α-Helical peptides on plasma-treated polymers promote ciliation of airway epithelial cells.

Airway respiratory epithelium forms a physical barrier through intercellular tight junctions, which prevents debris from passing through to the internal environment while ciliated epithelial cells expel particulate-trapping mucus up the airway. Polymeric solutions to loss of airway structure and integrity have been unable to fully restore functional epithelium. We hypothesised that plasma treatment of polymers would permit adsorption of α-helical peptides and that this would promote functional differentiation of airway epithelial cells. Five candidate plasma compositions are compared; Air, N2, H2, H2:N2 and Air:N2. X-ray photoelectron spectroscopy shows changes in at% N and C 1s peaks after plasma treatment while electron microscopy indicates successful adsorption of hydrogelating self-assembling fibres (hSAF) on all samples. Subsequently, adsorbed hSAFs support human nasal epithelial cell attachment and proliferation and induce differentiation at an air-liquid interface. Transepithelial measurements show that the cells form tight junctions and produce cilia beating at the normal expected frequency of 10-11 Hz after 28 days in culture. The synthetic peptide system described in this study offers potential superiority as an epithelial regeneration substrate over present "gold-standard" materials, such as collagen, as they are controllable and can be chemically functionalised to support a variety of in vivo environments. Using the hSAF peptides described here in combination with plasma-treated polymeric surfaces could offer a way of improving the functionality and integration of implantable polymers for aerodigestive tract reconstruction and regeneration.

Host macrophage response to injectable hydrogels derived from ECM and α-helical peptides.

Tissue engineering materials play a key role in how closely the complex architectural and functional characteristics of native healthy tissue can be replicated. Traditional natural and synthetic materials are superseded by bespoke materials that cross the boundary between these two categories. Here we present hydrogels that are derived from decellularised extracellular matrix and those that are synthesised from de novo α-helical peptides. We assess in vitro activation of murine macrophages to our hydrogels and whether these gels induce an M1-like or M2-like phenotype. This was followed by the in vivo immune macrophage response to hydrogels injected into rat partial-thickness abdominal wall defects. Over 28 days we observe an increase in mononuclear cell infiltration at the hydrogel-tissue interface without promoting a foreign body reaction and see no evidence of hydrogel encapsulation or formation of multinucleate giant cells. We also note an upregulation of myogenic differentiation markers and the expression of anti-inflammatory markers Arginase1, IL-10, and CD206, indicating pro-remodelling for all injected hydrogels. Furthermore, all hydrogels promote an anti-inflammatory environment after an initial spike in the pro-inflammatory phenotype. No difference between the injected site and the healthy tissue is observed after 28 days, indicating full integration. These materials offer great potential for future applications in regenerative medicine and towards unmet clinical needs. STATEMENT OF SIGNIFICANCE: Materials play a key role in how closely the complex architectural and functional characteristics of native healthy tissue can be replicated in tissue engineering. Here we present injectable hydrogels derived from decellularised extracellular matrix and de novo designed α-helical peptides. Over 28 days in the rat abdominal wall we observe an increase in mononuclear cell infiltration at the hydrogel-tissue interface with no foreign body reaction, no evidence of hydrogel encapsulation and no multinucleate giant cells. Our data indicate pro-remodelling and the promotion of an anti-inflammatory environment for all injected hydrogels with evidence of full integration with healthy tissue after 28 days. These unique materials offer great potential for future applications in regenerative medicine and towards designing materials for unmet clinical needs.

Silsesquioxane polymer as a potential scaffold for laryngeal reconstruction.

Cancer, disease and trauma to the larynx and their treatment can lead to permanent loss of structures critical to voice, breathing and swallowing. Engineered partial or total laryngeal replacements would need to match the ambitious specifications of replicating functionality, outer biocompatibility, and permissiveness for an inner mucosal lining. Here we present porous polyhedral oligomeric silsesquioxane-poly(carbonate urea) urethane (POSS-PCUU) as a potential scaffold for engineering laryngeal tissue. Specifically, we employ a precipitation and porogen leaching technique for manufacturing the polymer. The polymer is chemically consistent across all sample types and produces a foam-like scaffold with two distinct topographies and an internal structure composed of nano- and micro-pores. While the highly porous internal structure of the scaffold contributes to the complex tensile behaviour of the polymer, the surface of the scaffold remains largely non-porous. The low number of pores minimise access for cells, although primary fibroblasts and epithelial cells do attach and proliferate on the polymer surface. Our data show that with a change in manufacturing protocol to produce porous polymer surfaces, POSS-PCUU may be a potential candidate for overcoming some of the limitations associated with laryngeal reconstruction and regeneration.

