Dual-Functional Hydrazide-Reactive and Anhydride-Containing Oligomeric Hydrogel Building Blocks.

Biomimetic hydrogels are advanced biomaterials that have been developed following different synthetic routes. Covalent postfabrication functionalization is a promising strategy to achieve efficient matrix modification decoupled of general material properties. To this end, dual-functional macromers were synthesized by free radical polymerization of maleic anhydride with diacetone acrylamide (N-(1,1-dimethyl-3-oxobutyl)acrylamide) and pentaerythritol diacrylate monostearate. Amphiphilic oligomers (Mn < 7.5 kDa) with anhydride contents of 7-20% offered cross-linking reactivity to yield rigid hydrogels with gelatinous peptides (E = 4-13 kPa) and good cell adhesion properties. Mildly reactive methyl ketones as second functionality remained intact during hydrogel formation and potential of covalent matrix modification was shown using hydrazide and hydrazine model compounds. Successful secondary dihydrazide cross-linking was demonstrated by an increase of hydrogel stiffness (>40%). Efficient hydrazide/hydrazine immobilization depending on solution pH, hydrogel ketone content as well as ligand concentration for bioconjugation was shown and reversibility of hydrazone formation was indicated by physiologically relevant hydrazide release over 7 days. Proof-of-concept experiments with hydrazido-functionalized hyaluronan demonstrated potential for covalent aECM immobilization. The presented dual-functional macromers have perspective as reactive hydrogel building blocks for various biomedical applications.

Melanocytes from the outer root sheath of human hair and epidermal melanocytes display improved melanotic features in the niche provided by cGEL, oligomer-cross-linked gelatin-based hydrogel.

Non-invasively based cell treatments of depigmented skin disorders are largely limited by means of cell sampling as much as by their routes of application. Human melanocytes cultivated from the outer root sheath of hair follicle (HUMORS) are among the cell types that fit the non-invasive concept by being cultivated out of a minimal sample: hair root. Eventual implementation of HUMORS as a graft essentially depends on a choice of suitable biocompatible, biodegradable carrier that would mechanically and biologically support the cells as transient niche and facilitate their engraftment. Hence, the melanotic features of follicle-derived HUMORS and normal human epidermal melanocytes (NHEM) in engineered scaffolds based on collagen, the usual leading candidate for graft material for a variety of skin transplantation procedures were tested. Hydrogel named cGEL, an enzymatically degraded bovine gelatin chemically cross-linked with an oligomeric copolymer synthesized from pentaerythritol diacrylate monostearate (PEDAS), maleic anhydride (MA), and N-isopropylacrylamide (NiPAAm) or diacetone acrylamide (DAAm), was used. The cGEL provided a friendly three-dimensional (3D) cultivation environment for human melanocytes with increased melanin content of the 3D cultures in comparison to Collagen Cell Carrier® (CCC), a commercially available bovine decellularized collagen membrane, and electrospun polycaprolactone (PCL) matrices. One of the cGEL variants fostered not only a dramatic increase in melanin production but also a significant enhancement of melanotic gene PAX3, PMEL, TYR, and MITF expression in comparison to that of both CCC full-length collagen and PCL scaffolds, providing a clearly superior melanocyte niche that may be a suitable candidate for grafting carriers. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3115-3126, 2016.

Polycaprolactone fiber meshes provide a 3D environment suitable for cultivation and differentiation of melanocytes from the outer root sheath of hair follicle.

Melanocytes differentiated from the stem cells of human hair follicle outer root sheath (ORS) have the potential for developing non-invasive treatments for skin disorders out of a minimal sample: of hair root. With a robust procedure for melanocyte cultivation from the ORS of human hair follicle at hand, this study focused on the identification of a suitable biocompatible, biodegradable carrier as the next step toward their clinical implementation. Polycaprolactone (PCL) is a known biocompatible material used for a number of medical devices. In this study, we have populated electrospun PCL fiber meshes with normal human epidermal melanocytes (NHEM) as well as with hair-follicle-derived human melanocytes from the outer root sheath (HUMORS) and tested their functionality in vitro. PCL fiber meshes evidently provided a niche for melanocytes and supported their melanotic properties. The cells were tested for gene expression of PAX3, PMEL, TYR and MITF, as well as for proliferation, expression of melanocyte marker proteins tyrosinase and glycoprotein 100 (gp100), L-DOPA-tautomerase enzymatic activity and melanin content. Reduced mitochondrial activity and PAX-3 gene expression indicated that the three-dimensional PCL scaffold supported differentiation rather than proliferation of melanocytes. The monitored melanotic features of both the NHEM and HUMORS cultivated on PCL scaffolds significantly exceeded those of two-dimensional adherent cultures.

Highly adjustable biomaterial networks from three-armed biodegradable macromers.

