Tissue Culture Polystyrene Control Research Articles

Thermoresponsive Alginate-Graft-pNIPAM/Methyl Cellulose 3D-Printed Scaffolds Promote Osteogenesis In Vitro.

In this work, a sodium alginate-based copolymer grafted by thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) chains was used as gelator (Alg-g-PNIPAM) in combination with methylcellulose (MC). It was found that the mechanical properties of the resulting gel could be enhanced by the addition of MC and calcium ions (Ca2+). The proposed network is formed via a dual crosslinking mechanism including ionic interactions among Ca2+ and carboxyl groups and secondary hydrophobic associations of PNIPAM chains. MC was found to further reinforce the dynamic moduli of the resulting gels (i.e., a storage modulus of ca. 1500 Pa at physiological body and post-printing temperature), rendering them suitable for 3D printing in biomedical applications. The polymer networks were stable and retained their printed fidelity with minimum erosion as low as 6% for up to seven days. Furthermore, adhered pre-osteoblastic cells on Alg-g-PNIPAM/MC printed scaffolds presented 80% viability compared to tissue culture polystyrene control, and more importantly, they promoted the osteogenic potential, as indicated by the increased alkaline phosphatase activity, calcium, and collagen production relative to the Alg-g-PNIPAM control scaffolds. Specifically, ALP activity and collagen secreted by cells were significantly enhanced in Alg-g-PNIPAM/MC scaffolds compared to the Alg-g-PNIPAM counterparts, demonstrating their potential in bone tissue engineering.

Self-assembly of gelatin and collagen in the polyvinyl alcohol substrate and its influence on cell adhesion, proliferation, shape, spreading and differentiation.

Culture substrates display profound influence on biological and developmental characteristic of cells cultured in vitro. This study investigates the influence of polyvinyl alcohol (PVA) substrates blended with different concentration of collagen or/and gelatin on the cell adhesion, proliferation, shape, spreading, and differentiation of stem cells. The collagen/gelatin blended PVA substrates were prepared by air drying. During drying, blended collagen or/and gelatin can self-assemble into macro-scale nucleated particles or branched fibrils in the PVA substrates that can be observed under the optical microscope. These collagen/gelatin blended substrates revealed different surface topography, z-average, roughness, surface adhesion and Young's modulus as examined by the atomic force microscope (AFM). The results of Fourier transform infrared spectroscopy (FTIR) analysis indicated that the absorption of amide I (1,600-1,700cm-1) and amide II (1,500-1,600cm-1) groups increased with increasing collagen and gelatin concentration blended and the potential of fibril formation. These collagen or/and gelatin blended PVA substrates showed enhanced NIH-3T3 fibroblast adhesion as comparing with the pure PVA, control tissue culture polystyrene, conventional collagen-coated and gelatin-coated wells. These highly adhesive PVA substrates also exhibit inhibited cell spreading and proliferation. It is also found that the shape of NIH-3T3 fibroblasts can be switched between oval, spindle and flattened shapes depending on the concentration of collagen or/and gelatin blended. For inductive differentiation of stem cells, it is found that number and ration of neural differentiation of rat cerebral cortical neural stem cells increase with the decreasing collagen concentration in the collagen-blended PVA substrates. Moreover, the PVA substrates blended with collagen or collagen and gelatin can efficiently support and conduct human pluripotent stem cells to differentiate into Oil-Red-O- and UCP-1-positive brown-adipocyte-like cells via ectodermal lineage without the addition of mitogenic factors. These results provide a useful and alternative platform for controlling cell behavior in vitro and may be helpful for future application in the field of regenerative medicine and tissue engineering.

Bioinspired Collagen/Gelatin Nanopillared Films as a Potential Implant Coating Material.

Collagen-based Sharpey’s fibers are naturally located between alveolar bone and tooth, and they have critical roles in a well-functioning tooth such as mechanical stability, facile differentiation, and disease protection. The success of Sharpey’s fibers in these important roles is due to their unique location, vertical alignment with respect to tooth surface, as well as their micronanofiber architecture. Inspired by these structures, herein, we introduce the use of nanoporous anodic aluminum oxide molds in a drop-casting setup to fabricate biopolymeric films possessing arrays of uniform Collagen:Gelatin (Col:Gel) nanopillars. Obtained structures have diameters of ∼90 nm and heights of ∼300 nm, yielding significantly higher surface roughness values compared to their flat counterparts. More importantly, the nanostructures were parallel to each other but perpendicular to the underlying film surface imitating the natural collagenous structures of Sharpey’s fibers regarding nanoscale morphology, geometrical orientation, as well as biochemical content. Viability testing showed that the nanopillared Col:Gel films have high cell viabilities (over 90%), and they display significantly improved attachment (ca. ∼ 2 times) and mineralization for Saos-2 cells when compared to flat Col:Gel films and Tissue Culture Polystyrene (TCPS) controls, plausibly due to their largely increased surface roughness and area. Hence, such Sharpey’s fiber-inspired bioactive nanopillared Col:Gel films can be used as a dental implant coating material or tissue engineering platform with enhanced cellular and osteogenic properties.

