3T3 Swiss albino mouse cells are often used in biotechnological applications. These cells can grow adherently on suitable surfaces. In our study, they were grown on different titanium substrates, comparing commercially available titanium sheets of grade 1 and grade 2, respectively, with Ti64 which was 3D printed with different porosity in order to identify potential substitutes for common well-plates, which could - in case of 3D printed substrates - be produced in various shapes and dimensions and thus broaden the range of substrates for cell growth in biotechnology and tissue engineering. In addition, thin layers of poly(acrylonitrile) (PAN) nanofibers were electrospun on these substrates to add a nanostructure. The common titanium sheets showed lower cell cover factors than common well plates, which could not be improved by the thin nanofibrous coating. However, the Ti sheets with nanofiber mat coatings showed higher cell adhesion and proliferation than pure PAN nanofiber mats. The 3D printed Ti64 substrates prepared by laser metal fusion, on the other hand, enabled significantly higher proliferation of (66 ± 8)% cover factor after three days of cell growth than well plates which are usually applied as the gold standard for cell cultivation ((48 ± 11)% cover factor under identical conditions). Especially the Ti64 samples with higher porosity showed high cell adhesion and proliferation. Our study suggests investigating such porous Ti64 samples further as a potential future optimum for cell adhesion and proliferation.

Department of Machining Assembly and Engineering Metrology Faculty of Mechanical Engineering VSB Technical University of Ostrava 708 00 Ostrava Poruba Czech Republic

Faculty of Engineering and Mathematics Bielefeld University of Applied Sciences and Arts 33619 Bielefeld Germany

Faculty of Mechatronics and Mechanical Engineering Kielce University of Technology 25 314 Kielce Poland

Zobrazit více v PubMed

Trizio I., Rizzi V., Gristina R., Sardella E., Cosma P., Francioso E., von Woedtke T., Favia P. Plasma generated RONS in cell culture medium for in vitro studies of eukaryotic cells on Tissue Engineering scaffolds. Plasma Process. Polym. 2017;14

Przekora A. The summary of the most important cell-biomaterial interactions that need to be considered during in vitro biocompatibility testing of bone scaffolds for tissue engineering applications. Mater. Sci. Eng. C. 2019;97:1036–1051. PubMed

Lücking T.H., Sambale F., Schnaars B., Bulnes-Abundis D., Beutel S., Scheper T. 3D-printed individual labware in biosciences by rapid prototyping: in vitro biocompatibility and applications for eukaryotic cell cultures. Eng. Life Sci. 2015;15:57–64.

Tegel H., Dannemeyer M., Kanje S., et al. High throughput generation of a resource of the human secretome in mammalian cells. N. Biotech. 2020;58:45–54. PubMed

Sun T., Kwok W.C., Chua K.J., Lo T.-M., Potter J., Yew W.S., Chesnut J.D., Hwang I.Y., Chang M.W. Development of a proline-based selection system for reliable genetic engineering in Chinese hamster ovary cells. ACS Synth. Biol. 2020;9:1864–1872. PubMed

Wells E., Song L.Q., Greer M., Luo Y., Kurian V., Ogunaike B., Robinson A.S. Media supplementation for targeted manipulation of monoclonal antibody galactosylation and fucosylation. Biotechnol. Bioeng. 2020;117:3310–3321. PubMed

Musini A., Pokala B., Zahoorulah S.M.D., Giri A. Cytotoxicity of Salacia oblonga extracts against cancer and normal cells and isolation of bioactive compounds. Res. J. Biotechnol. 2020;15:105–109.

Wang Y.X., Puthussery J.V., Yu H.R., Verma V. Synergistic and antagonistic interactions among organic and metallic components of the ambient particulate matter (PM) for the cytotoxicity measured by Chinese hamster ovary cells. Sci. Total Environ. 2020;736 PubMed

Louie S., Lakkyreddy J., Castellano B.M., et al. Insulin degrading enzyme (IDE) expressed by Chinese hamster ovary (CHO) cells is responsible for degradation of insulin in culture media. J. Biotechnol. 2020;320:44–49. PubMed

Chatel A. A brief history of adherent cell culture: where we come from and where we should go. BioProcess Int. 2019;17:44–49.

