An IR femtosecond pulsed laser was used for micropatterning of biomineral containing chitosan membranes, aiming to enhance bone mineralization and angiogenesis. Pre and post irradiation materials have been characterized with XRD, SEM and spectroscopic techniques

Anastasiou, A

El-Raif, M

Giannoudis, PV

Iqbal, N

Jha, A

Maddi, C

English

Boccaccini

Crossref

Fabrication of Multi-Layered Bone Scaffolds using Femtosecond Pulsed Lasers

White Rose Research Online

This is a repository copy of Fabrication of Multi-Layered Bone Scaffolds using Femtosecond Pulsed Lasers.White Rose Research Online URL for this paper:http://eprints.whiterose.ac.uk/146035/Version: Accepted VersionConference or Workshop Item:Iqbal, N orcid.org/0000-0002-2801-707X, Anastasiou, A, Maddi, C et al. (3 more authors) (2019) Fabrication of Multi-Layered Bone Scaffolds using Femtosecond Pulsed Lasers. In: Biophotonics Congress: Optics in the Life Sciences Congress 2019, Tucson, Arizona, USA. This is an author produced version of presentation made at the Biophotonics Congress 2019 and published by the Optical Society of America. One print or electronic copy may bemade for personal use only. Systematic reproduction and distribution, duplication of any material in this paper for a fee or for commercial purposes, or modifications of the content of this paper are prohibited.eprints@whiterose.ac.ukhttps://eprints.whiterose.ac.uk/Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. OSA Biophotonics Congress: Optics in the Life Sciences Conference Loews Ventana Canyon Resort, Tucson, Arizona, United States 14 April 2019 – 17 April 2019   Fabrication of Multi-Layered Bone Scaffolds using Femtosecond Pulsed Lasers  Neelam Iqbal1, Antonios Anastasiou1, Chiranjeevi Maddi1, Mostafa El-Raif2, Peter V Giannoudis3 and Animesh Jha1 1 School of Chemical and Processing Engineering, Engineeri g Building, University of Leeds, LS2 9JT, UK 2 Division of Oral Biology, Leeds Dental School, University of Leeds, UK 3 Department of Trauma and Orthopaedic Surgery, Leeds General Infirmary, UK pm15ni@leeds.ac.uk  Abstract: An IR femtosecond pulsed laser was used for micropatterning of biomineral containing chitosan membranes, aiming to enhance bone mineralization and angiogenesis. Pr  and post irradiation materials have been characterized with XRD, SEM and spectroscopic techniques.  OCIS codes: 310.6845, 310.6870, 320.7090  1.  Introduction Bone is a complex living tissue with significant metabolic and regenerative acti ities, which are disrupted when the tissue is damaged. Healing of fractured bone is a complicated process therfore; an ideal bone scaffold should have: i) exceptional osteogenic potential in order to promote new bone formation; ii) load-bearing properties; iii) appropriate microstructure for promoting angiogenesis for circulation of nutrients and iv) antibacterial resistance to avoid infections that lead to failure of the surgery. Research efforts have been focused on development of the “perfect” biomaterial, targeting to achieve all the aforementioned properties with a single material system; e.g. blocks of calcium phosphates, coated Ti-implants [1], polymers [2] etc. The aim of our research is to establish a novel approach, for the development, fabrication and implantation of bone scaffolds, based on the fundamentals of advanced manufacturing using femtosecond lasers.  In the present work we discuss the fabrication of a multi-layered scaffold which has the potential to promote both bone mineralization and the formation of intrinsic vasculature. To achieve this we developed two types of chitosan mineral loaded samples. The Type-1 sample, aims to enhance osteogenic potential, it is loaded with calcium phosphate minerals (e.g. -calcium pyrophosphate) and is fabricated through freeze drying in order to achieve a highly porous honeycomb structure. The ype-2 samples are based on membrane geometries, fabricated through casting of chitosan solutions loaded with CeO2 and Ce2O3 nanoparticles which are known for their excellent antibacterial and angiogenetic properties already observed in systems for in vitro and also in vivo models [3]. A Coherent Libra-S-1K (100fs) femtosecond laser (1 kHz repetition rate), at a w velength of 800 nm, was utilised to fabricate microchannel networks and patterns on the Type-2 membrane surface . Micropatterning and microchannels are expected to promote guided cellular growth leading eventually to the formation of vasculature. In addition, the increased capillary forces in the channels will allow for better circulation of the nutritional components which are necessary for the formation of new blood vessels. The scaffold can be constructed through a numbering up approach where several layers of Type-1 and Type-2 membranes are piled up together in order to mimic the natural structure of cancellous bone.   2.  Materials & Characterizations     Fig. 1. (a) XRD analysis of cerium oxide nanoparticles; (b) TEM image of cerium oxide nanoparticles with particle size between 10-2nm  Cerium oxide nanoparticles were formed using a hydroxide mediated approach. A 0.3 M aqueous solution of sodium hydroxide was added dropwise to 1 M aqueous cerium nitrate hexahydrate solution at 25°C. The mixture was left under continuous stirring for 24 hrs. The nanoparticles were collected by filtration and washed several times with distilled water. They were subsequently frozen at -80°C for 24 hrs and the  placed into a freeze drier for 24 hrs. X-Ray powder diffraction and TEM (fig.1) were used to verify the formation of the cerium oxide nanoparticles.  For the synthesis of both Type-1 and Type-2 materials, 6g of chitosan flakes was dissolved in a 2% v/v aqueous acetic acid solution under continuous mixing for 24 hrs. Type-1 samples were synthesised by adding to the chitosan solution different quantities of calcium phosphate minerals (i.e. 20, 30 and 40% w/w). After freeze drying, porous membranes have been obtained with pore sizes in the range of 42 µm and 94 µm (fig.2a) which are appropriate for bone regeneration (the apparent porosities are shown in fig.2b). For Type-2 membranes, CeO2 nanoparticles have been added to chitosan solution and the mixture left to dry until the formation of a compact membrane.     Fig. 2. (a) Hitachi SU8230 SEM image of Type-1 freeze dried sample containing 30 wt% calcium phosphate min rals; (b) calculated apparent density of freeze dried calcium phosphate-chitosan samples.  3. Femtosecond Pulse Laser & Cell Study   Experiments were carried out to identify the ablation threshold of Type-2 materials; this was determined to be 5.4 J/cm2. For the fabrication of channels and micropatterns the initial average powerof the laser was set at 40 mW and a a b b b then consecutive channels were formed by gradually increasing the power in steps of 20 mW per line (maximum power used was 220 mW). As it was expected, the channel width was increased by increasing the power of the laser in a range between 30 m and 140 µm. Fabrication of microchannels on the membrane surfaces were uccessful, as can be seen in fig.3 (a).          Fig. 3. (a) Hitachi SU8230 SEM image of regular and periodic microchannels fabricated with the aid of a femtosecond laser operating at 800 nm; (b) Optical microscope image of inital osteoblast contact test on chitosan-calcium phospahte membrane  Initial tests verified that both Type-1 and Type-2 samples do not present any toxicological risks. Osteoblast cells (cell line G292) were grown on and close to our samples as shown in fig.3b. Further experiments are still under process in order to confirm that cell growth and proliferation occurs within the micropatterns/channels.   4.  References [1] Oh, S.-H., et al., Growth of nano-scale hydroxyapatite using chemically treated titanium oxide nanotubes. Biomaterials, 2005. 26(24): p. 4938-4943. [2] Das, S., et al., The induction of angiogenesis by cerium oxide nanoparticles through the modulation of oxygen in intracellular environments. Biomaterials, 2012. 33(31): p. 7746-7755. [3] Boccaccini, A.R., et al., Polymer/bioactive glass nanocomposites for biomedical applications: A review. Composites Science and Technology, 2010. 70(13): p. 1764-76.       a 