Binary colloidal crystals (BCCs) modulate the retina-related gene expression of hBMSCs – A preliminary study

Surface topography-induced lineage commitment of human bone marrow stem cells (hBMSCs) has been reported. However, this effect on hBMSC differentiation toward retinal pigment epithelium (RPE)-like cells has not been explored. Herein, a family of cell culture substrates called binary colloidal crystals (BCCs) was used to stimulate hBMSCs into RPE-like cells without induction factors. Two BCCs, named SiPS (silica (Si)/polystyrene (PS)) and SiPSC (Si/carboxylated PS), having similar surface topographies but different surface chemistry was used for cell culture. The result showed that cell proliferation was no difference between the two BCCs and tissue culture polystyrene (TCPS) control. However, the cell attachment, spreading area, and aspect ratio between surfaces were significantly changed. For example, cells displayed more elongated on SiPS (aspect ratio ~7.0) than those on SiPSC and TCPS (~2.0). The size of focal adhesions on SiPSC (~1.6 µm2) was smaller than that on the TCPS (~2.5 µm2). qPCR results showed that hBMSCs expressed higher RPE progenitor genes (i.e., MITF and PAX6) on day 15, and mature RPE genes (i.e., CRALBP and RPE65) on day 30 on SiPS than TCPS. On the other hand, the expression of optical vesicle or neuroretina genes (i.e., MITF and VSX2) was upregulated on day 15 on SiPSC compared to the TCPS. This study reveals that hBMSCs could be modulated into different cell subtypes depending on the BCC combinations. This study shows the potential of BCCs in controlling stem cell differentiation.

Compatibility and function of human induced pluripotent stem cell derived cardiomyocytes on an electrospun nanofibrous scaffold, generated from an ionomeric polyurethane composite.

Synthetic scaffolds are needed for generating organized neo-myocardium constructs to promote functional tissue repair. This study investigated the biocompatibility of an elastomeric electrospun degradable polar/hydrophobic/ionic polyurethane (D-PHI) composite scaffold with human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The composite material was electrospun to generate scaffolds, with nanofibres oriented in aligned or random directions. These features enabled the authors to evaluate the effect of characteristic elements which mimic that of the native extracellular matrix (alignment, chemical heterogeneity, and fiber topography) on hiPSC-CMs activity. The functional nature of the hiPSC-CM cultured on gelatin and Matrigel-coated scaffolds were assessed, investigating the influence of protein interactions with the synthetic substrate on subsequent cell phenotype. After 7 days of culture, high hiPSC-CM viability was observed on the scaffolds. The cells on the aligned scaffold were elongated and demonstrated aligned sarcomeres that oriented parallel to the direction of the fibers, while the cells on random scaffolds and a tissue culture polystyrene (TCPS) control did not exhibit such an organized morphology. The hiPSC-CMs cultured on the scaffolds and TCPS expressed similar levels of cardiac troponin-T, but there was a higher expression of ventricular myosin light chain-2 on the D-PHI composite scaffolds versus TCPS, indicating a higher proportion of hiPSC-CM exhibiting a ventricular cardiomyocyte like phenotype. Within 7 days, the hiPSC-CMs on aligned scaffolds and TCPS beat synchronously and had similar conductive velocities. These preliminary results show that aligned D-PHI elastomeric scaffolds allow hiPSC-CMs to demonstrate important cardiomyocytes characteristics, critical to enabling their future potential use for cardiac tissue regeneration.

Differentiation of adipose tissue-derived stem cells towards vascular smooth muscle cells on modified poly(L-lactide) foils