Functionalized α-Helical Peptide Hydrogels for Neural Tissue Engineering.

Trauma to the central and peripheral nervous systems often lead to serious morbidity. Current surgical methods for repairing or replacing such damage have limitations. Tissue engineering offers a potential alternative. Here we show that functionalized α-helical-peptide hydrogels can be used to induce attachment, migration, proliferation and differentiation of murine embryonic neural stem cells (NSCs). Specifically, compared with undecorated gels, those functionalized with Arg-Gly-Asp-Ser (RGDS) peptides increase the proliferative activity of NSCs; promote their directional migration; induce differentiation, with increased expression of microtubule-associated protein-2, and a low expression of glial fibrillary acidic protein; and lead to the formation of larger neurospheres. Electrophysiological measurements from NSCs grown in RGDS-decorated gels indicate developmental progress toward mature neuron-like behavior. Our data indicate that these functional peptide hydrogels may go some way toward overcoming the limitations of current approaches to nerve-tissue repair.

Assessing cellular response to functionalized α-helical peptide hydrogels.

α-Helical peptide hydrogels are decorated with a cell-binding peptide motif (RGDS), which is shown to promote adhesion, proliferation, and differentiation of PC12 cells. Gel structure and integrity are maintained after functionalization. This opens possibilities for the bottom-up design and engineering of complex functional scaffolds for 2D and 3D cell cultures.

Structural changes to resorbable calcium phosphate bioceramic aged in vitro.

This work investigates the effect of mammalian cell culture conditions on 3D printed calcium phosphate scaffolds. The purpose of the studies presented was to characterise the changes in scaffold properties in physiologically relevant conditions. Differences in crystal morphologies were observed between foetal bovine serum-supplemented media and their unsupplemented analogues, but not for supplemented media containing tenocytes. Scaffold porosity was found to increase for all conditions studied, except for tenocyte-seeded scaffolds. The presence of tenocytes on the scaffold surface inhibited any increase in scaffold porosity in the presence of extracellular matrix secreted by the tenocytes. For acellular conditions the presence or absence of sera proteins strongly affected the rate of dissolution and the distribution of pore diameters within the scaffold. Exposure to high sera protein concentrations led to the development of significant numbers of sub-micron pores, which was otherwise not observed. The implication of these results for cell culture research employing calcium phosphate scaffolds is discussed.

Effect of cold-setting calcium- and magnesium phosphate matrices on protein expression in osteoblastic cells.

Bone loss due to accidents or tissue diseases requires replacement of the structure by either autografts, allografts, or artificial materials. Reactive cements, which are based on calcium phosphate chemistry, are commonly used in nonload bearing areas such as the craniofacial region. Some of these materials are resorbed by the host under physiological conditions and replaced by bone. The aim of this study was to test different calcium and magnesium cement composites in vitro for their use as bone substitution material. Phase composition of calcium deficient hydroxyapatite (Ca(9) (PO(4) )(5) HPO(4) OH), brushite (CaHPO(4) ·2H(2) O), and struvite (MgNH(4) PO(4) ·6H(2) O) specimens has been determined by means of X-ray diffraction, and compressive strength was measured. Cell growth and activity of osteoblastic cells (MG 63) on the different surfaces was determined, and the expression of bone marker proteins was analyzed by western blotting. Cell activity normalized to cell number revealed higher activity of the osteoblasts on brushite and struvite when compared to hydroxyapatite and also the expression of osteoblastic marker proteins was highest on brushite scaffolds. While brushite sets under acidic conditions, formation of struvite occurs under physiological pH, similar to hydroxyapatite cements, providing the possibility of additional modifications with proteins or other active components.