Hydroxyapatite-decorated Fmoc-hydrogel as a bone-mimicking substrate for osteoclast differentiation and culture.

Hydrogels are water-swollen networks with great potential for tissue engineering applications. However, their use in bone regeneration is often hampered due to a lack of materials' mineralization and poor mechanical properties. Moreover, most studies are focused on osteoblasts (OBs) for bone formation, while osteoclasts (OCs), cells involved in bone resorption, are often overlooked. Yet, the role of OCs is pivotal for bone homeostasis and aberrant OC activity has been reported in several pathological diseases, such as osteoporosis and bone cancer. For these reasons, the aim of this work is to develop customised, reinforced hydrogels to be used as material platform to study cell function, cell-material interactions and ultimately to provide a substrate for OC differentiation and culture. Here, Fmoc-based RGD-functionalised peptide hydrogels have been modified with hydroxyapatite nanopowder (Hap) as nanofiller, to create nanocomposite hydrogels. Atomic force microscopy showed that Hap nanoparticles decorate the peptide nanofibres with a repeating pattern, resulting in stiffer hydrogels with improved mechanical properties compared to Hap- and RGD-free controls. Furthermore, these nanocomposites supported adhesion of Raw 264.7 macrophages and their differentiation in 2D to mature OCs, as defined by the adoption of a typical OC morphology (presence of an actin ring, multinucleation, and ruffled plasma membrane). Finally, after 7 days of culture OCs showed an increased expression of TRAP, a typical OC differentiation marker. Collectively, the results suggest that the Hap/Fmoc-RGD hydrogel has a potential for bone tissue engineering, as a 2D model to study impairment or upregulation of OC differentiation. STATEMENT OF SIGNIFICANCE: Altered osteoclasts (OC) function is one of the major cause of bone fracture in the most commonly skeletal disorders (e.g. osteoporosis). Peptide hydrogels can be used as a platform to mimic the bone microenvironment and provide a tool to assess OC differentiation and function. Moreover, hydrogels can incorporate different nanofillers to yield hybrid biomaterials with enhanced mechanical properties and improved cytocompatibility. Herein, Fmoc-based RGD-functionalised peptide hydrogels were decorated with hydroxyapatite (Hap) nanoparticles to generate a hydrogel with improved rheological properties. Furthermore, they are able to support osteoclastogenesis of Raw264.7 cells in vitro as confirmed by morphology changes and expression of OC-markers. Therefore, this Hap-decorated hydrogel can be used as a template to successfully differentiate OC and potentially study OC dysfunction.

Raman Spectroscopy to Monitor Post-Translational Modifications and Degradation in Monoclonal Antibody Therapeutics.

Monoclonal antibodies (mAbs) represent a rapidly expanding market for biotherapeutics. Structural changes in the mAb can lead to unwanted immunogenicity, reduced efficacy, and loss of material during production. The pharmaceutical sector requires new protein characterization tools that are fast, applicable in situ and to the manufacturing process. Raman has been highlighted as a technique to suit this application as it is information-rich, minimally invasive, insensitive to water background and requires little to no sample preparation. This study investigates the applicability of Raman to detect Post-Translational Modifications (PTMs) and degradation seen in mAbs. IgG4 molecules have been incubated under a range of conditions known to result in degradation of the therapeutic including varied pH, temperature, agitation, photo, and chemical stresses. Aggregation was measured using size-exclusion chromatography, and PTM levels were calculated using peptide mapping. By combining principal component analysis (PCA) with Raman spectroscopy and circular dichroism (CD) spectroscopy structural analysis we were able to separate proteins based on PTMs and degradation. Furthermore, by identifying key bands that lead to the PCA separation we could correlate spectral peaks to specific PTMs. In particular, we have identified a peak which exhibits a shift in samples with higher levels of Trp oxidation. Through separation of IgG4 aggregates, by size, we have shown a linear correlation between peak wavenumbers of specific functional groups and the amount of aggregate present. We therefore demonstrate the capability for Raman spectroscopy to be used as an analytical tool to measure degradation and PTMs in-line with therapeutic production.

Quantification of protein glycation using vibrational spectroscopy.

Glycation is a protein modification prevalent in the progression of diseases such as Diabetes and Alzheimer's, as well as a byproduct of therapeutic protein expression, notably for monoclonal antibodies (mAbs). Quantification of glycated protein is thus advantageous in both assessing the advancement of disease diagnosis and for quality control of protein therapeutics. Vibrational spectroscopy has been highlighted as a technique that can easily be modified for rapid analysis of the glycation state of proteins, and requires minimal sample preparation. Glycated samples of lysozyme and albumin were synthesised by incubation with 0.5 M glucose for 30 days. Here we show that both FTIR-ATR and Raman spectroscopy are able to distinguish between glycated and non-glycated proteins. Principal component analysis (PCA) was used to show separation between control and glycated samples. Loadings plots found specific peaks that accounted for the variation - notably a peak at 1027 cm-1 for FTIR-ATR. In Raman spectroscopy, PCA emphasised peaks at 1040 cm-1 and 1121 cm-1. Therefore, both FTIR-ATR and Raman spectroscopy found changes in peak intensities and wavenumbers within the sugar C-O/C-C/C-N region (1200-800 cm-1). For quantification of the level of glycation of lysozyme, partial least squares regression (PLSR), with statistical validation, was employed to analyse Raman spectra from solution samples containing 0-100% glycated lysozyme, generating a robust model with R2 of 0.99. We therefore show the scope and potential of Raman spectroscopy as a high throughput quantification method for glycated proteins in solution that could be applied in disease diagnostics, as well as therapeutic protein quality control.