The aim of our research was to study the behaviour of adipose tissue-derived stem cells (ADSCs) and vascular smooth muscle cells (VSMCs) on variously modified poly(L-lactide) (PLLA) foils, namely on pristine PLLA, plasma-treated PLLA, PLLA grafted with polyethylene glycol (PEG), PLLA grafted with dextran (Dex), and the tissue culture polystyrene (PS) control. On these materials, the ADSCs were biochemically differentiated towards VSMCs by a medium supplemented with TGFβ1, BMP4 and ascorbic acid (i.e. differentiation medium). ADSCs cultured in a non-differentiation medium were used as a negative control. Mature VSMCs cultured in both types of medium were used as a positive control. The impact of the variously modified PLLA foils and/or differences in the composition of the medium were studied with reference to cell adhesion, growth and differentiation. We observed similar adhesion and growth of ADSCs on all PLLA samples when they were cultured in the non-differentiation medium. The differentiation medium supported the expression of specific early, mid-term and/or late markers of differentiation (i.e. type I collagen, αSMA, calponin, smoothelin, and smooth muscle myosin heavy chain) in ADSCs on all tested samples. Moreover, ADSCs cultured in the differentiation medium revealed significant differences in cell growth among the samples that were similar to the differences observed in the cultures of VSMCs. The round morphology of the VSMCs indicated worse adhesion to pristine PLLA, and this sample was also characterized by the lowest cell proliferation. Culturing VSMCs in the differentiation medium inhibited their metabolic activity and reduced the cell numbers. Both cell types formed the most stable monolayer on plasma-treated PLLA and on the PS control. The behaviour of ADSCs and VSMCs on the tested PLLA foils differed according to the specific cell type and culture conditions. The suitable biocompatibility of both cell types on the tested PLLA foils seems to be favourable for vascular tissue engineering purposes.

Osteogenic differentiation of bone marrow mesenchymal stem cells on chitosan/gelatin scaffolds: gene expression profile and mechanical analysis

In the present study we explore the extracellular matrix (ECM) produced by human bone marrow mesenchymal stem/stromal cells (BM-MSCs) induced to undergo osteogenic differentiation within porous chitosan/gelatin (CS:Gel) scaffolds by investigating their multiple gene expression profile and mechanical behavior. Initially, the efficiency of the BM-MSCs osteogenic differentiation within the constructs was confirmed by the significant rise in the expression of the osteogenesis associated genes DLX5, RUNX2, ALP and OSC. In line with these findings, OSC and Col1A1 protein expression was also detected in BM-MSCs on the CS:Gel scaffolds at day 14 of osteogenic differentiation. We then profiled, for the first time, the expression of 84 cell adhesion and ECM molecules using PCR arrays. The arrays, which were conducted at day 14 of osteogenic differentiation, demonstrated that 49 genes including collagens, integrins, laminins, ECM proteases, catenins, thrombospondins, ECM protease inhibitors and cell-cell adhesion molecules were differentially expressed in BM-MSCs seeded on scaffolds compared to tissue culture polystyrene control. Moreover, we performed dynamic mechanical analysis of the cell-loaded scaffolds on days 0, 7 and 14 to investigate the correlation between the biological results and the mechanical behavior of the constructs. Our data demonstrate a significant increase in the stiffness of the constructs with storage modulus values of 2 MPa on day 7, compared to 0.5 MPa on day 0, following a drop of the stiffness at 0.8 MPa on day 14, that may be attributed to the significant increase of specific ECM protease gene expression such as MMP1, MMP9, MMP11 and MMP16 at this time period.

Ionically and Enzymatically Dual Cross-Linked Oxidized Alginate Gelatin Hydrogels with Tunable Stiffness and Degradation Behavior for Tissue Engineering.

Hydrogels that allow for the successful long-term in vitro culture of cell-biomaterial systems to enable the maturation of tissue engineering constructs are highly relevant in regenerative medicine. Naturally derived polysaccharide-based hydrogels promise to be one material group with enough versatility and chemical functionalization capability to tackle the challenges associated with long-term cell culture. We report a marine derived oxidized alginate, alginate dialdehyde (ADA), and gelatin (GEL) system (ADA-GEL), which is cross-linked via ionic (Ca2+) and enzymatic (microbial transglutaminase, mTG) interaction to form dually cross-linked hydrogels. The cross-linking approach allowed us to tailor the stiffness of the hydrogels in a wide range (from <5 to 120 kPa), without altering the initial ADA and GEL hydrogel chemistry. It was possible to control the degradation behavior of the hydrogels to be stable for up to 30 days of incubation. Increasing concentrations of mTG cross-linker solutions allowed us to tune the degradation behavior of the ADA-GEL hydrogels from fast (<7 days) to moderate (14 days) and slow (>30 days) degradation kinetics. The cytocompatibility of mTG cross-linked ADA-GEL was assessed using NIH-3T3 fibroblasts and ATDC-5 mouse teratocarcinoma cells. Both cell types showed highly increased cellular attachment on mTG cross-linked ADA-GEL in comparison to Ca2+ cross-linked hydrogels. In addition, ATDC-5 cells showed a higher proliferation on mTG cross-linked ADA-GEL hydrogels in comparison to tissue culture polystyrene control substrates. Further, the attachment of human umbilical vein endothelial cells (HUVEC) on ADA-GEL (+) mTG was confirmed, proving the suitability of mTG+Ca2+ cross-linked ADA-GEL for several cell types. Summarizing, a promising platform to control the properties of ADA-GEL hydrogels is presented, with the potential to be applied in long-term cell culture investigations such as cartilage, bone, and blood-vessel engineering, as well as for biofabrication.

