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Self-assembling peptide hydrogels offer a novel 3-dimensional platform for many applications in cell culture and tissue engineering but are not compatible with current methods of RNA isolation; owing to interactions between RNA and the biomaterial. This study investigates the use of two techniques based on two different basic extraction principles: solution-based extraction and direct solid-state binding of RNA respectively, to extract RNA from cells encapsulated in four β-sheet forming self-assembling peptide hydrogels with varying net positive charge. RNA-peptide fibril interactions, rather than RNA-peptide molecular complexing, were found to interfere with the extraction process resulting in low yields. A column-based approach relying on RNA-specific binding was shown to be more suited to extracting RNA with higher purity from these peptide hydrogels owing to its reliance on strong specific RNA binding interactions which compete directly with RNA-peptide fibril interactions. In order to reduce the amount of fibrils present and improve RNA yields a broad spectrum enzyme solution—pronase—was used to partially digest the hydrogels before RNA extraction. This pre-treatment was shown to significantly increase the yield of RNA extracted, allowing downstream RT-qPCR to be performed.

Calcinosis cutis, defined as sub-epidermal deposition of calcium salts, is a major clinical problem in patients with systemic sclerosis (SSc), affecting 20-40% of patients. A number of recognised associates of calcinosis have been identified, including disease duration, digital ischaemia and acro-osteolysis. Yet to date, the pathogenesis of SSc-related calcinosis remains unknown and currently there is no effective disease-modifying pharmacotherapy. Following onset of SSc, there are marked changes in the extracellular matrix (ECM) of the skin, notably a breakdown in the microfibrillar network and accumulation of type I collagen. Our hypothesis is that these pathological changes reflect a changing cellular phenotype and result in a primed microenvironment for soft tissue calcification, with SSc fibroblasts adopting a pro-osteogenic profile and specific ′driving forces′ promoting tissue mineralisation. Considering the role of the ECM in disease progression may help elucidate the mechanism(s) behind SSc-related calcinosis and inform the development of future therapeutic interventions.

Continuous optimization of in vitro analytical techniques is ever more important, especially given the development of new materials for tissue engineering studies. In particular, isolation of cellular components for downstream applications is often hindered by the presence of biomaterials, presenting a major obstacle in understanding how cell-matrix interactions influence cell behavior. Here, we describe an approach for western blot analysis of cells that have been encapsulated in self-assembling peptide hydrogels (SAPHs), which highlights the need for complete solubilization of the hydrogel construct. We demonstrate that both the choice of buffer and multiple cycles of sonication are vital in obtaining complete solubilization, thereby enabling the detection of proteins otherwise lost to SAP aggregation. Moreover, we show that the presence of self-assembling peptides (SAPs) does not interfere with the standard immunoblotting technique, offering the potential for use in more full-scale proteomic studies.

BackgroundCutis calcinosis, defined as sub-epidermal deposition of calcium salts, is a painful, disabling, disfiguring, problem in 20-40% of patients with systemic sclerosis. Its pathogenesis remains poorly understood and currently there is no effective disease-modifying pharmacotherapy. One potential strategy is topical application of calcium chelating agents, able to penetrate the epidermal barrier and dissolve subcutaneous calcinotic deposits in situ. To date, the only compound reported for this application has been sodium thiosulfate (STS), with often-contradictory results.ObjectivesTo test the hypothesis, in pre-clinical studies, that polycarboxylic acids can induce calcium dissolution without skin toxicity, with the long-term aim of developing an effective topical treatment for cutis calcinosis.MethodsWe compared the metal ion-chelating agents citric acid (CA) and ethylenediaminetetraacetic acid (EDTA) - polycarboxylic acids with well-characterised chelation profiles – to STS for their ability to chelate calcium, without inducing cytotoxicity or inflammation (pro-inflammatory cytokine expression and release), using in vitro 2D (keratinocyte [HaCaT]; fibroblast ([HCA2]) and recombinant human epidermal (RHE) models. The resultant data was subsequently used to predict therapeutic concentrations for assessment in a validated skin irritation model (SkinEthicTM; Episkin SA) and to assay maximal percutaneous absorption. At relative dermal concentrations, the dissolution performance of each chelator was further assessed using two different models of calcinosis: 1) pharmaceutical dissolution of a hydroxyapatite (HAp) tablet (1) and; 2) dissolution of a calcified extracellular matrix laid down by mineralising SaOS2 in vitro monolayer culture (2).ResultsIncubation with CA, EDTA and STS induced cytotoxicity in both in vitro cell lines studied at concentrations of >10 mM; only EDTA (10 mM) resulted in inflammatory cytokine release (IL8) from cells at these higher concentrations (cf positive control, Lipopolysaccharide 10 mg/mL). When applied topically to RHE models as near-saturated solutions, none of the chelators were categorised as skin irritants. Due to differences in their relative aqueous solubility, higher concentrations of CA (1600 mM) and STS (1200 mM) could be delivered through the RHE model than EDTA (200 mM). Using a simple linear regression model, the rate of compound absorption was: CA, 0.43 ± 0.05; STS, 0.26 ± 0.03 and; EDTA, 0.05 ± 0.01 g/L/hr. At each time-point, the cumulative concentration of compound in the receptor media was CA > STS > EDTA. Incubation with chelators had no effect on the integrity of the RHE by standard histology. Based on the rate of percutaneous absorption, the dissolution performance of each chelator was tested at relative dermal concentrations for phosphate dissolution (nmoles) of HAp (CA, 9.61 ±0.97; EDTA, 5.38 ± 0.28; 3.78 ± 0.58) and in the calcified in vitro model (Figure 1; CA, 3285 ± 105, STS, 947 ± 95, EDTA, 1174 ± 89), showing the superiority of CA in both model systems.Figure 1.Dissolution of a calcified extracellular matrix by citric acidConclusionOverall, this study highlights the promise of polycarboxylic acids, particularly CA, to target subcutaneous calcification, which are neither toxic nor inflammatory to the skin. Specifically, we have identified CA as a potentially more efficacious alternative to STS for the topical treatment of cutis calcinosis.References[1]Fei F, Gallas A, Chang YC, Rao Y, Hunter AC, Winpenny REP, Herrick AL, Lockyer NP, Blanford CF. 2017. Quartz crystal microbalance assay of clinical calcinosis samples and their synthetic models differentiates the efficacy of chelation-based treatments. ACS Appl Mater Interfaces, 9(33):27544-27552[2]Wang QG, Wimpenny I, Dey RE, Zhong X, Youle PJ, Downes S, Watts DC, Budd PM, Hoyland JA, Gough JE. 2018. The unique calcium chelation property of poly(vinyl phosphonic acid-co-acrylic acid) and effects on osteogenesis in vitro. J Biomed Mater Res Part A 2018:106A:168–179Disclosure of InterestsKyle Burgess: None declared, Richard Winpenny: None declared, Alberto Saiani: None declared, Ariane Herrick Speakers bureau: Janssen, Consultant of: Arena, Boehringer-Ingelheim, Camurus, CSL-Behring, Gesynta, Grant/research support from: Gesynta, Rachel Watson Consultant of: NAOS, AbbVie, Grant/research support from: Walgreens Alliance Boots.