Richardson S.A., Rawlings T.M., Muter J., Walker M., Brosens J.J., Cameron N.R., Eissa A.M. Covalent attachment of fibronectin onto emulsion-templated porous polymer scaffolds enhances human endometrial stromal cell adhesion, infiltration and function. Macromol. Biosci. 2019;19 PubMed

Gstraunthaler G., Lindl T. seventh ed. Springer; Berlin: 2013. Zell- und Gewebekultur.

Whitford W.G., Hardy J.C., Cadwell J.J.S. Single-use, continuous processing of primary stem cells. BioProcess Int. 2014;12:26–32.

Simon M. Bioreactor design for adherent cell culture. The bolt-on bioreactor project, part 1: volumetric productivity. BioProcess Int. 2015;13:28–33.

YekrangSafakar A., Hamel K.M., Mehrnezhad A., Jung J.W., Park P., D K. Development of rolled scaffold for high-density adherent cell culture. Biomed. Microdevices. 2020;22:4. PubMed

Meuwly F., Papp F., Ruffieux P.A., Bernard A.R., Kadouri A., von Stockar U. Use of glucose consumption rate (GCR) as a tool to monitor and control animal cell production processes in packed-bed bioreactors. J. Biotechnol. 2006;122:122–129. PubMed

Meuwly F., Loviat F., Ruffieux P.A., Bernard A.R., Kadouri A., von Stockar U. Oxygen supply for CHO cells immobilized on a packed-bed of Fibra-Cel(R) disks. Biotechnol. Bioeng. 2006;93:791–800. PubMed

Sölmann S., Rattenholl A., Blattner H., Ehrmann G., Gudermann F., Lütkemeyer D., Ehrmann A. Mammalian cell adhesion on different 3D printed polymers with varying sterilization methods and acidic treatment. AIMS Bioeng. 2020;8:25–35.

Li X.Y., Wang Y., Guo M., Wang Z.L., Shao N.N., Zhang P.B., Chen X.S., Huang Y.B. Degradable three dimensional-printed polylactic acid scaffold with long-term antibacterial activity. ACS Sustainable Chem. Eng. 2018;6:2047–2054.

Melcová V., Svoradová K., Mencik P., Kontárová S., Rampichová M., Hedvicáková V., Sovková V., Prikryl R., Vojtova L. FDM 3D printed composites for bone tissue engineering based on plasticized poly(3-hydroxybutyrate)/poly(d,l-lactide) blends. Polymers. 2020;12:2806. PubMed PMC

Kreß S., Schaller-Ammann R., Feiel J., Priedl J., Kasper C., Egger D. 3D printing of cell culture devices: assessment and prevention of the cytotoxicity of photopolymers for stereolithography. Materials. 2020;13:3011. PubMed PMC

Mamun A. Review of possible applications of nanofibrous mats for wound dressings. Tekstilec. 2019;62:89–100.

Aljawish A., Muniglia L., Chevalot I. Growth of human mesenchymal stem cells (MSCs) on films of enzymatically modified chitosan. Biotechnol. Prog. 2016;32:491–500. PubMed

Klinkhammer K., Seiler N., Grafahrend D., Gerardo-Nava J., Mey J., Brook G.A., Möller M., Dalton P.D., Klee D. Deposition of electrospun fibers on reactive substrates for in vitro investigations. Tissue Eng. Part C. 2009;15:77–85. PubMed

Ehrmann A. Non-toxic crosslinking of electrospun gelatin nanofibers for tissue engineering and biomedicine—a review. Polymers. 2021;13:1973. PubMed PMC

Wortmann M., Freese N., Sabantina L., Petkau R., Kinzel F., Gölzhäuser A., Moritzer E., Hüsgen B., Ehrmann A. New polymers for needleless electrospinning from low-toxic solvents. Nanomaterials. 2019;9:52. PubMed PMC

Kyrylenko S., Kornienko V., Gogotsi O., Oleshko O., Kolesnyk M., Mishchenko O., Zahorodna V., Buranich V., Pogrebnjak A., Zozulia Y., Balitskyi V., Pogorielov M., Baginskiy I. Sumy; Ukraine: 2020. Bio-functionalization of Electrospun Polymeric Nanofibers by Ti3C2Tx MXene. 2020 IEEE 10th International Conference Nanomaterials: Applications & Properties (NAP) 02BA10-1-02BA10-5.