P11. Facile aqueous based method to improve mineralization onto PEEK

BACKGROUND CONTEXT Spinal fusion is the standard surgical care for numerous pathologic conditions of the spine, including degenerative disc disease, spondylolisthesis, scoliosis, instability of the spine and spinal fractures. Poly(ether ether ketone) (PEEK), due to its properties of artifact free imaging, radiolucency, and an elastic modulus similar to cortical bone, has been the material of choice for interbody implants. While these advantages arise from the physical and mechanical properties of PEEK, its inert, hydrophobic surface and lack of functional groups which promote cell attachment and growth create the potential for poor surgical outcomes. The aforementioned negative material properties of PEEK may limit osseointegration of PEEK-based implants in clinical and pre-clinical settings leading to eventual poor spinal fusion. Thus, enchaining PEEK's biological properties by suitable surface modification strategies remain critical to expand its application in the clinic. The modification and derivatization of PEEK and PEEK-based spinal implant devices to improve osseointegration properties has been widely explored including physical surface modification and changes to the polymers surface properties. Methods to increase the surface area of PEEK such as laser or acid etching as well as salt crystal templating result in additional regions for bone growth. Chemical derivatization of PEEK adds cell binding functional groups including techniques to introduce sulfate, alcohol, polymers, or metallic layers. Both chemical and physical approaches have shown some clinical improvements but are challenging to scale-up or may damage the mechanical integrity of the PEEK materials. PURPOSE Determine if a covalent surface modification strategy could improve PEEK cell growth. Determine if modification of PEEK improved cell binding compared to unmodified PEEKStudy how modified PEEK's properties impact early and late phase markers of osseointegration. STUDY DESIGN/SETTING The study was designed to compare the ability of surface modified PEEK to support cell binding and growth compared to unmodified PEEK and positive control tissue culture polystyrene(TCPS). Cell binding was studied using scanning electron microscopy and dye membrane labeled cells. Early stage osteogenic proliferation was measured using alkaline phosphatase (ALP) staining. Mineralization, a late phase osteogenic marker, was studied via Alizarin red staining. OUTCOME MEASURES Cell growth: number of cell per image field and cell morphological assessmentALP staining: Colorimetric assessment of ALP substrate converted into product Alizarin red staining: Quantification of Alizarin red stain absorbed onto cell mineral deposits. METHODS PEEK discs were reacted with a arginine memetic compound and characterized to determine percent nitrogen incorporation using x-ray photoelectron spectroscopy and morphological assessment using scanning electron microscopy. Cell binding: C2C12 cells were labeled with a green fluorescent dye and incubated with PEEK, modified PEEK or TCPS. ALP Assay: C2C12 cells were stimulated with BMP2 supplemented media and incubated with PEEK, modified PEEK or TCPS ALP expression was evaluated after 72 hours. Mineralization assay: MC3T3 cells were stimulated with BMP2 supplemented media incubated with PEEK, modified PEEK or TCPS. Mineral deposits were stained with Alizarin red after 28 days. RESULTS Both ALP and Alizarin red assays showed our novel surface modification produced significant enhancement in osteoblastic activity. Measurement of absorbance related to ALP expression was significantly higher with modified PEEK samples (7463±722) compared to standard PEEK (3183±942), p=0.003. Similarly, quantification of alizarin red was significantly higher with modified PEEK samples (0.403±0.06) when compared to standard PEEK (0.057±0.02), p=0.001. Notably, there was no significant difference between modified PEEK samples and the tissue culture polystyrene with respect to either ALP or Alizarin red expression. Through the attachment of novel cell binding groups to the ketone repeat unit of the PEEK backbone, using mild aqueous based reaction conditions, we demonstrate through a series of cell-based experiments dramatically improved rates of cell adhesion and proliferation in vitro and vastly improved early (two times greater, p=0.003) and late stage markers of osteoblast maturation (almost seven times greater, p=0.001) late stage mineral deposition with results statistically equivalent to data acquired from positive tissue culture polystyrene. CONCLUSIONS An ideal strategy for the creation of next generation PEEK materials would render the material surface with functional groups that promote cell binding and growth using a process that is (a) solution based to evenly react at complex medical device surfaces while avoiding labor intensive material manipulation, (b) employs facile reaction conditions to avoid damaging material mechanical integrity, (c) utilizes materials that are non-toxic and covalently modify the surface. We have recently demonstrated dramatically improved rates of cell adhesion and proliferation in vitro and vastly improved markers of osteoblast maturation at both early and late time points. In vivo evaluation studies for changes to osseointegration to compare the novel surface modified PEEK to translational PEEK devices are being planned. FDA DEVICE/DRUG STATUS Unavailable from authors at time of publication.

Chitosan/gelatin scaffolds support bone regeneration.

