, Volume 69, Issue 4, pp 601–616 | Cite as

Optimization of ultraviolet ozone treatment process for improvement of polycaprolactone (PCL) microcarrier performance

  • Nurhusna Samsudin
  • Yumi Zuhanis Has-Yun HashimEmail author
  • Mohd Azmir Arifin
  • Maizirwan Mel
  • Hamzah Mohd. SallehEmail author
  • Iis Sopyan
  • Dzun Noraini Jimat
Original Article


Growing cells on microcarriers may have overcome the limitation of conventional cell culture system. However, the surface functionality of certain polymeric microcarriers for effective cell attachment and growth remains a challenge. Polycaprolactone (PCL), a biodegradable polymer has received considerable attention due to its good mechanical properties and degradation rate. The drawback is the non-polar hydrocarbon moiety which makes it not readily suitable for cell attachment. This report concerns the modification of PCL microcarrier surface (introduction of functional oxygen groups) using ultraviolet irradiation and ozone (UV/O3) system and investigation of the effects of ozone concentration, the amount of PCL and exposure time; where the optimum conditions were found to be at 60,110.52 ppm, 5.5 g PCL and 60 min, respectively. The optimum concentration of carboxyl group (COOH) absorbed on the surface was 1495.92 nmol/g and the amount of gelatin immobilized was 320 ± 0.9 µg/g on UV/O3 treated microcarriers as compared to the untreated (26.83 ± 3 µg/g) microcarriers. The absorption of functional oxygen groups on the surface and the immobilized gelatin was confirmed with the attenuated total reflectance Fourier transformed infrared spectroscopy (ATR-FTIR) and the enhancement of hydrophilicity of the surface was confirmed using water contact angle measurement which decreased (86.93°–49.34°) after UV/O3 treatment and subsequently after immobilization of gelatin. The attachment and growth kinetics for HaCaT skin keratinocyte cells showed that adhesion occurred much more rapidly for oxidized surfaces and gelatin immobilized surface as compared to untreated PCL.


Microcarrier Polycaprolactone (PCL) Ultra violet ozone (UV/O3Gelatin immobilization Surface modification 



The authors are grateful to the Ministry of Higher Education Malaysia, for financing the research project (PRGS 11-001-0001) under the Prototype Development Research Grant Scheme (PRGS) and to the Department of Biotechnology Engineering, International Islamic University Malaysia for their support.


