remodeling proteins, including HA synthases 1 and 2 and MMP9. Our results highlight the importance of matrix stiffness in influencing brain tumor cell behavior in 3D. Such biomimetic hydrogels could provide a 3D in vitro model for elucidating tumor-niche interactions in a controlled manner.

Glioblastoma multiforme (GBM), the most prevalent primary brain cancer, is characterized by diffuse infiltration of tumor cells into brain tissue, which severely complicates surgical resection and likely gives rise to the almost universal tumor recurrence. This diffuse infiltration is frequently guided by anatomical “tracks” in the brain in the form of blood vessels or white matter tracts, which give rise to the highest migration speeds observed in vivo. Despite this observation, little is known about the biophysical and biochemical mechanisms through which these tissue interfaces promote invasive motility, which in turn may derive from a lack of appropriate culture paradigms. To address this need, we developed a culture system in which tumor cells are sandwiched between a ventral fibronectin-coated dorsal surface representing vascular basement membrane and a dorsal hyaluronic acid (HA) surface representing brain parenchyma. We find that inclusion of the dorsal HA surface induces formation of adhesive complexes and significantly slows cell migration relative to a free fibronectin-coated surface. This retardation is amplified by inclusion of integrin binding peptides in the dorsal layer and expression of CD44, suggesting that it acts through biochemically specific mechanisms rather than simple physical confinement. Moreover, both the reduction in migration speed and assembly of dorsal adhesions depend on myosin activation and the stiffness of the ventral layer, implying that mechanochemical feedback directed by the ventral layer can influence adhesive signaling at the dorsal surface.

Patients with glioblastoma multiforme (GBM), the most aggressive form of primary brain tumor, have a poor prognosis due to a rapid diffuse infiltration of tumor cells into normal parenchyma. The biochemical and biophysical interactions between tumor cells and brain extracellular matrix play an important role in the rapid progression of the tumor. Platforms to replicate the tumor microenvironment are a critical topic in the field of cancer research, and offer unique opportunities to engage next generation genomic tools. These technologies may serve as diagnostic platforms for clinical assessment of therapeutic strategies using patient-specific biopsies. As a result, they will turn into a powerful clinical tool to characterize the tumor microenvironment, and the associated intercellular signaling network in individual patients, enabling personalized therapy. We have developed a versatile gelatin-based biomaterial platform to present combinations of mechanical, structural, and cellular cues inspired by the native glioblastoma microenvironment. Strategies to decorate these biomaterials with biomolecular cues (e.g. hyaluronic acid), common glioma mutations (e.g. EGFR) and chemokines that regulate cell motility, proliferation and survival (e.g. CXCL12) demonstrate an impact in their response to microenvironment. Moreover, spatial and temporal gradients regulate the cell proliferation, migration, and differentiation during cancer. Therefore, we use a microfluidic approach to fabricate patterned biomaterials that have the ability to examine transitions between defined environments (e.g., glioma core, periphery and neural tissue). We developed a series of gelatin and hyaluronic acid (HA) macromers in order to create libraries of composite hydrogel structures. Hydrogels containing brain-mimetic HA show significant impact on GBM malignancy metrics in comparison to 2D culture or through the use of 3D GelMA hydrogels. Glioma cell clusters were observed exclusively in HA containing gels as well as HA-dose dependent gene expression patterns. Using this tool we aim to generate a brain tumor biochip to examine how the heterogeneities within the tumor microenvironment impact glioma malignant transformation, growth, and the formation of immunomodulatory zones that limit therapeutic efficacy. We combined this technology with clinical specimens and data obtained from diagnosis of patients in order to prognosticate the cell dynamics in tumor progression and lead, as a result, to the design of personalized therapy.

To investigate the possibility of treating multidrug-resistant tumors with targeted chemo-photothermal treatment, we conducted in vitro and in vivo studies using a doxorubicin (DOX)-resistant DLD-1 cell line (DLD-1/DOX) and nude mice with human xenograft tumors, respectively. The chemo-photothermal treatment consisted of DOX-loaded-poly(lactic-co-glycolic acid)-Au half-shell nanoparticles with targeting moieties of anti-death receptor-4 monoclonal antibody conjugated to the Au surface. The cells or xenografted tumors were exposed to near infrared light for 10 min, which caused an increase in temperature to 45oC. Chemo-photothermal treatment resulted in a large reduction in the rate of tumor xenograft growth on DLD-1/DOX tumor-bearing mice with a much smaller dose of DOX than conventional DOX chemotherapy. These results demonstrate that targeted chemo-photothermal treatment can provide high therapeutic efficacy and low toxicity in the treatment of multidrug- resistant tumors.