Chitosan/Gelatin (CS:Gel) scaffolds were fabricated by chemical crosslinking with glutaraldehyde or genipin by freeze drying. Both crosslinked CS:Gel scaffold types with a mass ratio of 40:60% form a gel-like structure with interconnected pores. Dynamic rheological measurements provided similar values for the storage modulus and the loss modulus of the CS:Gel scaffolds when crosslinked with the same concentration of glutaraldehyde vs. genipin. Compared to genipin, the glutaraldehyde-crosslinked scaffolds supported strong adhesion and infiltration of pre-osteoblasts within the pores as well as survival and proliferation of both MC3T3-E1 pre-osteoblastic cells after 7 days in culture, and human bone marrow mesenchymal stem cells (BM-MSCs) after 14 days in culture. The levels of collagen secreted into the extracellular matrix by the pre-osteoblasts cultured for 4 and 7 days on the CS:Gel scaffolds, significantly increased when compared to the tissue culture polystyrene (TCPS) control surface. Human BM-MSCs attached and infiltrated within the pores of the CS:Gel scaffolds allowing for a significant increase of the osteogenic gene expression of RUNX2, ALP, and OSC. Histological data following implantation of a CS:Gel scaffold into a mouse femur demonstrated that the scaffolds support the formation of extracellular matrix, while fibroblasts surrounding the porous scaffold produce collagen with minimal inflammatory reaction. These results show the potential of CS:Gel scaffolds to support new tissue formation and thus provide a promising strategy for bone tissue engineering.

Laser-synthesized nanocrystalline, ferroelectric, bioactive BaTiO3/Pt/FS for bone implants.

The goal of our study is to design BaTiO3 ferroelectric layers that will cover metal implants and provide improved osseointegration. We synthesized ferroelectric BaTiO3 layers on Pt/fused silica substrates, and we studied their physical and bio-properties. BaTiO3 and Pt layers were prepared using KrF excimer laser ablation at substrate temperature Ts in the range from 200°C to 750°C in vacuum or under oxygen pressure of 10 Pa, 15 Pa, and 20 Pa. The BaTiO3/Pt and Pt layers adhered well to the substrates. BaTiO3 films of crystallite size 60-140 nm were fabricated. Ferroelectric loops were measured and ferroelectricity was also confirmed using Raman scattering measurements. Results of atomic force microscopy topology and the X-ray diffraction structure of the BaTiO3/Pt/fused silica multilayers are presented. The adhesion, viability, growth, and osteogenic differentiation of human osteoblast-like Saos-2 cells were also studied. On days 1, 3, and 7 after seeding, the lowest cell numbers were found on non-ferroelectric BaTiO3, while the values on ferroelectric BaTiO3, on non-annealed and annealed Pt interlayers, and on the control tissue culture polystyrene dishes and microscopic glass slides were similar, and were usually significantly higher than on non-ferroelectric BaTiO3. A similar trend was observed for the intensity of the fluorescence of alkaline phosphatase, a medium-term marker of osteogenic differentiation, and of osteocalcin, a late marker of osteogenic differentiation. At the same time, the cell viability, tested on day 1 after seeding, was very high on all tested samples, reaching 93-99%. Ferroelectric BaTiO3 films deposited on metallic bone implants through a Pt interlayer can therefore markedly improve the osseointegration of these implants in comparison with non-ferroelectric BaTiO3 films.

Template-assisted fabrication of tunable aspect ratio, biocompatible iron oxide pillar arrays

The extensive use of iron oxide nanomaterials in biomedical applications has prompted the development of a novel substrate for evaluating cell behaviour. This study examines the fabrication of tuneable length iron oxide pillar arrays using the porous nanochannels of anodic aluminium oxide membranes, and evaluates the biocompatibility of the substrate. The electroformed iron pillars were found to conform to the template channels with slightly larger iron oxide pillar diameters, due to the presence of an oxide shell. The biocompatibility was then confirmed with WST-1 proliferation and viability assay of cultured KT98 murine neural/progenitor stem cells on the surface of the pillar array; with no significant difference observed between viable cells after seven days of culture on iron oxide pillars, flat iron oxide, and tissue culture polystyrene. The physical properties of the pillar arrays were linked to the adhesion and spreading of the cells, and found that cells cultured on the pillar arrays had reduced spreading in comparison to tissue culture polystyrene control. In addition, it was found that protein expression was unaffected by culture on iron oxide substrates. The results of this study indicate that iron oxide pillar arrays are suitable to extended cell studies.

