This research was funded by Instituto de Salud Carlos III, Ministerio de Econom&#237;a y Competitividad, co-funded by the ESF European Social Fund and the ERDF European Regional Development Fund (MS16/00124; CP16/00124; PI17/01605), the Direcci&#243; General d&#39;Investigaci&#243;, Conselleria d&#39;Investigaci&#243;, Govern Balear (FPI/2046/2017), and PROGRAMA JUNIOR del projecte TALENT PLUS, construyendo SALUD, generando VALOR (JUNIOR01/18), financed by the sustainable tourism tax of the Balearic Islands.

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
<ol>
	<li>Larger bodies removal
	<ol>
		<li>Thaw PL at room temperature.</li>
		<li>Centrifuge PL at 1,500 x g for 15 min at 4 &#176;C. Discard the pellet as it contains ...

<ol>
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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+a+nanostructured+titanium+oxide+layer+with+self-assembled+monolayers+for+biomedical+applications:+Surface+properties+and+biomechanical+behaviors.">The potential of a nanostructured titanium oxide layer with self-assembled monolayers for biomedical applications: Surface properties and biomechanical behaviors.</a> Applied Sciences. 10 (2), 590 (2020).</li><li>Jemat, A., Ghazali, M. J., Razali, M., Otsuka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modifications+and+their+effects+on+titanium+dental+implants.">Surface modifications and their effects on titanium dental implants.</a> BioMed Research International. 2015, 791725 (2015).</li><li>Damiati, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+surface+topography+and+coating+on+osteogenesis+and+bacterial+attachment+on+titanium+implants.">Impact of surface topography and coating on osteogenesis and bacterial attachment on titanium implants.</a> Journal of Tissue Engineering. 9, 2041731418790694 (2017).</li><li>Chen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembled+human+adipose-derived+stem+cell-derived+extracellular+vesicle-functionalized+biotin-doped+polypyrrole+titanium+with+long-term+stability+and+potential+osteoinductive+ability.">Self-assembled human adipose-derived stem cell-derived extracellular vesicle-functionalized biotin-doped polypyrrole titanium with long-term stability and potential osteoinductive ability.</a> ACS Applied Materials &amp; Interfaces. 11 (49), 46183-46196 (2019).</li><li>Wei, F., Li, M., Crawford, R., Zhou, Y., Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome-integrated+titanium+oxide+nanotubes+for+targeted+bone+regeneration.">Exosome-integrated titanium oxide nanotubes for targeted bone regeneration.</a> Acta Biomaterialia. 86, 480-492 (2019).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+influence+the+behavior+of+human+mesenchymal+stem+cells+on+titanium+surfaces.">Exosomes influence the behavior of human mesenchymal stem cells on titanium surfaces.</a> Biomaterials. 230, 119571 (2020).</li><li>Lozano-Ramos, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-exclusion+chromatography-based+enrichment+of+extracellular+vesicles+from+urine+samples.">Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples.</a> Journal of Extracellular Vesicles. 4, 27369 (2015).</li><li>Th&#233;ry, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+information+for+studies+of+extracellular+vesicles+2018+(MISEV2018):+a+position+statement+of+the+International+Society+for+Extracellular+Vesicles+and+update+of+the+MISEV2014+guidelines.">Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.</a> Journal of Extracellular Vesicles. 7 (1), 1535750 (2018).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+extracellular+vesicles+from+adult+schistosoma+japonicum.">Isolation and characterization of extracellular vesicles from adult schistosoma japonicum.</a> Journal of Visualized Experiments: JoVE. (135), e57541 (2018).</li><li>JoVE. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Methods+in+Cellular+and+Molecular+Biology.+Using+a+Hemacytometer+to+Count+Cells.">Basic Methods in Cellular and Molecular Biology. Using a Hemacytometer to Count Cells.</a> JoVE Science Education Database. , (2021).</li><li>Chouirfa, H., Bouloussa, H., Migonney, V., Falentin-Daudr&#233;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+titanium+surface+modification+techniques+and+coatings+for+antibacterial+applications.">Review of titanium surface modification techniques and coatings for antibacterial applications.</a> Acta Biomaterialia. 83, 37-54 (2019).</li><li>C&#243;rdoba, A., Monjo, M., Hierro-Oliva, M., Gonz&#225;lez-Mart&#237;n, M. L., Ramis, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinspired+quercitrin+nanocoatings:+A+fluorescence-based+method+for+their+surface+quantification,+and+their+effect+on+stem+cell+adhesion+and+differentiation+to+the+osteoblastic+lineage.">Bioinspired quercitrin nanocoatings: A fluorescence-based method for their surface quantification, and their effect on stem cell adhesion and differentiation to the osteoblastic lineage.</a> ACS Applied Materials and Interfaces. 7 (30), 16857-16864 (2015).</li></ol>

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</su...

