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In vitro evaluation of the biocompatibility of nanofibrous yarns from an oxidative stress perspective

Periodic Reporting for period 1 - BIOXYARN (In vitro evaluation of the biocompatibility of nanofibrous yarns from an oxidative stress perspective)

Reporting period: 2015-12-01 to 2017-11-30

The aim of the BIOXYARN project was to investigate the biocompatibility of curcumin-loaded electrospun yarns in vitro from an oxidative stress point of view and to investigate their potential for wound repair applications.

Fibres and textiles have been used as biomaterials for thousands of years, mainly as sutures and in dressings for wound care. Recently, it has become of increasing interest to use fibres as implantable materials to support the repair of damaged tissues and organs. In particular, the emergence of nanoscale electrospun fibres has allowed the fabrication of scaffolds that mimic the architecture of native biological tissues. Through their physical cues, these materials have the ability to promote cell adhesion, proliferation, and differentiation. However, further improvements of the material may be possible, such as to minimise the foreign body reaction and to accelerate the repair response. Targeting oxidative stress by incorporating antioxidant molecules is a particularly promising strategy. Oxidative stress is known to be one of the key pathophysiological elements in both the foreign body reaction and the tissue healing processes.

Among candidates antioxidant molecules, curcumin has shown great potential in multiple studies, which have included clinical trials. Curcumin, found in turmeric (commonly used as a spice), exhibits strong antioxidant, anti-inflammatory and antimicrobial properties. Although several studies have studied electrospun fibres loaded with curcumin for anticancer and antimicrobial applications, wound healing applications have been little explored, in particular with regard to the concentrations that are relevant for incorporation.

In BIOXYARN, we have explored a wide range of curcumin concentrations in electrospun filaments and have assessed the potential of the resulting materials for soft tissue repair applications. Our main results show that, at high concentrations (above 1% in weight to weight of polymer), curcumin induces pathological levels of oxidants in the culture medium. This inhibits the proliferation or even causes the apoptosis of normal human dermal fibroblasts (NHDFs). On the other hand, low concentrations of curcumin (below 0.01%) stimulate both the metabolic activity and the proliferation of NHDFs. Moreover, while high concentration affect the physicochemical properties of the filaments, low concentrations have no influence meaning that their incorporation is straightforward. Overall, our findings suggest that materials loaded with concentrations a hundred and thousand times lower than currently used (usually above 1%) have better potential for tissue repair.
This one-year project was carried out in partnership with three different institutions: the Rudjer Boskovic Institute (RBI, host), the University of Zagreb (Technical Textile Faculty, TTF) and the University of Oxford (NDORMS).

To achieve the aim, three secondary objectives were set:

1. Manufacture electrospun yarns loaded with curcumin at different concentrations
Polymer solutions with various amounts of antioxidant (curcumin) were prepared and were subsequently electrospun into continuous filaments at NDORMS. The main polymer used was polydioxanone (PDO), a degradable polyester already widely used in clinics (mostly as suture material). PDO electrospun filaments were successfully prepared with various amounts of curcumin: 0%, 0.001%, 0.01%, 0.1%, 1% and 10% (weight to weight of PDO).

2. Measure the physicochemical properties of the yarns and determine the release profiles of the antioxidant.
The physio-chemical characterisation of the filaments was performed with the various methods such as electron microscopy, tensile testing and mass spectrometry. We have shown that high concentrations of curcumin (≥1%) led to smaller fibre diameters, resulting in improved mechanical properties of the filament and faster release. Interestingly, smaller concentrations did not influence the main properties of PDO filaments.

3. Evaluate the biocompatibility of electrospun yarns using a human fibroblast model cultured in induced oxidative stress conditions and assess the cellular response.
For this objective, we evaluated the biocompatibility of electrospun yarns by cultured NHDFs in induced oxidative stress conditions. Our results showed that filaments with high concentration of curcumin (1% and 10%) inhibited the proliferation of NHDFs or caused apoptosis. Filaments with 10% curcumin were also shown to increase the cell antioxidant activity, indicating the presence of higher amounts of ROS, a result of curcumin’s toxicity. At lower concentrations (< 0.1% in the filaments), curcumin stimulated the proliferation of NHDFs compared to the no filament control.

Overall, these findings suggested that PDO filament loaded with low amounts of curcumin, in particular below 0.1%, are more promising materials for stimulating tissue repair than those loaded with high concentrations . It also highlighted the need to explore lower concentrations for other polymers (currently researchers are mostly working with values above 1%), in particular those similar to PDO, such as polycaprolactone and other degradable polyesters.

A manuscript presenting these findings has been submitted for publication (January 2017). We have also published an original review in Biomaterials (IF: 8.4) another top ranked journal, in which we highlight the link between oxidative stress and the fate of biomaterials. In addition, we have presented part of our results at two international conferences (BSTE 2016 and Electrospin2016).
It is worth mentioning that NDORMS is about to begin a clinical trial with PDO electrospun sutures (Bioyarn project). This could become a platform for the translation of curcumin-loaded PDO filaments into clinics. We hope to continue the development and evaluation of antioxidant-loaded electrospun materials as a collaboration between RBI, TTF and NDORMS.
With BIOXYARN, we show for the first time that very low concentration incorporated in materials, a hundred to a thousand times lower than currently proposed, may have better potential for tissue repair applications. With this study, we highlight that more care should be taken when adding curcumin in biomaterials since large amount may results in inhibition of proliferation or cell apoptosis.

This one-year research project provides crucial information for researchers designing materials for tissue repair applications involving curcumin or similar molecules. Antioxidant-loaded electrospun materials may find applications for the repair of damaged soft tissues, such as tendon and ligaments, as sutures or patches. Soft tissue repair surgery is an important and common area of clinical practice in most surgical disciplines. In our aging population, it also represents a substantial and growing social and economic burden. However, surgical repairs still fail to produce competent and robust tissue regenerates despite advances in surgical techniques. New materials, which better harness endogenous repair capabilities, may improve the success of repair and patient outcomes. This would not only benefit to patients, by improving their quality of life, but it will also have a positive impact on the society and the economy, by decreasing the costs associated to repeated surgeries and time off work.
Polydioxanone electrospun filaments with various amounts of curcumin