Christian Kascholke

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.

Dual-component collagenous peptide/reactive oligomer hydrogels as potential nerve guidance materials – from characterization to functionalization

Toward a new generation of improved nerve guidance conduits (NGCs), novel biomaterials are required to address pressing clinical shortcomings in peripheral nerve regeneration (PNR) and to promote biological performance. A dual-component hydrogel system formed by cross-linking reaction between maleic anhydride groups in an oligomeric building block for cross-linking of free amine functionalities in partially hydrolyzed collagen is formulated for continuous processing and NGC fabrication. The influence of the gelation base is optimized for processing from a double syringe delivery system with a static mixer. A hydrophilic low-concentrated base was introduced to control network formation and to utilize highly reactive macromers for gelation. Cross-linking extent and building block conversion were improved and homogenous monoliths were fabricated. Chemically derivatized hydrogels were obtained by conversion of a fraction of anhydride groups in the oligomeric precursor with monovalent primary amine-containing grafting molecules prior to gelation. Network stability in functionalized hydrogels was maintained and cationic moieties were implement to the gel that promoted in vitro cell attachment and spreading irrespective of mechanical stiffness. A molding strategy was introduced that allowed for fabrication of flexible tubular conduits in tunable dimensions and with chemically patterned structures. These hydrogel-based conduits hold promise for the next generation NGCs with integrated chemical cues for PNR.

Biodegradable and adjustable sol-gel glass based hybrid scaffolds from multi-armed oligomeric building blocks

Biodegradability is a crucial characteristic to improve the clinical potential of sol-gel-derived glass materials. To this end, a set of degradable organic/inorganic class II hybrids from a tetraethoxysilane(TEOS)-derived silica sol and oligovalent cross-linker oligomers containing oligo(d,l-lactide) domains was developed and characterized. A series of 18 oligomers (Mn: 1100-3200Da) with different degrees of ethoxylation and varying length of oligoester units was established and chemical composition was determined. Applicability of an established indirect rapid prototyping method enabled fabrication of a total of 85 different hybrid scaffold formulations from 3-isocyanatopropyltriethoxysilane-functionalized macromers. In vitro degradation was analyzed over 12months and a continuous linear weight loss (0.2-0.5wt%/d) combined with only moderate material swelling was detected which was controlled by oligo(lactide) content and matrix hydrophilicity. Compressive strength (2-30MPa) and compressive modulus (44-716MPa) were determined and total content, oligo(ethylene oxide) content, oligo(lactide) content and molecular weight of the oligomeric cross-linkers as well as material porosity were identified as the main factors determining hybrid mechanics. Cytocompatibility was assessed by cell culture experiments with human adipose tissue-derived stem cells (hASC). Cell migration into the entire scaffold pore network was indicated and continuous proliferation over 14days was found. ALP activity linearly increased over 2weeks indicating osteogenic differentiation. The presented glass-based hybrid concept with precisely adjustable material properties holds promise for regenerative purposes. STATEMENT OF SIGNIFICANCE Adaption of degradation kinetics toward physiological relevance is still an unmet challenge of (bio-)glass engineering. We therefore present a glass-derived hybrid material with adjustable degradation. A flexible design concept based on degradable multi-armed oligomers was combined with an established indirect rapid prototyping method to produce a systematic set of porous sol-gel-derived class II hybrid scaffolds. Mechanical properties in the range of cancellous bone were narrowly controlled by hybrid composition. The oligoester introduction resulted in significantly increased compressive moduli. Cytocompatible hybrids degraded in physiologically relevant time frames and a promising linear and controllable weight loss profile was found. To our knowledge, our degradation study represents the most extensive long-term investigation of sol-gel-derived class II hybrids. Due to the broad adjustability of material properties, our concept offers potential for engineering of biodegradable hybrid materials for versatile 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.