Platelet-Derived Extracellular Vesicle Functionalization of Ti Implants

Summary

Here, we present a method for the isolation of Extracellular Vesicles (EVs) derived from the platelet lysates (PL) and their use for coating titanium (Ti) implant surfaces. We describe the drop casting coating method, the EVs release profile from the surfaces, and in vitro biocompatibility of EVs coated Ti surfaces.

Abstract

Extracellular Vesicles (EVs) are biological nanovesicles that play a key role in cell communication. Their content includes active biomolecules such as proteins and nucleic acids, which present great potential in regenerative medicine. More recently, EVs derived from Platelet Lysate (PL) have shown an osteogenic capability comparable to PL. Besides, biomaterials are frequently used in orthopedics or dental restoration. Here, we provide a method to functionalize Ti surfaces with PL-derived EVs in order to improve their osteogenic properties.

EVs are isolated from PL by size exclusion chromatography, and afterward Ti surfaces are functionalized with PL-EVs by drop casting. Functionalization is proven by EVs release and its biocompatibility by the lactate dehydrogenase (LDH) release assay.

Introduction

EVs are membrane vesicles (30-200 nm) secreted by any cell and play a key role in cell-to-cell communication by delivering their cargo. They contain a variety of active biomolecules that may include nucleic acids, growth factors, or bioactive lipids¹. For these reasons, EVs have been evaluated for their potential use in therapeutics. In terms of orthopedics and bone regeneration, EVs from different sources have been tested. Among them, platelet-derived EVs have been shown to induce a differentiation effect on stem cells while maintaining a low cytotoxic profile^2,3. Therefore, further re....

Protocol

Platelet Lysate (PL) is obtained as previously described in compliance with institutional guidelines³ using fresh buffy coats provided by the IdISBa Biobank as starting material. Their use for the current project was approved by its Ethics Committee (IB 1995/12 BIO).

1. EVs isolation from PL

1. Larger bodies removal
   1. Thaw PL at room temperature.
   2. Centrifuge PL at 1,500 x g for 15 min at 4 °C. Discard the pellet as it contains cell debris.
   3. Collect the supernatant and perform two consecutive centrifugations at 10,000 x g for 30 min at 4 °C.
      NO....

Results

The method presented in this article allows obtaining EVs functionalized titanium discs. EVs are physically bonded to the surface, which allows a sustained release over time. The amount of EVs released can be measured by NTA on Day 2, 6, 10, and 14. The first measurements, on Day 2, show that around 10⁹ EVs are released, followed by a sustained release on day 6 (~10⁸ EVs); day 10 (~10⁷ EVs), and day 14 (~10⁷ EVs). This confirms a sustained release, despite showing a decrease in.......

Discussion

This protocol aims to provide clear instructions for EVs functionalization onto Ti surfaces. The method presented is based on a drop casting strategy, which is a physisorption type of functionalization. Poor bibliography exists regarding EVs functionalization on Ti surfaces, although there are few studies showing different advantages by immobilizing EVs on Ti¹⁰. Anyway, some of the strategies explored include biochemical binding⁸, polymeric entrapment⁹

Materials

Name	Company	Catalog Number	Comments
0,8 µm syringe filter	Sartorius	16592K
1.5 mL Centrifuge tube	SPL life sciences	PLC60015
1mL syringe	BD	303174
96-well culture plate	SPL life sciences	PLC30096
Absolut ethanol	Scharlau	ET0006005P	Used to prepare 20 %  ethanol with Milli-Q® water
AKTA purifier System	GE Healthcare	8149-30-0014
Allegra X-15R Centrifuge	Beckman Coutler	392934	SX4750A swinging rotor
Centrifuge 5430 R	Eppendorf	5428000210	F-45-48-11 rotor
Conical Tube, Conical Bottom, 50ml	SPL life sciences	PLC50050
Cytotoxicity Detection Kit (LDH)	Roche	11644793001
Disposable Syringes 10 ml	Becton Dickinson	BDH307736
DMEM Low Glucose Glutamax	GIBCO	21885025
Dulbecco's PBS (1x)	Capricorn Scientific	PBS-1A
Fetal Bovine Serum (FBS) Embrionic Certified	GIBCO	16000044
Filtropur S 0.2 µm syringe filter	Sarstedt	83.1826.001
HiPrep 16/60 Sephacryl S-400 HR	GE Healthcare	28-9356-04	Precast columns
human umbilical cord-derived mesenchymal stem cells (hUC-MSC)	IdISBa Biobank
Nanodrop 2000 spectrophotometer	ThermoFisher	ND-2000
NanoSight NS300 nanoparticle tracking analysis	Malvern	NS300	Device with embedded laser at λ= 532 nm and camera sCMOS
Needle	Terumo	946077135
Nitric acid 69,5%	Scharlau	AC16071000
Optima L-100 XP Ultracentrifuge	Beckman Coulter	8043-30-1124	SW-32Ti Rotor
Penicillin-Streptomycin Solution 100X	Biowest	L0022
pH Test strips 4.5-10.0	Sigma	P-4536
Platelet Lysate (PL)	IdISBa Biobank		Obtained from  buffy coats discarded after blood donation
Polypropylene centrifuge tubs	Beckman Coutler	326823
Power wave HT	BioTek	10340763
Screw cap tube, 15 ml, (LxØ): 120 x 17 mm, PP, with print	Sarstedt	62554502
Sodium hidroxide	Sharlau	SO04251000
Titanium implants replicas	Implantmedia, SA	NA	Titanium grade IV. Diameter: 6,2 mm. Height: 1,95 mm
Trypsin-EDTA 1 X	Biowest	L0930
Tryton X100	Sigma	T8787