A synthetic extracellular matrix (sECM) from hyaluronic acid (HA) affords highly reproducible, manufacturable, approvable, and affordable biomaterials. These injectable clinical materials are being developed for clinical use to deliver autologous and allogeneic cells to sites in need of repair. Tissue repair targeted by us, our collaborators and partners include heart, kidney, brain, liver, fat, bone, cartilage, and vocal folds. The HA materials offer a cell-friendly niche for cell retention and proliferation, as well as being permissive and conducive to angiogenesis and nutrient flow.

We report increased stimulation of dendritic cells via heterodimers of immunostimulants formed at a discrete molecular distances. Many vaccines present spatially organized agonists to immune cell receptors. These receptors cluster suggesting that signaling is increased by spatial organization and receptor proximity, but this has not been directly tested for multiple, unique receptors. In this study we probe the spatial aspect of immune cell activation using heterodimers of two covalently attached immunostimulants.

As a technology to investigate T cell - antigen presenting cell interactions, we have created spatially structured surfaces that mimic nanoscale features of the Immunological Synapse. We show that biomimetic nanoarrays functionalised with antibody fragments engage with human CD4+ T cells to form an "artificial immune synapse" and demonstrate that the level of T cell signalling can be controlled by varying the spacing of ligand anchoring points on the nanoscale.
Although it is well known that TCR-ligand engagements plays a key role in activation of T Cells, there is some controversy over the minimum stimulation required to induce signalling clusters on the submicron scale, and structurally important features have been described on the micro- and nano- lengthscales. To investigate the effect of such structures in controlling signalling, we have prepared biomimetic surfaces that present antibodies and antibody fragments with nanoscale spacing using nanopatterned gold arrays. Using the technique of diblock copolymer micelle nanolithography, arrays of gold nanoparticles with spacings ranging from 25-104nm have been formed. By controlling the nanoarray structure, and the biological ligands bound to the surface, we investigate how the spatial distribution of selected stimulatory ligands affects cell activation and downstream signalling. The nanoarrays have been succesfully functionalised with biologically complex antibody F(ab')2 fragments, bound through the di-sulphide hinge region to the gold nanoparticle.
Specifically, we assess CD4+ engagement with these biomimetic nanopatterns, showing that the degree of signalling decreases with increasing spacing of anti-CD3 ligands. Parallel studies of NK cell stimulation by CD16 binding nanoarrays showed intriguingly similar results. These results indicate that immune cell activation can be driven by the spatial organisation of receptor-ligand engagement on a sub-100nm scale. The elucidation of these signalling and activation requirements will help mechanistic understanding of the activation process, and direct immunotherapy towards new nanoscale targets.

Immune cell stimulation has been shown to be enhanced by co-treatment of multiple agonists. One such pairing, Toll-like receptor (TLR) 4 phospholipid agonists with TLR 7/8 imidazoquinoline agonists, have shown synergistic increased in cell surface markers and cytokines indicative of dendritic cell (DC) activation. We have designed caged agonists of TLR 4 and 7/8 to probe the mechanism of the observed synergy. Protecting agonists with orthogonal photo-labile groups allows for spatially- and temporally-controlled release of agonists within a single cell. Agonist moieties critical for TLR binding and activation, the phosphate of monophosphoryl Lipid A (MPLA) and primary amine of resiquimod, were protected with photo-labile nitrophenyl and coumarin groups, respectively. We show that caged compounds elicit minimal stimulation in a model macrophage cell line, and that upon photo-deprotection, the parent compounds reform and regain activity. For activation of single cells and precise deprotection at the organelle level, two-photon confocal microscopy was employed. Immune stimulation, assessed by direct observation of endocytosis in bone marrow-derived dendritic cells (BMDCs), again was knocked out with the cage and regained following exposure with the respective wavelength. We demonstrate that a spatio-temporal relationship exists between TLR 4 and 7/8 activation pathways and that this synergy can be exploited for creating multi-functional, robust materials for immune cell programming.