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 profile2,3. Therefore, further research is required to explore the possibility of combining EVs with biomaterials in order to use them in daily clinical practice.
Titanium-based biomaterials are widely used as scaffolds for bone healing clinical interventions due to their mechanical properties, high biocompatibility, and long-term durability4. Nevertheless, Ti implants are a bioinert material and, therefore, present a poor capability for bonding with the surrounding bone tissue5. For this reason, titanium modifications are being studied in order to improve their performance by achieving a more functional microenvironment on its surface4,6,7. In this sense, EVs can be anchored to titanium by chemical8 or physical interactions9,10. Immobilized EVs derived from stem cells or macrophages enhance the bioactivity of Ti by promoting cellular adhesion and proliferation thereby inducing an osteogenic effect8,9,10.
This article will focus on a drop casting strategy for coating Ti surfaces with PL-derived EVs in detail. In addition, we will evaluate EVs release profile from the coated surface over time and confirm its cellular biocompatibility in vitro.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0,8 &#181;m syringe filter</td>
			<td>Sartorius</td>
			<td>16592K</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Centrifuge tube</td>
			<td>SPL life sciences</td>
			<td>PLC60015</td>
			<td></td>
		</tr>
		<tr>
			<td>1mL syringe</td>
			<td>BD</td>
			<td>303174</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well culture plate</td>
			<td>SPL life sciences</td>
			<td>PLC30096</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolut ethanol</td>
			<td>Scharlau</td>
			<td>ET0006005P</td>
			<td>Used to prepare 20 %&#160; ethanol with Milli-Q&#174; water</td>
		</tr>
		<tr>
			<td>AKTA purifier System</td>
			<td>GE Healthcare</td>
			<td>8149-30-0014</td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra X-15R Centrifuge</td>
			<td>Beckman Coutler</td>
			<td>392934</td>
			<td>SX4750A swinging rotor</td>
		</tr>
		<tr>
			<td>Centrifuge 5430 R</td>
			<td>Eppendorf</td>
			<td>5428000210</td>
			<td>F-45-48-11 rotor</td>
		</tr>
		<tr>
			<td>Conical Tube, Conical Bottom, 50ml</td>
			<td>SPL life sciences</td>
			<td>PLC50050</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytotoxicity Detection Kit (LDH)</td>
			<td>Roche</td>
			<td>11644793001</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Syringes 10 ml</td>
			<td>Becton Dickinson</td>
			<td>BDH307736</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Low Glucose Glutamax</td>
			<td>GIBCO</td>
			<td>21885025</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS (1x)</td>
			<td>Capricorn Scientific</td>
			<td>PBS-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS) Embrionic Certified</td>
			<td>GIBCO</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Filtropur S 0.2 &#181;m syringe filter</td>
			<td>Sarstedt</td>
			<td>83.1826.001</td>
			<td></td>
		</tr>
		<tr>
			<td>HiPrep&#160;16/60 Sephacryl&#160;S-400 HR</td>
			<td>GE Healthcare</td>
			<td>28-9356-04</td>
			<td>Precast columns</td>
		</tr>
		<tr>
			<td>human umbilical cord-derived mesenchymal stem cells (hUC-MSC)</td>
			<td>IdISBa Biobank</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop 2000 spectrophotometer</td>
			<td>ThermoFisher</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoSight NS300 nanoparticle tracking analysis</td>
			<td>Malvern</td>
			<td>NS300</td>
			<td>Device with embedded laser at &#955;= 532 nm and camera sCMOS</td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>Terumo</td>
			<td>946077135</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitric acid 69,5%</td>
			<td>Scharlau</td>
			<td>AC16071000</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima L-100 XP Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>8043-30-1124</td>
			<td>SW-32Ti Rotor</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution 100X</td>
			<td>Biowest</td>
			<td>L0022</td>
			<td></td>
		</tr>
		<tr>
			<td>pH Test strips 4.5-10.0</td>
			<td>Sigma</td>
			<td>P-4536</td>
			<td></td>
		</tr>
		<tr>
			<td>Platelet Lysate (PL)</td>
			<td>IdISBa Biobank</td>
			<td></td>
			<td>Obtained from&#160; buffy coats discarded after blood donation</td>
		</tr>
		<tr>
			<td>Polypropylene centrifuge tubs</td>
			<td>Beckman Coutler</td>
			<td>326823</td>
			<td></td>
		</tr>
		<tr>
			<td>Power wave HT</td>
			<td>BioTek</td>
			<td>10340763</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw cap tube, 15 ml, (Lx&#216;): 120 x 17 mm, PP, with print</td>
			<td>Sarstedt</td>
			<td>62554502</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hidroxide</td>
			<td>Sharlau</td>
			<td>SO04251000</td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium implants replicas</td>
			<td>Implantmedia, SA</td>
			<td>NA</td>
			<td>Titanium grade IV. Diameter: 6,2 mm. Height: 1,95 mm</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 1 X</td>
			<td>Biowest</td>
			<td>L0930</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryton X100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
	</tbody>
</table>

platelet-derived extracellular vesicle functionalization of ti implants

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.

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...

Watch this Scientific Journal Video about Platelet-Derived Extracellular Vesicle Functionalization of Ti Implants at JoVE.com

Platelet-Derived Extracellular Vesicle Functionalization of Ti Implants

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.

Extracellular Vesicles (EVs) are biological nanovesicles that play a key role in cell communication. Their content includes active biomolecules such as ...

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.

platelet-derived-extracellular-vesicle-functionalization-ti

Research

JoVE Journal

Bioengineering

2.2K Views.  University of the Balearic Islands. 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 be...