Biocompatible material platforms with adjustable properties and option for chemical modification are warranted for site-specific biomedical applications. To this end, three-armed biodegradable macromers of well-defined chemical characteristics were prepared from trivalent alcohols with different degrees of ethoxylation and different lengths of oligoester domains. A platform of 15 different macromers was established. The macromers were designed to exhibit different hydrophilicities and molecular weights and contained various types of oligoesters such as d,l-lactide, l-lactide and ε-caprolactone. Macromers chemical composition was determined and molecular weights ranged from 900 to 3000 Da. Thermally induced cross-linking of methacrylated macromers was monitored by oscillation rheology. A novel variant of the solid lipid templating technique was established to fabricate macroporous tissue engineering scaffolds from these macromers. Scaffold properties were thoroughly investigated regarding mechanical properties, compositional analysis including methacrylic double bond conversion, microstructure and porosity. Material properties could be controlled by macromer chemistry. By variation of the fabrication procedure and processing parameters scaffold porosity was increased up to 88%. Basic cytocompatibility was assessed including indirect and direct contact methods. The established macromers hold promise for various biomedical purposes. STATEMENT OF SIGNIFICANCE: Specific biomedical applications require tailored biomaterials with defined properties. We established a macromer platform for preparation of tissue engineering scaffolds with adjustable chemical and mechanical characteristics. Macromers were composed of trivalent core alcohols with different degrees of ethoxylation to which biodegradable domains - lactide or ε-caprolactone - were oligomerized before final methacrylation. The solid lipid templating technique was adapted to fabricate macroporous scaffolds with controlled pore structure and porosity from the developed macromers, which can also be processed by solid freeform fabrication techniques. The material platform relies on clinically established chemistries of the biodegradable domains and the macromer concept enables the fabrication of networks in which cross-polymerization kinetics, mechanical properties and surface hydrophobicity is predefined by macromer chemistry. Cytocompatibility was confirmed by indirect and direct cell contact experiments.

Gelatin-based biomaterial engineering with anhydride-containing oligomeric cross-linkers.

Chemically cross-linked gelatin hydrogels are versatile cell-adhesive hydrogel materials that have been established for a variety of biomedical applications. The most prominent cross-linker is glutaraldehyde, which, however, has been described to cause compatibility problems and loss of microscopic but relevant structural features. A recently developed oligomeric cross-linker that contains anhydride functionalities was evaluated as cross-linker for the fabrication of gelatin-based hydrogels and microparticles. In a fast curing reaction, hydrogels composed of gelatin and oligomeric cross-linker were fabricated with good conversion over a wide concentration range of constituents and with cross-linkers of different anhydride contents. Hydrogel properties, such as dry weight and mechanics, could be controlled by hydrogel composition and rheological properties correlated to elastic moduli from 1 to 10 kPa. The gels were shown to be cytocompatible and promoted cell adhesion. In soft formulations, cells migrated into the hydrogel bulk. Gelatin microparticles prepared by a standard water-in-oil emulsion technique were also treated with the novel oligomers, and cross-linking degrees matching those obtained with glutaraldehyde were obtained. At the same time, fewer interparticular cross-links were observed. Fluorescein-derivatized cross-linkers yielded labeled microparticles in a concentration-dependent manner. The oligomeric cross-linkers are presented as an efficient and possibly more functional and compatible alternative to glutaraldehyde. The engineered hydrogel materials hold potential for various biomedical applications.

Open porous microscaffolds for cellular and tissue engineering by lipid templating.

Porous microspheres fabricated from biodegradable polymers have great potential as microscaffolds in tissue engineering applications, especially for novel strategies such as microtissue fabrication in vitro and microtissue assembly in vivo. Fabrication techniques for microparticulate scaffolds with surface and bulk pore sizes relevant for effective cell intrusion, however, are scarce. This study presents two techniques for the fabrication of open porous microscaffolds from poly(lactide-co-glycolide) in which lipid templating is used for pore formation and combined with either dispersion spraying or a double emulsion technique to determine the size and shape of the particulate structures generated. Both techniques yield microscaffolds with an average size of between 500 and 800 µm, high bulk porosities and open surface pores larger than 50 µm in diameter. Microscaffold morphology was investigated microscopically, particle size distribution was determined and porosity was quantified by intrusion measurements. Particle size and morphology was controlled by the processing parameters and the content and type of lipid porogen. Efficient extraction of the lipid template was shown by thermal analysis. Microscaffold cytocompatibility and in vitro cell culture performance was evaluated with L929 fibroblasts and rat adipose-derived stromal cells (ADSC), respectively. Extracts of different formulations were cytocompatible. Rat ADSC proliferated on the microscaffolds and were differentiated along the adipogenic lineage.