Tissue and organ development is regulated by a complex interplay of multiple extracellular cues, and emulating the process with synthetic scaffolds remains challenging. One challenge is the independent control in a single scaffold of soluble signals, bound signals, and mechanical properties. We describe here a two-component core-shell microcapsule construct 150-200 µm in diameter that consists of a polymeric alginate core and a shell comprised of peptide amphiphile (PA) nanofibers. We demonstrate that the release of bone morphogenetic protein 4 (BMP-4) from the polymer core can be tuned by changing the degree of calcium cross-linking of alginate. At the same time the PA nanofiber shell on the capsule surface provides a substrate for cellular attachment without affecting BMP-4 release. Moreover, using C2C12 premyoblasts, we show that cell proliferation and spreading on PA substrates can be modulated by molecularly tuning the bioactivity of nanofibers displaying the fibronectin-derived RGDS epitope. Combining the tailorable PA design and tunable BMP-4 release afforded an independent control over C2C12 cell distribution and osteogenic differentiation on the microcapsule surface. The microcapsules investigated could be easily injected and thus serve as a novel bioactive scaffold for cell therapies in regenerative medicine.

INTRODUCTION
The availability of forming technology able to mass produce porous polymeric microspheres with diameters ranging from 150 to 300 µm is significant for some biomedical applications where tissue augmentation is required1,2. Moreover, appropriate assembly of microspheres into scaffolds is an important challenge to enable direct usage of the as-formed structures in treatments of chronic wound such as fistula3. This specified range of microspheres has the advantage of large enough interstices between the packed spherical particles for migration of cells into scaffold for tissue regeneration.
EXPERIMENTAL METHOD
Poly Lactic-Co-Glylolic Acid (PLGA, 50:50 co-polymer) was used to generate the required size of microspheres. Different concentrations (5, 10 and 15 wt%) of PLGA solutions were prepared by combining appropriate amounts of polymer in Dimethyl Carbonate (DMC). Electrospraying of the polymer solutions was conducted by adopting single-axial cone-jetting to produce the hollow microspheres with different size ranges. The applied voltage was varied according to the flow rate used in order to obtain a stable cone jet and produce monodisperse microspheres. When the required size of microspheres was achieved with the 15% concentration of the polymer solution at the specific flow-rate and range of voltage supply, the single-axial cone-jet mode spraying was performed once again to electrospray the polymer solution for 1800s into a metallic beaker filled with liquid nitrogen. Once the required size of microspheres was collected in liquid nitrogen, the frozen products were immediately placed into freeze-dryer in order to remove the residual solvent from the structure of microspheres (TIPS).
RESUTLS AND DISCUSSION
The required size of microspheres for the development of the desired scaffold was obtained at a flow rate 225µl/min after collection in liquid nitrogen. Due to thermal phase separation, when the frozen microspheres were placed in freeze dryer, the residual solvent was removed from the structure of particles, and this led to porosity on the surface of microspheres.
During the procedure of electrospraying, flow rate was changed, therefore in order to obtain stable cone-jet and monodisperse particles, voltage supply was also varied.
CONCLUSION
Electrospray of PLGA in DMC, a low toxicity solvent with moderate conductivity and low dielectric constant, followed by freeze drying generated porous microspheres within the required diameter range of 150-300 µm suitable for use as minimally invasive, in situ forming scaffolds.
REFERENCES
1. H. Keshaw, N. Thapar, A.J. Burns, N. Mordan, J.C. Knowles, A. Forbes, R.M. Day, Acta Biomaterialia 6 (2010) 1158-1166
2. J.J. Blaker, J.C. Knowles, R.M. Day, ActaBiomater 4 (2008) 364-272
3. J.J. Blaker, J. Pratten, D. Ready, J.C. Knowles, A. Forbes, R.M. Day, Journal of Alimentary Pharmacology & Therapeutics 28 (2008) 614-622

