Insulin released from titanium discs with insulin coatings-Kinetics and biological activity

Abstract

Local administration of insulin from a titanium surface has been demonstrated to increase bone formation in non-diabetic rats. The authors hypothesized that insulin was released from the titanium surface and with preserved biological activity after the release. Thus, in the present in vitro study, human recombinant insulin was immobilized onto titanium discs, and the insulin release kinetics was evaluated using Electro-chemiluminescence immunoassay. Neutral Red uptake assay and mineralization assay were used to evaluate the biological effects of the released insulin on human osteoblast-like MG-63 cells. The results confirmed that insulin was released from titanium surfaces during a six-week period. Etching the disc prior to insulin coating, thickening of the insulin coating and incubation of the discs in serum-enriched cell culture medium increased the release. However, longer storage time decreased the release of insulin. Furthermore, the released insulin had retained its biological activity, as demonstrated by the significant increase in cell number and a stimulated mineralization process, upon exposure to released insulin. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1847-1854, 2017.

Effects of implant‐delivered insulin on bone formation in osteoporotic rats

“…Therefore, a reproducible thickness of <50 Å of insulin on the reference silica sample was used. However, to increase the amount of immobilized insulin, that is, the coating thickness, it might be advantageous to use a surface with different topography (micro‐pores) or hydrophilicity …”

“…It is possible that gene expression analyses of the peri‐implant bone would have shown different results, as compared to those obtained for the implant‐adherent cells. Furthermore, we have previously demonstrated that approximately 25% of initially immobilized insulin is released to the surroundings after 1 h of incubation of the implant in cell culture medium . The amount of insulin that remains on the surface in vivo after 3 weeks is presently unknown.…”

“…Thereafter, additional insulin was bound by incubation in the zero‐length cross‐linker N ‐(3‐dimethylaminopropyl)‐ N '‐ethylcarbodiimide (0.2 M EDC; Sigma Aldrich), in 0.05 M N ‐hydroxysulfosuccinimide sodium salt (NHS; Sigma Aldrich), followed by rinsing with PBS and an additional insulin incubation. This cycle was repeated multiple times, as described elsewhere . To avoid denaturation of the surface‐bound proteins, the coating procedure was carried out no later than 1 week prior to surgery.…”

“…The thickness of the immobilized insulin on the Si‐samples ( n = 5) was measured at three different sites on each sample using null ellipsometry (HeNe laser‐equipped Auto‐EL III; Rudolph Research Analytical, Hackettstown, NJ), programmed for measurements in air. The immobilized insulin thickness was calculated according to the McCrackin evaluation algorithm and converted to an approximate adsorbed amount per unit area using de Feijter's formula, as reported in previous studies . The assumed refractive index of insulin was n f =1.465, with 1 nm ellipsometric thickness of the adsorbed molecules being equivalent to approximately 0.12 μg/cm 2 .…”

“…The affected trabecular bone is associated with decreased osteoblast cell numbers and thickness, and increased trabecular spacing . It has been suggested that insulin stimulates osteoblast proliferation and differentiation, as well as the production of bone matrix proteins, and enhances bone matrix mineralization . In 2005, Gandhi and colleagues described the first attempt to improve bone healing (in a fracture model), using a diabetic rat model and locally delivered insulin with palmitic acid as the carrier .…”