Authors would like to thank Dr. Andre Walder and Nicolas Perrin for manufacturing titanium implants, K. Benmlih for the build-up of the Teflon molds and Dr. G. Prevost for his help with animal experiments. We also acknowledge the Region Alsace and PMNA (Pole Materiaux et Nanosciences d'Alsace) for financial contribution.

1. Preparation of Micropore Gradients in Macroporous Metallic Implants
<ol>
	<li>Clean the implants (such as implants made of medical grade titanium beads with a size range of 400-500 &mu;m, Neyco SAS, France) with ethanol and then sonicate in acetone for 15 min.</li>
	<li>Design and manufacture Teflon molds according to the implant size and shape (For standard experiments, cylindrical molds of 1.5 cm diameter with a height of 2 cm are used). Molds should be modular, so that the certain parts can be removed during extr...

<ol>
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P., 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=Bone+ingrowth+in+porous+titanium+implants+produced+by+3D+fiber+deposition.">Bone ingrowth in porous titanium implants produced by 3D fiber deposition.</a> Biomaterials. 28, 2810-2820 (2007).</li><li>Schultz, P., 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=Polyelectrolyte+multilayers+functionalized+by+a+synthetic+analogue+of+an+anti-inflammatory+peptide,+alpha-MSH,+for+coating+a+tracheal+prosthesis.">Polyelectrolyte multilayers functionalized by a synthetic analogue of an anti-inflammatory peptide, alpha-MSH, for coating a tracheal prosthesis.</a> Biomaterials. 26, 2621-2630 (2005).</li><li>M&#252;ller, S., 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=VEGF-Functionalized+Polyelectrolyte+Multilayers+as+Proangiogenic+Prosthetic+Coatings.">VEGF-Functionalized Polyelectrolyte Multilayers as Proangiogenic Prosthetic Coatings.</a> Advanced Functional Materials. 18, 1767-1775 (2008).</li><li>Mills, R. 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NE Vrana is an employee of Protip SAS.

Pore gradients are important tools in interface tissue engineering and the system described here can be used alone or in conjunction with metallic implants to form pore gradient to study cell migration. The system does not necessitate any extra setting or extra equipment except a chemical fume hood to handle organic solvents, thus it can be applied in biology laboratories. Similar polymers such as Poly(glycolic acid) (PGA), Poly(lactic-co-glycolic) acid (PLGA) and Poly(caprolactone) (PCL) can be used with slight modifica...