Delivery of biological effectors into cells is a technological challenge of significant importance for diagnosis and treatment of disease and for fundamental biological studies. Vertical nanowire (NW) arrays have shown promise as a potential universal platform for drug delivery. In contrast to conventional techniques which rely on biochemical pathways, vertical NW platforms physically penetrate the cell membrane, enabling biomolecules to be directly introduced to the cytoplasm to avoid endosomal degradation routes. However, the NW arrays are restricted to in vitro applications because the NWs are attached to a planar 2D substrate. In this work, we grow ZnO NWs on suspended nanoparticles to produce "spiky particles", which can be used for biomolecular delivery into cells by utilizing the sharp NWs' cell membrane penetration ability. Naturally occurring particle phagocytosis by cells encourages spiky particle engulfment, and once the cell membrane is penetrated by the NWs, the biomolecules bound on the particle surface can be directly released into the cytosol. The particle surface is protected by a thin layer of alumina to reduce the cytotoxicity of ZnO materials, and is further functionalized with amino groups to allow noncovalent binding of biomolecules. We demonstrate synthesis of spiky particles with tunable particle size, NW length and NW density. The spiky particle-cell interface and interaction studies suggest that cells actively engulf the spiky particles. The cytotoxicity of spiky particles at high doses is mainly due to the ZnO content, and can be reduced by the thin alumina protection layer. Cell viability is found to be similar for treatments with spiky particles compared to plain particles. In addition, spiky particle-mediated DNA plasmid transfection was demonstrated. By combining the advantages of the NW penetration ability and the suspension nature of nanoparticles, the spiky particles present a direct cell membrane penetrant vehicle for drug delivery, with potential applications in vivo.

The use of nanomaterials in medicine is a growing field. Thereby drug delivery with nanoparticles is coming into focus, as it promises the possibility of targeted and controlled delivery of medicine to diseased cells, for example with mesoporous silica nanoparticles (MSN). MSN offer a large surface area and pore volume, a defined and tunable pore size, and various functionalization possibilities of the inner and outer surface. [1] Here we demonstrate a modular toolbox which allows the sequential covalent attachment of different functionalities to the surface of colloidal MSN. In the presented system, poly(2-vinylpyridine) (PVP) was used as a pH-responsive cap system. To solubilize the particles in a neutral environment, polymeric poly(ethylene glycol) (PEG) was anchored to the cap system. To demonstrate the possibility for targeting, folic acid was attached to PEG and the functionality was demonstrated in vitro and in first in vivo studies in mice. After uptake of the MSN by cancer cells, endosomal entrapment represents a further bottle-neck. With the presented system we were able to deliver membrane permeable drugs which can diffuse through the endosomal membrane after opening of the polymer shell. Membrane-impermeable cargos could be delivered with an additionally attached photosensitizer similar to our previous published system. [2,3] Furthermore stability tests in cell culture medium revealed an enhanced stability of the system due to the coating and first in vivo studies in mice revealed a good tolerability of the MSN.
Acknowledgement
The authors are grateful for funding from DFG through the SFB 749, the NIM cluster and the “Fonds der Chemischen Industrie”.
References:
[1] V. Cauda, A. Schlossbauer, J. Kecht, A. Zuerner, T. Bein, J. Am. Chem. Soc. 2009, 131, 11361.
[2] S. A. Mackowiak, A. Schmidt, V. Weiss, C. Argyo, C. von Schirnding, T. Bein, C. Braeuchle, Nano Lett. 2013, 13, 2576.
[3] A. Schlossbauer, A.M. Sauer, V. Cauda, A. Schmidt, H. Engelke, U. Rothbauer, K. Zolghadr, H. Leonhardt, C. Braeuchle, T. Bein, Adv. Healthcare Mater. 2012, 3, 316.

Islet cell transplantation is considered as a promising treatment for type 1 diabetes. To avoid immune response after transplantation, we decided to use hydrogel system because hydrogels are expected to be the most excellent cell carriers. Hydrogel contains a lot of water in its structure, so it can prevent bioactive agents such as drugs, proteins and cells from denaturation. Also, to minimize surgical process, hydrogels which can be injected into body using syringe are required and a lot of researches have been reported. In this study, we designed injectable hydrogel by mixing oppositely charged nanoparticles together. There are many kinds of biopolymers and peptides, so we made charged nanoparticles using this polymers. For example, it is well known that gelatin has a lot of RGD sequences in its contents, so it is expected that the gelatin containing hydrogel can enhance cell adhesion. Among a lot of candidates, we used gelatin, hyaluronic acid, heparin and glycol chitosan. The size of nanoparticles were successfully controlled around 200 nm. Gelatin from porcine skin and glycol chitosan nanoparticles showed positively charges and gelatin from bovine skin, heparin and hyaluronic acid nanoparticles exhibited negatively charges. By blending oppositely charged nanoparticles, we confirmed the gelatin behavior and also found that the hydrogel using this system is injectable by shear thinning property. Finally, we applied this hydrogel into islet cell carrier to control the high glucose level by in vivo experiment.

