Anti-fibronectin aptamers improve the colonization of chitosan films modified with D-(+) Raffinose by murine osteoblastic cells

Abstract

The aim of the present study was to investigate how the enrichment of chitosan films with anti-fibronectin aptamers could enhance scaffold colonization by osteoblasts, by improving their adhesion and accelerating their proliferation. Chitosan discs were enriched with excess of anti-fibronectin aptamer. Aptamer adsorption on chitosan was monitored by measuring aptamer concentration in the supernatant by spectrophotometry, as well as its release, while functionalization was confirmed by labelling aptamers with a DNA intercalating dye. Chitosan samples were then characterized morphologically with atomic force microscopy and physically with contact angle measurement. Chitosan enrichment with fibronectin was then investigated by immunofluorescence and Bradford assay. 2% chitosan discs were then enriched with increasing doses of aptamers and used as culture substrates for MC3T3-E1 cells. Cell growth was monitored by optical microscopy, while cell viability and metabolic activity were assessed by chemiluminescence and by Resazurin Sodium Salt assay. Cell morphology was investigated by cytofluorescence and by scanning electron microscopy. Chitosan films efficiently bound and retained aptamers. Aptamers did not affect the amount of adsorbed fibronectin, but affected osteoblasts behavior. Cell growth was proportional to the amount of aptamer used for the functionalization, as well as aptamers influenced cell morphology and their adhesion to the substrate. Our results demonstrate that the enrichment of chitosan films with aptamers could selectively improve osteoblasts behavior. Furthermore, our results support further investigation of this type of functionalization as a suitable modification to ameliorate the biocompatibility of biomaterial for hard tissue engineering applications.

Surface modification of chitosan films with a fibronectin fragment–DNA aptamer complex to enhance osteoblastic cell activity: A mass spectrometry approach probing evidence on protein behavior

“…As in the field of biomaterials, the ESI‐MS and MS/MS data obtained in our study presented, as key feature, the ability of a simple HDX approach to provide useful and direct insights into the structural dynamics of a FN fragment involved in chitosan surface functionalization in the presence or absence of a selective aptamer. As previously reported aptamers did not affect the amount of adsorbed FN, but presumably influenced its conformation enhancing its biological activity toward osteoblasts adhesion and growth. Here, FN peptides involved in interactions with aptamers were identified and the MS data indicated the improved functionality of FN on chitosan when aptamers were used as selective spacer.…”

“…In order to improve the ability of chitosan to act as a substrate for cell growth, the preparation of chitosan films in the presence of phosphates and D‐(+)‐raffinose was described, which positively contributed to the growth of WI‐38 and HUVEC cells on this biomaterial. The same model however was not very efficient in supporting the growth of osteoblastic cells, so further functionalization of these hydrogels was performed to improve their biocompatibility by exploiting the binding affinity of chitosan for sulfur‐containing components, such as thiol‐modified gelatin, or by means of aptamers …”

“…A previous work by Parisi et al described the positive effect of the functionalization of chitosan films with anti‐fibronectin aptamers on their colonization by murine osteoblastic cells. Aptamers significantly increased the selective adsorption of fibronectin on chitosan hydrogels, which resulted in improved adhesion and growth of MC3T3‐E1 cells.…”

Physicochemical and pharmacokinetic properties of polymeric films loaded with cisplatin for the treatment of malignant pleural mesothelioma

“…It is then clear that innovation in the field of loco-regional drug administration is a viable strategy to increase the effectiveness of chemotherapy and to reduce toxic side effects for lung cancer patients undergoing chemotherapy (38). In the case of the two materials studied in this research, chitosan, despite being a material used often for implants and for tissue engineering (39) and considered biocompatible and relatively non pro-inflammatory (40), showed a strong foreign body reaction and released cisplatin at an extremely low rate in vitro as well as in vivo. However, recently it has been reported that chitosan can show mild to moderate chronic inflammation (41) and foreign body reaction (42) in the case of crosslinked chitosan derivatives.…”

Selective protein adsorption for implantable biomaterials: A new road to bioactivity

“…To bypass these issues, aptamers were then proposed. One such approach was reported by Galli, et al [18] and Parisi, et al [19]. In these papers, anti-Fibronectin aptamers were used as docking points on hyaluronic acid-polyethylenglycole hydrogels and chitosan hydrogels respectively.…”