Currently available metallic implants are suitable for load-bearing applications, but their non-degradability necessitates designs which ensure a strong interface with the tissue surrounding them 1. By providing structures that facilitate cellular in-growth and colonization in vivo, the lifetime of metallic implants can be prolonged 2. Openly porous metallic implants are promising materials for tissue interface engineering and also for ensuring good colonization of the implants. They have been actively used as orthopedic implants and also as tracheal implants 3-5. However, there are still problems that need to be solved such as the precise control over cell movement in the pore areas. Failure to control this process might lead to incomplete colonization in one end and restenosis in the other. Also further functionalization of these implants is necessary for achieving higher functions such as, delivery of growth factors, directed vascularization and simultaneous movement of different cell types 6-8. For tracheal implants, this is crucial as the colonization of the implant by a vascularized tissue is desirable. However, the uncontrolled tissue in-growth to the lumen of trachea is undesirable because it decreases implant patency.
One possibility to control cell movement is size exclusion. By knowing the size of the target cells and their ability to interact with a given synthetic polymer it is possible to develop gradients of pores which can effectively determine the depth of cell movement. For example by creating a pore architecture that is large enough for the entry of connective tissue cells such as fibroblasts extraluminally, but small enough (less than 10 &mu;m) to prevent their movement intraluminally an effective control over colonization of a tubular implant can be achieved.
From available pore creation methods such as freeze-drying, particle leaching, gas foaming 9,10; the easiest to adapt method for fast formation of pore gradients with minimal amount of necessary equipments is freeze-extraction 11. In this method, a polymer solution is frozen in a binary mixture of an organic solvent and water. Afterwards, the solvent is exchanged via extraction by a miscible pre-chilled liquid such as ethanol. Freezing and extraction conditions determine the shape and size of the pores and if the extraction is done in a way where the movement of the extraction solution can be controlled, pore size and shape can be directionally modulated.
Second step for multicellular tissues is the formation of porous barriers between different cell types to control their interaction. This is also necessary for the availability of different microenvironments for different cell types depending on their requirements 12,13. Trachea is a tubular organ that connects larynx with bronchi. It has an inner pseudostratified ciliary epithelium lining with interdispersed goblet cells which produce mucus. The 3D structure and stability of trachea is maintained by cartilage in the shape of C-rings. Thus, in an artificial trachea there should be a defined junction between the connective tissue and the ciliary epithelial layer. While a 3D structure is necessary for the connective tissue part, the migration of epithelial cells requires a basement membrane-like surface to achieve directional movement and closure of the wound. Polyelectrolyte multilayer films (PEMs) are one possible option to obtain basement membrane mimics. Layer-by-layer method (LbL) is a versatile process to obtain thin and functional surface coatings. It is based on electrostatic interactions of two oppositely charged polyelectrolytes and their build-up in a sequential manner to obtain nanoscale surface coatings whose properties can be varied by simply changing variables such as polyelectrolyte species, pH, layer number, addition of a capping layer, crosslinking etc. One of the main advantages of the LbL method is its ability to conform to the topography of the underlying substrate. Thus, under controlled conditions this method can also be used for obtaining surface coverage of porous structures. If collagen is used as one of the polyelectrolytes it is possible to obtain nanofibrillar structures that can mimic the surface of basement membrane. The hydrophobicity of titanium enables development of such structures and fibrillarity can be preserved in thick coatings 14. This way attachment and movement of cell on the surface can also be controlled. By using freeze-extraction and LbL film coating sequentially, a structure where cell movement can be controlled laterally, longitudinally and circumferentially can be obtained 15.
Here we describe two novel modification methods for titanium implants by using their hydrophobic behavior which can be extended to modification of various porous implants: i) formation of gradients of micropores within the macroporous titanium implants with hydrophobic, synthetic polymers ii) formation of a thick polymeric film layer on the implant surface that supports cell growth and lining formation by polyelectrolyte multilayers. These methods can be used sequentially or separately. They provide structures that ensure controlled migration and spatial organization of different cell types in multicellular tissues 16,17. For the specific case of trachea, the desired outcome for the implant would be the colonization by fibrovascular tissue within the micropore gradients without restenosis and the formation of the inner lining of ciliated epithelial cells on the polyelectrolyte multilayers.