Engineered nano- and microparticles that co-deliver antigen and immunostimulatory molecules are of interest as next-generation subunit vaccines and so-called ‘smart&’ adjuvants, given their ability to mimic pathogens while avoiding toxicity and anti-vector immune responses. To demonstrate that mesoporous oxide nanoparticles warrant development as particulate vaccines and adjuvants, we co-loaded mesoporous silica nanoparticles (MSNPs) with a model protein antigen, ovalbumin (OVA), and an immunostimulatory RNA (isRNA) known to activate Toll-like receptor (TLR) 7 and TLR8 and then encapsulated cargo-loaded MSNPs in a supported lipid bilayer (SLB) that we further modified with various targeting and phagosomolytic moieties. We found that high-surface-area MSNPs are able to encapsulate 50-60 wt% of OVA or isRNA individually and simultaneously encapsulate ~30 wt% of both OVA and isRNA, capacities that exceed those of state-of the-art liposomes and polymeric nanoparticles by up to 100-fold. We, furthermore, found that a SLB composition of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) modified with 5 wt% of a single-chain antibody fragment (scFv) against DEC-205 triggers efficient uptake of MSNPs by bone marrow-derived dendritic cells (BMDCs) and that the degree of silica condensation in the MSNP core can be controlled to tailor antigen release from burst (100% in 12 hours) to sustained (2-5% per day for several weeks) rates. We employed fluorescently-labeled OVA and isRNA to demonstrate that incorporating phagosomolytic peptides (e.g. ‘H5WYG&’) and/or lipids (e.g. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, or DOPE) in the SLB enables phagosomal release of isRNA and cytosolic dispersion of OVA, which, in turn, trigger DC maturation (determined by staining for CD40, CD80, and CD86) and cross-presentation of OVA-derived peptides (determined by staining for MHC class I H-2K^b molecules complexed with the OVA-derived peptide, SIINFEKL). Furthermore, DCs pulsed with DEC-205-targeted MSNPs loaded with OVA and isRNA induce vigorous in vitro proliferation of OVA-specific CD8⁺ T cells, whereas DCs pulsed with free OVA or OVA complexed with Imject Alum trigger weak T cell responses. Upon immunization of C57Bl/6 mice, MSNPs loaded with OVA and isRNA and targeted to DEC-205, additionally, induce high-titer (>10⁵), OVA-specific IgG responses that are 100- and 10,000-fold higher than the titers achieved using OVA complexed to Imject Alum and free OVA, respectively. Importantly, these MSNPs also trigger OVA-specific CD8⁺ T cell responses, the magnitude of which is higher for MSNPs with sustained release kinetics than for MSNPs with burst release kinetics. Our results indicate that mesoporous oxide nanoparticles are an important class of antigen delivery vehicles and warrant further development.

Recent studies demonstrate that pluripotent cells recognize topographical cues in their local environment through mechanotransduction, allowing them identify and adapt to their niche. Much effort has focused on fabricating bioscaffolds that can provide similar cues to control stem cell fate. Not surprisingly, scaffolds with nanoscale topography, mimicking the extracellular matrices naturally encountered by cells, provide the greatest success this regard.
One intense area of research is on the topographical control of neural stem cell (NSC) fate. Recent studies show that scaffolds with flat, isotropic surfaces promote astrocyte differentiation, while anisotropic scaffolds with a high degree nanotopography promote neuron differentiation. This ability to control NSC fate without chemical treatment is a very useful tool for the study of NSCs and their differentiated cell types. However, most surfaces developed to drive NSC differentiation are designed to promote differentiation to only one cell type.
A scaffold system capable of promoting the differentiation of NSCs into multiple cell types would be a very useful in vitro model, especially if the cell types could be individually patterned into custom arrangements to recreate neural structures of multiple cell types. Such a model would allow for the study of complex relationships between neural cells types in a controlled in vitro setting. To accomplish this, a scaffold would require a combination of micron-scale features, for cell patterning, and nano-scale topography, for control of NSC fate. With this in mind, we have developed a novel technique, termed micropatterned nanotemplating, to creating such a scaffold. We are developing this new scaffold to create custom patterns of neurons and astrocytes as a tool to study their interactions in a highly controlled in vitro environment.