Wehlage D., Blattner H., Sabantina L., Böttjer R., Grothe T., Rattenholl A., Gudermann F., Lütkemeyer D., Ehrmann A. Sterilization of PAN/gelatine nanofibrous mats for cell growth. Tekstilec. 2019;62:78–88.

Wehlage D., Blattner H., Mamun A., Kutzli I., Diestelhorst E., Rattenholl A., Gudermann F., Lütkemeyer D., Ehrmann A. Cell growth on electrospun nanofiber mats from polyacrylonitrile (PAN) blends. AIMS Bioeng. 2020;7:43–54.

Lakra R., Kiran M.S., Korrapati P.S. Electrospun gelatin–polyethylenimine blend nanofibrous scaffold for biomedical applications. J. Mater. Sci. Mater. Med. 2019;30:129. PubMed

Malik S., Khan A., Ali N., Ali F., Rahdar A., Mulla S.I., Nguyen T.A., Bilal M. Elsevier; 2022. Electrospun Cellulose Composite Nanofibers and Their Biotechnological Applications. Nanotechnology in Paper and Wood Engineering; pp. 329–348.

Ajalli N., Pourmadadi M., Yazdian F., Abdouss M., Rashedi H., Rahdar A. PVA based nanofiber containing GO modified with Cu nanoparticles and loaded curcumin; high antibacterial activity with acceleration wound healing. Curr. Drug Deliv. 2023;20:1569–1583. PubMed

Norouzi F., Pourmadadi M., Yazdian F., Khoshmaram K., Mohammadnejad J., Sanati M.H., Chogan F., Rahdar A., Baino F. PVA-based nanofibers containing chitosan modified with graphene oxide and carbon quantum dot-doped TiO2 enhance wound healing in a rat model. J. Funct. Biomater. 2022;13:300. PubMed PMC

Foroushani P.H., Rahmani E., Alemzadeh I., Vossoughi M., Purmadadi M., Rahdar A., Díez-Pascual A.M. Curcumin sustained release with a hybrid chitosan-silk fibroin nanofiber containing silver nanoparticles as a novel highly efficient antibacterial wound dressing. Nanomaterials. 2022;12:3426. PubMed PMC

Rahmani E., purmadadi M., Zandi N., Rahdar A., Baino F. pH-responsive PVA-based nanofibers containing GO modified with Ag nanoparticles: physico-chemical characterization, wound dressing, and drug delivery. Micromachines. 2022;13:1847. PubMed PMC

Adell R., Eriksson B., Lekholm U., Branemark P.I., Jemt T. Long-term follow-up study of osseointegrated implants in the treatment of totally edentulous jaws. Int. J. Oral Maxillofac. Implants. 1990;5:347–359. PubMed

Ahn T.-K., Lee D.H., Kim T.-s., Jang G.C., Choi S.J., Oh J.B., Ye G.H., Lee S.C. In: Novel Biomaterials for Regenerative Medicine. Chun H., Park K., Kim C.H., Khang G., editors. Springer; Singapore: 2023. Modification of titanium implant and titanium dioxide for bone tissue engineering; pp. 355–368.

Lodish H., Berk A., Kaiser C.A., Krieger M., Scott M.P., Bretscher A., Ploegh H., Matsudaira P. W. H. Freeman and Company; New York: 2008. Molecular Cell Biology.

Kobayakawa J., Sato-Nishimori F., Moriyasu M., Matsukawa Y. G2-M arrest and antimitotic activity mediated by casticin, a flavonoid isolated from Viticis Fructus (Vitex rotundifolia Linne fil.) Cancer Lett. 2004;208:59–64. PubMed

Ölschläger V., Schrader A., Hockertz S. Comparison of primary human fibroblasts and keratinocytes with immortalized cell lines regarding their sensitivity to sodium dodecyl sulfate in a neutral red uptake cytotoxicity assay. Arzneimittelforschung. 2009;59:146–152. PubMed

Fronza M., Heinzmann B., Hamburger M., Laufer S., Merfort I. Determination of the wound healing effect of Calendula extracts using the scratch assay with 3T3 fibroblasts. J. Ethnopharmacol. 2009;126:463–467. PubMed

Evek GmbH Titanband Grade 1. 2023. https://evek.top/37-titan?en=titan-grade-1

Evek GmbH itanblech Grade 2 0.5mm Titanplatte 3.7035 Zuschnitt 100 mm bis 2000 mm. 2023. https://evek.top/titan/699-titanblech-grade-2-37035-05mm-4066435009731.html

Trumpf Additive Manufacturing: Pulver und Parameter. Available online: https://www.trumpf.com/de_DE/produkte/maschinen-systeme/additive-fertigungssysteme/pulver-und-parameter/, last accessed August 20, 2023.