Mechanically resilient injectable scaffolds for intramuscular stem cell delivery and cytokine release

A promising strategy for treating peripheral ischemia involves the delivery of stem cells to promote angiogenesis through paracrine signaling. Treatment success depends on cell localization, retention, and survival within the mechanically dynamic intramuscular (IM) environment. Herein we describe an injectable, in situ-gelling hydrogel for the IM delivery of adipose-derived stem/stromal cells (ASCs), specifically designed to withstand the dynamic loading conditions of the lower limb and facilitate cytokine release from encapsulated cells. Copolymers of poly(trimethylene carbonate)-b-poly(ethylene glycol)-b-poly(trimethylene carbonate) diacrylate were used to modulate the properties of methacrylated glycol chitosan hydrogels crosslinked by thermally-initiated polymerization using ammonium persulfate and N,N,N′,N′-tetramethylethylenediamine. The scaffolds had an ultimate compressive strain over 75% and maintained mechanical properties during compressive fatigue testing at physiological levels. Rapid crosslinking (<3 min) was achieved at low initiator concentration (5 mM). Following injection and crosslinking within the scaffolds, human ASCs demonstrated high viability (>90%) over two weeks in culture under both normoxia and hypoxia. Release of angiogenic and chemotactic cytokines was enhanced from encapsulated cells under sustained hypoxia, in comparison to normoxic and tissue culture polystyrene controls. When delivered by IM injection in a mouse model of hindlimb ischemia, human ASCs were well retained in the scaffold over 28 days and significantly increased the IM vascular density compared to untreated controls.

Morphological adaptations in breast cancer cells as a function of prolonged passaging on compliant substrates.

Standard tissue culture practices involve propagating cells on tissue culture polystyrene (TCP) dishes, which are flat, 2-dimensional (2D) and orders of magnitude stiffer than most tissues in the body. Such simplified conditions lead to phenotypical cell changes and altered cell behaviors. Hence, much research has been focused on developing novel biomaterials and culture conditions that more closely emulate in vivo cell microenvironments. In particular, biomaterial stiffness has emerged as a key property that greatly affects cell behaviors such as adhesion, morphology, proliferation and motility among others. Here we ask whether cells that have been conditioned to TCP, would still show significant dependence on substrate stiffness if they are first pre-adapted to a more physiologically relevant environment. We used two commonly utilized breast cancer cell lines, namely MDA-MB-231 and MCF-7, and examined the effect of prolonged cell culturing on polyacrylamide substrates of varying compliance. We followed changes in cell adhesion, proliferation, shape factor, spreading area and spreading rate. After pre-adaptation, we noted diminished differences in cell behaviors when comparing between soft (1 kPa) and stiff (103 kPa) gels as well as rigid TCP control. Prolonged culturing of cells on complaint substrates further influenced responses of pre-adapted cells when transferred back to TCP. Our results have implications for the study of stiffness-dependent cell behaviors and indicate that cell pre-adaptation to the substrate needs consideration.

Infused polymers for cell sheet release.

Tissue engineering using whole, intact cell sheets has shown promise in many cell-based therapies. However, current systems for the growth and release of these sheets can be expensive to purchase or difficult to fabricate, hindering their widespread use. Here, we describe a new approach to cell sheet release surfaces based on silicone oil-infused polydimethylsiloxane. By coating the surfaces with a layer of fibronectin (FN), we were able to grow mesenchymal stem cells to densities comparable to those of tissue culture polystyrene controls (TCPS). Simple introduction of oil underneath an edge of the sheet caused it to separate from the substrate. Characterization of sheets post-transfer showed that they retain their FN layer and morphology, remain highly viable, and are able to grow and proliferate normally after transfer. We expect that this method of cell sheet growth and detachment may be useful for low-cost, flexible, and customizable production of cellular layers for tissue engineering.

Efficient generation of smooth muscle cells from adipose-derived stromal cells by 3D mechanical stimulation can substitute the use of growth factors in vascular tissue engineering.

Occluding artery disease causes a high demand for bioartificial replacement vessels. We investigated the combined use of biodegradable and creep-free poly (1,3-trimethylene carbonate) (PTMC) with smooth muscle cells (SMC) derived by biochemical or mechanical stimulation of adipose tissue-derived stromal cells (ASC) to engineer bioartificial arteries. Biochemical induction of cultured ASC to SMC was done with TGF-β1 for 7d. Phenotype and function were assessed by qRT-PCR, immunodetection and collagen contraction assays. The influence of mechanical stimulation on non-differentiated and pre-differentiated ASC, loaded in porous tubular PTMC scaffolds, was assessed after culturing under pulsatile flow for 14d. Assays included qRT-PCR, production of extracellular matrix and scanning electron microscopy. ASC adhesion and TGF-β1-driven differentiation to contractile SMC on PTMC did not differ from tissue culture polystyrene controls. Mesenchymal and SMC markers were increased compared to controls. Interestingly, pre-differentiated ASC had only marginal higher contractility than controls. Moreover, in 3D PTMC scaffolds, mechanical stimulation yielded well-aligned ASC-derived SMC which deposited ECM. Under the same conditions, pre-differentiated ASC-derived SMC maintained their SMC phenotype. Our results show that mechanical stimulation can replace TGF-β1 pre-stimulation to generate SMC from ASC and that pre-differentiated ASC keep their SMC phenotype with increased expression of SMC markers.