Keloids are the type of scar formed by an overgrown fibrous tissue (mostly collagen type I or III) at the site of the healed skin. Since the scar usually extends beyond the borders of the original wound and tends to recur after excision, it is not easy to remove the keloids. In this work, we suggest the plasma treatment as a new way to remove (or reduce) the keloids in the cell levels. Recently, it was reported that the plasma treatment can selectively activate the apoptosis of the cancer cells without any serious damage to the normal cells [C.-H. Kim et al., Appl Phys Lett. 96, 243701 (2010)]. We hypothesized that the reactive oxygen species (ROS) are increased in the keloid-derived stem cells by the plasma treatment as in the cancer cells because of the similar biological properties. The ROS will increase the cell death of the keloid-derived stem cells. Two different cells of the keloid-derived stem cells and the hypertrophic scars, which is a control, were treated by the non-thermal dielectric barrier discharge plasma to avoid the heat effects. Both treated cells were characterized by Fourier-transform infrared (FT-IR) spectroscopy. In the keloid-derived stem cells, it was clearly observed increasing in the stretching mode of the O-H which indicates the O-H defect is strongly affected by the plasma treatment. To confirm the plasma effects on the keloid-derived stem cells, additional results including MTT assay, Raman spectroscopy, and atomic force microscopes will be reported.

Recent studies have shown that the interactions between chondrocytes and mesenchymal stem cells (MSCs) can be harnessed for cartilage repair, with enhanced MSC chondrogenesis, increased chondrocyte proliferation and cartilage matrix formation. To better predict the clinical outcomes of utilizing such cell-cell interactions for cartilage repair, it is important to understand the effects of microenvironmental factors on the ability of these cells to form neocartilage tissues. Culture conditions, such as soluble factors and oxygen tension, play important roles in modulating chondrogenesis. Due to its avascular nature, the local oxygen tension in native cartilage is low (1-7%). As such, the goal of this study is to examine the effects of soluble factors and oxygen tension on chondrocyte-stem cell interactions. In particular, neonatal chondrocytes (NCs) were chosen in this study due to their immunoprivileged property, highly proliferative nature, and the ability to produce abundant cartilage matrix. Adipose-derived stem cells (ADSCs) were used given their relative abundance and potential to differentiate into chondrogenic lineage. NCs and ADSCs were mixed co-cultured in 3D biomimetic hydrogels for 14 days in vitro under two oxygen tension (2 and 20% O2) and two culture medium conditions (GM: serum supplemented vs. CM: serum-free supplemented with TGF-β3). The extent of cell proliferation and cartilage matrix production (sGAG and collagen) were evaluated. Interaction synergy was quantified with interaction index, which is defined as the measured biochemical content in the mixed co-culture normalized by the expected value based the measured content in the controls and the initial cell ratio. Although varying oxygen tension affected cell proliferation and cartilage matrix synthesis of NCs, it did not affect the overall interaction synergy, as shown by the interaction index analyses. Under both oxygen tension, mixed co-culture in CM led to enhanced cell proliferation and cartilage matrix synthesis, as indicated by interaction index analyses (up to 2 folds increase). Culture medium composition was found to be a critical modulator of interaction synergy, and with positive synergy only observed in CM containing TGF-β3 alone, whereas negative synergy was observed in serum containing GM. The presence of TGF-β3 in CM changes the differentiation state of ADSCs, which will likely change the release of paracrine factors and affect their role as a trophic mediator, resulting in differences in interaction synergy between CM and GM groups. Our results showed that soluble factors, but not oxygen tension, play an important role in regulating the interactions between NCs and ADSCs, as well as the result