One way of controlling integration of implants is to do small surgical interventions during the period of their integration with the host in situ. In order to be able to decide on the timing of the interventions, it is important to have information on the systemic effects of the implant. C-Reactive Protein (CRP) has been used for monitoring of infection and inflammatory response in clinical settings. Chromogranin A (CGA) can also be used in a similar manner and might provide more accurate results to observe the level of inflammation 18. As a possible way of observing metallic implant integration in vivo, we present a continuous monitoring procedure of implant systemic effects by characterization of animal blood samples with High Pressure Liquid Chromatography (HPLC) and subsequent protein sequencing. Elaboration of this method can be used to evade regular end-point histological analysis. Histological cutting of metallic implants is a long, cumbersome and expensive process and can be only undertaken at specific time points. Because of this reason, well-designed blood tests providing robust information about the implant health would be possible routes to decrease animal experiments as mandated by the recent EU rules concerning animal experiments.
The methods presented here can be used to improve the performance of metallic implants via functionalization or to have an alternative way of monitoring the existing implants.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Dioxane</td>
 <td>Sigma-Aldrich</td>
 <td>360481</td>
 <td>Toxic material, Strictly under chemical hood</td>
 </tr>
 <tr>
 <td>PLLA 
 i. Poly(L-lactide) inherent viscosity ~0.5 dl/g 
 ii. Poly(L-lactide) inherent viscosity ~2.0 dl/g 
 </td>
 <td>Sigma-Aldrich</td>
 <td>94829, 81273</td>
 <td>The choice of molecular weight and inherent viscosity is application dependent.</td>
 </tr>
 <tr>
 <td>PRONOVA UP LVG (Sodium Alginate)</td>
 <td>Novamatrix</td>
 <td>4200006</td>
 <td>Low viscosity(20-200 mPa.s)</td>
 </tr>
 <tr>
 <td>Collagen type I (Bovine)</td>
 <td>Symatese</td>
 <td>CBPE2US100</td>
 <td></td>
 </tr>
 <tr>
 <td>Pen/Strep, Fungizone </td>
 <td>Promocell</td>
 <td>C42020</td>
 <td></td>
 </tr>
 <tr>
 <td>Genipin</td>
 <td>Wako</td>
 <td>0703021</td>
 <td></td>
 </tr>
 <tr>
 <td>Silicon nitride probes with aspring constant of 0.03 N.m-1. </td>
 <td>Bruker </td>
 <td>MSCT </td>
 <td></td>
 </tr>
 <tr>
 <td>Trifluoroacetic acid for HPLC ,&ge;99.0% </td>
 <td>Sigma-Aldrich</td>
 <td>302031</td>
 <td>Hazardous Material, Please follow MSDS carefully</td>
 </tr>
 <tr>
 <td>Acetonitrile, for HPLC ,&ge;99.9% </td>
 <td>Sigma-Aldrich</td>
 <td>34998</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcein-AM</td>
 <td>Invitrogen</td>
 <td>C3100MP </td>
 <td></td>
 </tr>
 <tr>
 <td>PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling </td>
 <td>Sigma-Aldrich</td>
 <td>PKH26GL</td>
 <td></td>
 </tr>
 <tr>
 <td>Rabbit C-Reactive Protein (CRP) ELISA kit</td>
 <td>Genway Bio</td>
 <td>GWB-9BF960 </td>
 <td></td>
 </tr>
 <tr>
 <td>DMSO, Bioreagent, &ge;99.7% </td>
 <td>Sigma-Aldrich</td>
 <td>D2650</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Multimode Nanoscope IV Atomic Force microscope</td>
 <td>Bruker</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Procise microsequencer </td>
 <td>Applied Biosystems </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ultima 3000 HPLC system </td>
 <td>Dionex</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Scanning Electron Microscope Hitachi TM 100</td>
 <td>Hitachi </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Confocal Scanning Laser Microscope Zeiss LSM 510 </td>
 <td>Zeiss </td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
 </table>
Table 1. List of Materials and Reagents.

multi-scale modification of metallic implants with pore gradients, polyelectrolytes and their indirect monitoring in vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Formation of pore gradients
By changing the concentration of the PLLA solution, it is possible to control the size of the pores on the extraluminal side of the implants. Pore size and shape was significantly affected by the presence of titanium implants (Figures 1a and 1b). Pore sizes ranged from 40-100 &mu;m and utilization of lower concentrations resulted in smaller pores. Whereas, in the intraluminal side pore size was governed by the restricted extraction and...

Watch this Scientific Journal Video about Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo at JoVE.com

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

multi-scale-modification-metallic-implants-with-pore-gradients

Research

JoVE Journal

Bioengineering

10.8K visualizações.  INSERM. Neste vídeo, vamos demonstrar técnicas de modificação de implantes metálicos porosos para melhorar a sua funcionalidade e para controlar a migração celular. As técnicas incluem o desenvolvimento de gradientes de poros para controlar o movimento de células em 3D e a produção da membrana basal imita para controlar o movimento de células em 2-D. Além disso, um método baseado em HPLC para o acompanhamento da integração do implante in vivo através de análise ...