Dental implant surface chemistry and energy alter macrophage activation invitro.

To determine the effects of dental implant surface chemistry and energy on macrophage activation invitro. Disks made from two clinically used implant materials (titanium [Ti], titanium zirconium alloy [TiZr]) were produced with two different surface treatments (sandblast/acid-etch [SLA], hydrophilic-SLA [modSLA]). Surface roughness, energy, and chemistry were characterized. Primary murine macrophages were isolated from 6- to 8-week-old male C57Bl/6 mice and cultured on test surfaces (Ti SLA, TiZr SLA, Ti modSLA, TiZr modSLA) or control tissue culture polystyrene. mRNA was quantified by quantitative polymerase chain reaction after 24h of culture. Pro- (IL-1β, IL-6, and TNF-α) and anti-inflammatory (IL-4, IL-10) protein levels were measured by ELISA after 1 or 3days of culture. Quantitatively, microroughness was similar on all surfaces. Qualitatively, nanostructures were present on modSLA surfaces that were denser on Ti than on TiZr. modSLA surfaces were determined hydrophilic (high-energy surface) while SLA surfaces were hydrophobic (low-energy surface). Cells on high-energy surfaces had higher levels of mRNA from anti-inflammatory markers characteristic of M2 activation compared to cells on low-energy surfaces. This effect was enhanced on the TiZr surfaces when compared to cells on Ti SLA and Ti modSLA. Macrophages cultured on TiZr SLA and modSLA surfaces released more anti-inflammatory cytokines. The combination of high-energy and altered surface chemistry present on TiZr modSLA was able to influence macrophages to produce the greatest anti-inflammatory microenvironment and reduce extended pro-inflammatory factor release.

Prebiotic chemistry inspired antimicrobial surfaces

Event Abstract Back to Event Prebiotic chemistry inspired antimicrobial surfaces Donna Menzies1, Mario Salwiczek1, Christopher D. Easton1, Yue Qu2, Trevor Lithgow3, Helmut Thissen1 and Richard A. Evans1 1 CSIRO, Manufacturing Flagship, Australia 2 Monash University, Department of Biochemistry & Molecular Biology, Australia 3 Monash University, Department of Microbiology, Australia Introduction: Prebiotic chemistry is associated with the chemical origin of life; how simple non-living molecules (such as HCN oligomers) formed amino acids, nucleic acids and proteins. Building on the knowledge collected in the field of prebiotic chemistry over more than 50 years, we have been able to show that polymeric coatings can be deposited using the p-toluene sulphonate form of aminomalonitrile (AMN, the trimer of HCN). The robust, adhesive polymeric surface coatings are formed via neutralisation and spontaneous polymerisation in slightly basic, aqueous conditions[1]. The resulting coatings are chemically complex, peptidic in nature with a high content of nitrogenous heterocycles. The AMN surfaces are non-cytotoxic and by incorporation of silver, we have additionally introduced antibacterial properties as measured against the biofilm forming bacterial strains S. epidermidis (gram-positive) and P. aeruginosa (gram-negative). Materials and Methods: Aminomalonitrile p-toluenesulphonate (AMN, Sigma-Aldrich) solutions were prepared in 100 mM phosphate buffer (pH 8.5, 10 mg/mL). Samples were incubated upside for 24 hours at 25 °C. Incorporation of silver was achieved by incubation in AgNO3 for 24 hours (1 nM to 100 mM), rinsed and incubated in water for a further 24 hours. XPS was used to determine the incorporation of Ag and atomic composition of the coatings. Quantification of biofilm formation on the AMN and Ag impregnated AMN coatings was performed via a crystal violet assay, (using S. epidermidis (ATCC 35984) and P. aeruginosa (ATCC 27853)) and cell toxicity and attachment of mouse fibroblasts (ATCC NCTC clone 929 adipose mouse) were observed via photomicroscopy and quantified using an MTS assay, after 24 hours. Results and Discussion: The AMN coatings have been shown to be highly nitrogenous, non-cytotoxic and facilitate attachment of L929 mouse fibroblast cells equivalent to tissue culture polystyrene control surfaces. As silver containing coatings are of particular interest in antimicrobial surface applications, we investigated the potential for silver impregnation of the AMN coating, with the nitrogenous nature of the polymeric surfaces expected to provide a suitable environment for the complexation of metal ions. XPS analysis of the AMN coated surfaces after incubation in AgNO3 indicated that silver was detected in the films when the silver nitrate concentration was >10-5 M (Figure 1A). Analysis of the Ag MVV Auger region of the XPS spectrum has indicated that some of the incorporated Ag exists in its reduced form, Ag(0), in coatings treated with higher concentrations of AgNO3 (0.01 and 0.1 M). The silver treated AMN surfaces were evaluated for their ability to prevent biofilm growth of two well-known biofilm forming bacterial strains S. epidermidis (gram-positive) and P. aeruginosa (gram-negative). The results show a concentration dependent anti-biofilm activity with biofilm growth sharply dropping off on surfaces that have been incubated with AgNO3 at a concentration greater than 10-4 M (Figure 1B). Conclusion: Prebiotic chemistry inspired polymeric coatings based on aminomalonitrile have shown to be highly biocompatible and to facilitate the growth and attachment of mammalian cells. Incubation in silver nitrate solutions impregnated the AMN coatings with silver, when a concentration of 10-4 M and above were used. This same concentration was found to be optimal in terms of exhibiting antibacterial effects against two biofilm forming bacterial strains, S.epidermidis and P.aeruginosa.