  1. Anderson MJ, Whitcomb PJ (2015) DOE simplified: practical tools for effective experimentation. CRC Press, FloridaCrossRefGoogle Scholar
  2. Bock A, Sann H, Schulze-Horsel J, Genzel Y, Reichl U, Möhler L (2009) Growth behavior of number distributed adherent MDCK cells for optimization in microcarrier cultures. Biotechnol Progr 25:1717–1731. doi: 10.1002/btpr.262 Google Scholar
  3. Callen BW, Lowenberg BF, Lugowski S, Sodhi RN, Davies JE (1995) Nitric acid passivation of Ti6Al4V reduces thickness of surface oxide layer and increases trace element release. J Biomed Mater Res 29:279–290CrossRefGoogle Scholar
  4. Chen DR, Bei JZ, Wang SG (2000) Polycaprolactone microparticles and their biodegradation. Polym Degrad Stab 67:455–459CrossRefGoogle Scholar
  5. Clark JT, Ruiz JD, Fan H, Brinker CJ, Swanson BI, Parikh AN (2000) A new application of UV-ozone treatment in the preparation of substrate- supported mesoporous thin films. Chem Mater 12:3879–3884CrossRefGoogle Scholar
  6. Darain F, Chan WY, Chian KS (2010) Performance of surface-modified polycaprolactone on growth factor binding, release, and proliferation of smooth muscle cells. Soft Mater 9:64–78. doi: 10.1080/1539445X.2010.520797 CrossRefGoogle Scholar
  7. Davidson MR, Mitchell SA, Bradley RH (2004) UV-ozone modification of plasma-polymerised acetonitrile films for enhanced cell attachment. Colloids Surf B 34:213–219CrossRefGoogle Scholar
  8. Deyrieux AF, Wilson VG (2007) In vitro culture conditions to study keratinocyte differentiation using the HaCaT cell line. Cytotechnology 54:77–83. doi: 10.1007/s10616-007-9076-1 CrossRefGoogle Scholar
  9. Goh TK-P, Zhang Z-Y, Chen AK-L, Reuveny S, Choolani M, Chan JKY, Oh SK-W (2013) Microcarrier culture for efficient expansion and osteogenic differentiation of human fetal mesenchymal stem cells. BioRes Open Access 2:84–97. doi: 10.1089/biores.2013.0001 CrossRefGoogle Scholar
  10. Gomathi N, Neogi S (2009) Surface modification of polypropylene using argon plasma: statistical optimization of the process variables. Appl Surf Sci 255:7590–7600. doi: 10.1016/j.apsusc.2009.04.034 CrossRefGoogle Scholar
  11. Hinkelmann K, Kempthorne O (2008) Design of experiment: introduction to experimental design, vol 2. Wiley, New YorkGoogle Scholar
  12. Hong Y, Gao C, Xie Y, Gong Y, Shen J (2005) Collagen-coated polylactide microspheres as chondrocyte microcarriers. Biomaterials 26:6305–6313. doi: 10.1016/j.biomaterials.2005.03.038 CrossRefGoogle Scholar
  13. Jacobson BS, Ryan US (1982) Growth of endothelial and HeLa cells on a new multipurpose microcarrier that is positive, negative or collagen coated. Tissue Cell 14:69–83. doi: 10.1016/0040-8166(82)90008-8 CrossRefGoogle Scholar
  14. Kemala T, Budianto E, Soegiyono B (2012) Preparation and characterization of microspheres based on blend of poly(lactic acid) and poly(ε-caprolactone) with poly(vinyl alcohol) as emulsifier. Arab J Chem 5:103–108. doi: 10.1016/j.arabjc.2010.08.003 CrossRefGoogle Scholar
  15. Khan W, Kapoor M, Kumar N (2007) Covalent attachment of proteins to functionalized polypyrrole-coated metallic surfaces for improved biocompatibility. Acta Biomater 3:541–549CrossRefGoogle Scholar
  16. Kim W-J, Kim S, Lee BS, Kim A, Ah CS, Huh C, Yun WS (2009) Enhanced protein immobilization efficiency on a TiO2 surface modified with a hydroxyl functional group. Langmuir 25:11692–11697. doi: 10.1021/la901615e CrossRefGoogle Scholar
  17. Kwon Y, Coleman MA, Camarero JA (2006) Selective immobilization of proteins onto solid supports through split-intein mediated protein trans-splicing. Angew Chem Int Ed 45:1726–1729CrossRefGoogle Scholar
  18. Ma Z, Gao C, Ji J, Shen J (2002) Protein Immobilization on the surface of poly-l-lactic acid films for improvement of cellular interactions. Eur Polym J 38:2279–2284CrossRefGoogle Scholar
  19. Macmanus LF, Walzak MJ, Mcintyre NS (1999) Study of ultraviolet light and ozone surface modification of polypropylene. J Polym Sci, Part A Polym Chem 37(14):2489–2501. doi: 10.1002/(SICI)1099-0518(19990715)37:14<2489:AID-POLA23>3.0.CO;2-G CrossRefGoogle Scholar
  20. Maia JL, Santana MHA (2004) The effect of some processing conditions on the characteristics of biodegradable microspheres obtained by an emulsion solvent evaporation process. Braz J 21:1–12Google Scholar
  21. Mano JF, Silva GA, Azevedo HS, Malafaya PB, Sousa RA, Silva SS et al (2007) Natural origin biodegradable systems in tissue engineering and regenerative medicine: present status and some moving trends. J R Soc Interfac R Soc 4:999–1030. doi: 10.1098/rsif.2007.0220 CrossRefGoogle Scholar
  22. Mitchell S, Poulsson HC, Davidson MR, Emmison N, Shard G, Bradley RH (2004) Cellular attachment and spatial control of cells using micro-patterned ultra-violet/ozone treatment in serum enriched media. Biomaterials 25:4079–4086. doi: 10.1016/j.biomaterials.2003.11.010 CrossRefGoogle Scholar
