Extracellular vesicles combination with biomaterials is a desirable approach for regenerative medicine studies. Thus, this study reports titanium functionalization with EVs as a practical tool for orthopedics and bone regeneration. The drop casting functionalization is an easy and low-cost method compared with other strategies such as polymeric entrapment or biochemical binding.
Begin the titanium surface functionalization by washing the titanium disks with deionized water in a glass beaker. Then discard the water before washing the disks with 70%ethanol. Decant the solution to sonicate the washed implants at 50 C for five minutes in deionized water.
After discarding the water incubate titanium implants in a 40%sodium hydroxide solution at 50 C for 10 minutes with agitation and then sonicate the implants in deionized water as demonstrated. Next, perform at least five washes of the implants with deionized water. Until the pH is neutral on the pH indicator.
Repeat the sonication and incubation process, as explained earlier, by replacing sodium hydroxide with 50%nitric acid. Perform the washes and sonication with deionized water before storing the implants in a 70%ethanol solution. Incubate the titanium implants in a 30%nitric acid solution for 30 minutes at room temperature with gentle agitation.
After incubation perform at least five washes with deionized water until the pH is neutral on pH indicators. Then incubate titanium implants overnight at room temperature in deionized water. The next day, dry off the implants under vacuum at 40 C for 10 minutes.
Working under the cell culture cabinet, place titanium implants in a 96-well plate with the machine side facing up. Arrange for the extracellular vesicles EVs drop casting by thawing EVs solution, and vortexing solution at a pulse of three seconds. After mixing, deposit 40 microliters of the EVs solution on the titanium surface to immobilize a maximum of 4 x 10^11 EVs per implant, according to the concentration determined by nanoparticle tracking analysis.
Next, place the plates under vacuum conditions at 37 C for approximately two hours or until drops are completely dry. Adjust the time of drying depending on the number of titanium implants and the water present in the vacuum chamber. The amount of EVs released from titanium disks was measured by nanoparticle tracking analysis.
On day 2, around 10^9 EVs were released, followed by a sustained release on days 6, 10 and 14. In vitro cell biocompatibility of the titanium EVs was assessed with lactate dehydrogenase, or LDH release assay, at 48 hours after cell seeding onto the implants. Titanium EVs exhibited a lower amount of LDH than the maximum accepted cytotoxic value.
Whereas, the titanium control group, displayed higher LDH activity levels. The most important step is the vacuum drying of the coated implant. It is important to make sure that all water is evaporated to get optimal physisorption.
