Sustained growth factor delivery from bioactive PNIPAM-grafted-chitosan/heparin multilayers as a tool to promote growth and migration of cells.

Delivery of growth factors (GFs) is challenging for regulation of cell proliferation and differentiation due to their rapid inactivation under physiological conditions. Here, a bioactive polyelectrolyte multilayer (PEM) is engineered by the combination of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and glycosaminoglycans to be used as reservoir for GF storage. PNIPAM-grafted-chitosan (PChi) with two degrees of substitution (DS) are synthesized, namely LMW* (DS 0.14) and HMW (DS 0.03), by grafting low (2 kDa) and high (10 kDa) molecular weight of PNIPAM on the backbone of chitosan (Chi) to be employed as polycations to form PEM with the polyanion heparin (Hep) at pH 4. Subsequently, PEMs are chemically crosslinked to improve their stability at physiological pH 7.4. Resulting surface and mechanical properties indicate that PEM containing HMW is responsive to temperature at 20 °C and 37 °C, while LMW is not. More importantly, Hep as terminal layer combined with HMW allows not only a better retention of the adhesive protein vitronectin but also a sustained release of FGF-2 at 37 °C. With the synergistic effect of vitronectin and matrix-bound FGF-2, significant promotion on adhesion, proliferation, and migration of 3T3 mouse embryonic fibroblasts is achieved on HMW-containing PEM compared to Chi-containing PEM and exogenously added FGF-2. Thus, PEM containing PNIPAM in combination with bioactive glycosaminoglycans like Hep represents a versatile approach to fabricate a GF delivery system for efficient cell culture, which can be potentially served as cell culture substrate for production of (stem) cells and bioactive wound dressing for tissue regeneration.

Synthesis of Thermoresponsive PNIPAM-Grafted Cellulose Sulfates for Bioactive Multilayers via Layer-by-Layer Technique.

The robust thermoresponsive and bioactive surfaces for tissue engineering by combining poly-N-isopropylacrylamide (PNIPAM) and cellulose sulfate (CS) remain highly in demand but not yet realized. Herein, PNIPAM-grafted cellulose sulfates (PCSs) with diverse degrees of substitution ascribed to sulfate groups (DSS) are synthesized for the first time. Higher sulfated PCS2 generally forms larger aggregates than lower sulfated PCS1 at their cloud point temperatures (TCP) of around 33 °C, whereas PCS1 leads to larger aggregates at body temperature (37 °C). Via the layer-by-layer (LbL) technique, biocompatible polyelectrolyte multilayers (PEMs) composed of PCSs as polyanions in combination with poly-l-lysine (PLL) or quaternized chitosan (QCHI) as polycations were fabricated. The resulting surfaces contained a more intermingled structure of polyanions with both polycations, while higher sulfated cellulose derivatives (CS2 and PCS2) displayed greater stability. Studies on toxicity and biocompatibility of PEM using 3T3 mouse fibroblasts showed a lower cytotoxicity of PEM with PCS2 and CS2 than PCS1 and CS1. Furthermore, the PEM using PCS2 particularly in combination with QCHI demonstrated excellent biocompatibility that is promising for new bioactive, thermoresponsive coatings on biomaterials and substrata for culturing adhesion-dependent cells.

Engineering of Stable Cross-Linked Multilayers Based on Thermo-Responsive PNIPAM-Grafted-Chitosan/Heparin to Tailor Their Physiochemical Properties and Biocompatibility.