  23. Murakami TN, Fukushima Y, Hirano Y (2003) Surface modification of polystyrene and poly (methyl methacrylate) by acti v e oxygen treatment. Colloid Surf B Biointerf 29:171–179CrossRefGoogle Scholar
  24. Murakami TN, Fukushima Y, Hirano Y, Tokuoka Y, Takahashi M, Kawashima N (2005) Modification of PS films by combined treatment of ozone aeration and UV irradiation in aqueous ammonia solution for the introduction of amine and amide groups on their surface. Appl Surf Sci 249:425–432. doi: 10.1016/j.apsusc.2004.12.017 CrossRefGoogle Scholar
  25. Prockop DJ, Udenfriend S (1960) A specific method for the analysis of hydroxyproline in tissues and urine. Anal Biochem 1:228–239CrossRefGoogle Scholar
  26. Ratner DR, Bryant JB (2004) Biomaterial: where we have been and where we are going. Annu Rev Biomed Eng 6:41–75CrossRefGoogle Scholar
  27. Sabino MA (2007) Oxidation of polycaprolactone to induce compatibility with other degradable polyesters. Polym Degrad Stab 92:986–996. doi: 10.1016/j.polymdegradstab.2007.03.010 CrossRefGoogle Scholar
  28. Seland H, Gustafson C-J, Johnson H, Junker JPE, Kratz G (2011) Transplantation of acellular dermis and keratinocytes cultured on porous biodegradable microcarriers into full-thickness skin injuries on athymic rats. Burns J Int Soc Burn Inj 37:99–108. doi: 10.1016/j.burns.2010.03.014 CrossRefGoogle Scholar
  29. Shen H, Hu X, Yang F, Bei J, Wang S (2007) Combining oxygen plasma treatment with anchorage of cationized gelatin for enhancing cell affinity of poly(lactide-co-glycolide). Biomaterials 28:4219–4230CrossRefGoogle Scholar
  30. Steynberg T, Visagie M, Mqoco T, Idicula A, Moolman S, Richter W, Joubert A (2012) Qualitative assessment of smooth muscle cells propagated on 2D-and 3D- polycaprolactone polymers via scanning electron microscope. Biomed Res 23:191–198Google Scholar
  31. Teare DOH, Emmison N, Bradley RH (2000) Cellular attachment to ultraviolet ozone modified polystyrene surfaces. Langmuir 16:2818–2824. doi: 10.1021/la9907533 CrossRefGoogle Scholar
  32. Teare DOH, Emmison N, Ton-That C, Bradley RH (2001) Effects of serum on the kinetics of CHO attachment to ultraviolet-ozone modified polystyrene surfaces. J Colloid Interface Sci 234:84–89. doi: 10.1006/jcis.2000.7282 CrossRefGoogle Scholar
  33. van der Velden-de Groot C (1995) Microcarrier technology, present status and perspective. Cytotechnology 18:51–56. doi: 10.1007/BF00744319 CrossRefGoogle Scholar
  34. Wu C (2002) Performance of an acrylic acid grafted polycaprolactone/starch composite: characterization and mechanical properties. J Appl Polym Sci 88:2888–2895Google Scholar
  35. Yanagisawa K, Murakami TN, Tokuoka Y, Ochiai A, Takahashi M, Kawashima N (2006) Immobilization and enzymatic activity of glucose oxidase on polystyrene surface modified with ozone aeration and UV irradiation in distilled water and/or aqueous ammonia solution. Colloids Surf B Biointerf 48:67–71. doi: 10.1016/j.colsurfb.2006.01.008 CrossRefGoogle Scholar
  36. Yang Y, Porte M, Marmey P, El Hai AJ, Amedee J, Baquey C (2003) Covalent bonding of collagen on poly(L-lactic acid) by gamma irradiation. Nucl Instrum Methods 207:165–174CrossRefGoogle Scholar
  37. Yuan S, Xiong G, Roguin A, Choong C (2012) Immobilization of gelatin onto poly(glycidyl methacrylate)-grafted polycaprolactone substrates for improved cell-material interactions. Biointerphases 7:30. doi: 10.1007/s13758-012-0030-1 CrossRefGoogle Scholar
  38. Yusilawati AN, Maizirwan M, Hamzah MS, Ng KH, Wong CS (2010) Surface modification of polystyrene beads by ultraviolet/ozone treatment and its effect on gelatin coating. Am J Appl Sci 7:724–731CrossRefGoogle Scholar
  39. Zhou W, Ma G, Su Z (2013) Microspheres for cell culture. In: Ma G, Su Z (eds) Microcapsules in biotechnology. Taylor and Francis, New YorkGoogle Scholar
  40. Zhu Y, Gao C, Liu X, Shen J (2002) Surface modification of polycaprolactone membrane via aminolysis and biomacromolecule immobilization for promoting cytocompatibility of human endothelial cells. Biomacromolecules 3:1312–1319CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  • Nurhusna Samsudin
    • 1
  • Yumi Zuhanis Has-Yun Hashim
    • 1
    • 2
    Email author
  • Mohd Azmir Arifin
    • 1
    • 3
  • Maizirwan Mel
    • 1
  • Hamzah Mohd. Salleh
    • 1
    • 2
    Email author
  • Iis Sopyan
    • 4
  • Dzun Noraini Jimat
    • 1
  1. 1.Department of Biotechnology Engineering, Kulliyyah of EngineeringInternational Islamic University MalaysiaKuala LumpurMalaysia
  2. 2.International Institute for Halal Research and Training (INHART)International Islamic University MalaysiaKuala LumpurMalaysia
  3. 3.Faculty of Engineering TechnologyUniversiti Malaysia PahangKuantanMalaysia
  4. 4.Department of Manufacturing and Material Engineering, Kulliyyah of EngineeringInternational Islamic University MalaysiaKuala LumpurMalaysia

Personalised recommendations