Titanium surface characteristics, including topography and wettability, alter macrophage activation

Biomaterial surface properties including chemistry, topography, and wettability regulate cell response. Previous studies have shown that increasing surface roughness of metallic orthopaedic and dental implants improved bone formation around the implant. Little is known about how implant surface properties can affect immune cells that generate a wound healing microenvironment. The aim of our study was to examine the effect of surface modifications on macrophage activation and cytokine production. Macrophages were cultured on seven surfaces: tissue culture polystyrene (TCPS) control; hydrophobic and hydrophilic smooth Ti (PT and oxygen-plasma-treated (plasma) PT); hydrophobic and hydrophilic microrough Ti (SLA and plasma SLA), and hydrophobic and hydrophilic nano-and micro-rough Ti (aged modSLA and modSLA). Smooth Ti induced inflammatory macrophage (M1-like) activation, as indicated by increased levels of interleukins IL-1β, IL-6, and TNFα. In contrast, hydrophilic rough titanium induced macrophage activation similar to the anti-inflammatory M2-like state, increasing levels of interleukins IL-4 and IL-10. These results demonstrate that macrophages cultured on high surface wettability materials produce an anti-inflammatory microenvironment, and this property may be used to improve the healing response to biomaterials. Statement of significanceMetals like titanium (Ti) are common in orthopaedics and dentistry due to their ability to integrate with surrounding tissue and good biocompatibility. Roughness- and wettability-increasing surface modifications promote osteogenic differentiation of stem cells on Ti. While these modifications increase production of osteoblastic factors and bone formation, little is known about their effect on immune cells. The initial host response to a biomaterial is controlled primarily by macrophages and the factors they secrete in response to the injury caused by surgery and the material cues. Here we demonstrate the effect of surface roughness and wettability on the activation and production of inflammatory factors by macrophages. Control of inflammation will inform the design of surface modification procedures to direct the immune response and enhance the success of implanted materials.

Boron nitride nanotubes and nanoplatelets as reinforcing agents of polymeric matrices for bone tissue engineering.

This study investigates the mechanical properties and in vitro cytotoxicity of one- and two-dimensional boron nitride nanomaterials-reinforced biodegradable polymeric nanocomposites. Poly(propylene fumarate) (PPF) nanocomposites were fabricated using crosslinking agent N-vinyl pyrrolidone and inorganic nanomaterials: boron nitride nanotubes (BNNTs) and boron nitride nanoplatelets (BNNPs) dispersed at 0.2 wt % in the polymeric matrix. The incorporation of BNNPs and BNNTs resulted in a ∼38 and ∼15% increase in compressive (Young's) modulus, and ∼31 and ∼6% increase in compressive yield strength compared to PPF control, respectively. The nanocomposites showed a time-dependent increased protein adsorption for collagen I protein. The cytotoxicity evaluation of aqueous BNNT and BNNP dispersions (at 1-100 μg/mL concentrations) using murine MC3T3 preosteoblast cells showed ∼73-99% viability. The cytotoxicity evaluation of media extracts of nanocomposites before crosslinking, after crosslinking, and upon degradation (using 1×-100× dilutions) showed dose-dependent cytotoxicity responses. Crosslinked nanocomposites showed excellent (∼79-100%) cell viability, cellular attachment (∼57-67%), and spreading similar to cells grown on the surface of tissue culture polystyrene control. The media extracts of degradation products showed a dose-dependent cytotoxicity. The favorable cytocompatibility results in combination with improved mechanical properties of BNNT and BNNP nanocomposites opens new avenues for further in vitro and in vivo safety and efficacy studies towards bone tissue engineering applications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 406-419, 2017.