The thermo-responsive poly(N-isopropylacrylamide) (PNIPAM) is ubiquitously applied in controlled drug release and tissue engineering. However, the lack of bioactivity of PNIPAM restricts its use in cell-containing systems being a thermo-responsive adhesive substratum with no regulating effect on cell growth and differentiation. In this study, integrating PNIPAM with chitosan into PNIPAM-grafted-chitosan (PNIPAM-Chi) allows a layer-by-layer assembly with bioactive heparin to fabricate PNIPAM-modified polyelectrolyte multilayers (PNIPAM-PEMs). Grafting PNIPAM chains of either 2 (LMW) or 10 kDa (HMW) on the chitosan backbone influences the cloud point (CP) temperature in the range from 31 to 33 °C. PNIPAM-Chi with either a higher molecular weight or a higher degree of substitution of PNIPAM chains exhibiting a significant increase in diameter above CP as ensured by dynamic light scattering is selected to fabricate PEM with heparin as a polyanion at pH 4. Little difference of layer growth is detected between the chosen PNIPAM-Chi used as polycations by surface plasmon resonance, while multilayers formed with HMW-0.02 are more hydrated and show striking swelling-and-shrinking abilities when studied with quartz crystal microbalance with dissipation monitoring. Subsequently, the multilayers are covalently cross-linked using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide to strengthen the stability of the systems under physiological conditions. Ellipsometry results confirm the layer integrity after exposure to the physiological buffer at pH 7.4 compared to those without cross-linking. Moreover, significantly higher adhesion and more spreading of C3H10T1/2 multipotent embryonic mouse fibroblasts on cross-linked PEMs, particularly with heparin terminal layers, are observed owing to the bioactivity of heparin. The slightly more hydrophobic surfaces of cross-linked PNIPAM-PEMs at 37 °C also increase cell attachment and growth. Thus, layer-by-layer constructed PNIPAM-PEM with cross-linking represents an interesting cell culture system that can be potentially employed for thermally uploading and controlled release of growth factors that further promotes tissue regeneration.

Recent Progress on Cellulose-Based Ionic Compounds for Biomaterials.

Glycans play important roles in all major kingdoms of organisms, such as archea, bacteria, fungi, plants, and animals. Cellulose, the most abundant polysaccharide on the Earth, plays a predominant role for mechanical stability in plants, and finds a plethora of applications by humans. Beyond traditional use, biomedical application of cellulose becomes feasible with advances of soluble cellulose derivatives with diverse functional moieties along the backbone and modified nanocellulose with versatile functional groups on the surface due to the native features of cellulose as both cellulose chains and supramolecular ordered domains as extractable nanocellulose. With the focus on ionic cellulose-based compounds involving both these groups primarily for biomedical applications, a brief introduction about glycoscience and especially native biologically active glycosaminoglycans with specific biomedical application areas on humans is given, which inspires further development of bioactive compounds from glycans. Then, both polymeric cellulose derivatives and nanocellulose-based compounds synthesized as versatile biomaterials for a large variety of biomedical applications, such as for wound dressings, controlled release, encapsulation of cells and enzymes, and tissue engineering, are separately described, regarding the diverse routes of synthesis and the established and suggested applications for these highly interesting materials.

Data of continuous harvest of stem cells via partial detachment from thermoresponsive nanobrush surfaces.

This data article contains two figures and one table supporting the research article entitled: "Continuous harvest of stem cells via partial detachment from thermoresponsive nanobrush surface" [1]. The table shows coating conditions of three copolymers, poly(styrene-co-acrylic acid) grafted with oligovitronectin, poly(styrene-co-N-isopropylacrylamide) and poly(styrene-co-polyethylene glycol methacrylate) to prepare thermoresponsive surface. XPS spectra show the nitrogen peak of the polystyrene surface coated with poly(styrene-co-acrylic acid) grafted with oligovitronectin. The surface coating density analyzed from sorption of poly(styrene-co-acrylic acid) grafted with oligovitronectin by UV-vis spectroscopy is also presented.

Continuous harvest of stem cells via partial detachment from thermoresponsive nanobrush surfaces.

Stem cell culture is typically based on batch-type culture, which is laborious and expensive. Here, we propose a continuous harvest method for stem cells cultured on thermoresponsive nanobrush surfaces. In this method, stem cells are partially detached from the nanobrush surface by reducing the temperature of the culture medium below the critical solution temperature needed for thermoresponse. The detached stem cells are harvested by exchange into fresh culture medium. Following this, the remaining cells are continuously cultured by expansion in fresh culture medium at 37 °C. Thermoresponsive nanobrush surfaces were prepared by coating block copolymers containing polystyrene (for hydrophobic anchoring onto culture dishes) with three types of polymers: (a) polyacrylic acid with cell-binding oligopeptides, (b) thermoresponsive poly-N-isopropylacrylamide, and (c) hydrophilic poly(ethyleneglycol)methacrylate. The optimal coating durations and compositions for these copolymers to facilitate adequate attachment and detachment of human adipose-derived stem cells (hADSCs) and embryonic stem cells (hESCs) were determined. hADSCs and hESCs were continuously harvested for 5 and 3 cycles, respectively, via the partial detachment of cells from thermoresponsive nanobrush surfaces.