Surmeneva M.A., Khrapov D., Prosolov K., Kozadayeva M., Koptyug A., Volkova A., Paveleva A., Surmenev R.A. The influence of chemical etching on porous structure and mechanical properties of the Ti6AL4V Functionally Graded Porous Scaffolds fabricated by EBM. Mater. Chem. Phys. 2022;275

Khrapov D., Koptyug A., Manabaev K., Léonard F., Mishurova T., Bruno G., Cheneler D., Loza K., Epple M., Surmenev R., Surmeneva M. The impact of post manufacturing treatment of functionally graded Ti6Al4V scaffolds on their surface morphology and mechanical strength. J. Mater. Res. Technol. 2020;9:1866–1881.

Szymczyk-Ziólkowska P., Hoppe V., Rusinska M., Gasiorek J., Ziólkowski G., Dydak K., Czajkowska J., Junka A. The impact of EBM-manufactured Ti6Al4V ELI alloy surface modifications on cytotoxicity toward eukaryotic cells and microbial biofilm formation. Materials. 2020;13:2822. PubMed PMC

Majhy B., Priyadarshini P., Sen A.K. Effect of surface energy and roughness on cell adhesion and growth – facile surface modification for enhanced cell culture. RSC Adv. 2021;11:15467–15476. PubMed PMC

Lv Y.T., Wang B.H., Liu G.H., Tang Y.J., Lu E.Y., Xie K.G., Lan C.G., Liu J., Qin Z.B., Wang L.Q. Metal material, properties and design methods of porous biomedical scaffolds for additive manufacturing: a review. Front. Bioeng. Biotechnol. 2021;9 PubMed PMC

Ferreira F.V., Otoni C.G., De France K.J., Barud H.S., Lona L.M.F., Cranston E.D., Rojas O.J. Porous nanocellulose gels and foams: breakthrough status in the development of scaffolds for tissue engineering. Mater. Today. 2020;37:126–141.

Kozior T., Trabelsi M., Mamun A., Sabantina L., Ehrmann A. Stabilization of electrospun nanofiber mats used for filters by 3D printing. Polymers. 2019;11:1618. PubMed PMC

Storck J.L., Brockhagen B., Grothe T., Sabantina L., Kaltschmidt B., Tuvshinbayar K., Braun L., Tanzli E., Hütten A., Ehrmann A. Stabilization and Carbonization of PAN Nanofiber Mats Electrospun on Metal Substrates. C. 2021;7:12.

Wortmann M., Layland A.S., Frese N., Kahmann U., Grothe T., Storck J.L., Blachowicz T., Grzybowski J., Hüsgen B., Ehrmann A. On the reliability of highly magnified micrographs for structural analysis in materials science. Sci. Rep. 2020;10 PubMed PMC

Shuai C.J., Yuan X., Yang W.J., Peng S.P., Qian G.W., Zhao Z.Y. Synthesis of a mace-like cellulose nanocrystal@Ag nanosystem via in-situ growth for antibacterial activities of poly-L-lactide scaffold. Carbohydr. Polym. 2021;262 PubMed

Qi F.W., Gao X.W., Shuai Y., Peng S.P., Deng Y.W., Yang S., Yang Y.W., Shuai C.J. Magnetic-driven wireless electrical stimulation in a scaffold. Compos. B Eng. 2022;237

Yang W.J., Zhong Y.C., He C.X., Peng S.P., Yang Y.W., Qi F.W., Feng P., Shuai C.J. Electrostatic self-assembly of pFe3O4 nanoparticles on graphene oxide: a co-dispersed nanosystem reinforces PLLA scaffolds. J. Adv. Res. 2020;24:191–203. PubMed PMC