Raif, EM

This is a repository copy of Fabrication of Multi-Layered Bone Scaffolds using Femtosecond Pulsed Lasers.White Rose Research Online URL for this paper:http://eprints.whiterose.ac.uk/155746/Version: Accepted VersionProceedings Paper:Iqbal, N, Anastasiou, A, Maddi, C et al. (3 more authors) (2019) Fabrication of Multi-Layered Bone Scaffolds using Femtosecond Pulsed Lasers. In: Proceedings of the Biophotonics Congress: Optics in the Life Sciences Congress 2019 (BODA, BRAIN, NTM, OMA, OMP). Biophotonics Congress: Optics in the Life Sciences Congress 2019 (BODA, BRAIN, NTM, OMA, OMP), 14-17 Apr 2019, Tucson, AZ, United States. Optical Society of America . https://doi.org/10.1364/BODA.2019.JT4A.45This is an author produced version of a paper published in Proceedings of the Biophotonics Congress: Optics in the Life Sciences Congress 2019 (BODA, BRAIN, NTM, OMA, OMP). Uploaded in accordance with the publisher's self-archiving policy.eprints@whiterose.ac.ukhttps://eprints.whiterose.ac.uk/Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. Fabrication of Multi-Layered Bone Scaffolds usingFemtosecond Pulsed LasersNeelam Iqbal1, Antonios Anastasiou1, Chiranjeevi Maddi1, Mostafa El-Raif2, Peter V Giannoudis3 andAnimesh Jha11 School of Chemical and Processing Engineering, Engineering Building, University of Leeds, LS2 9JT, UK2 Division of Oral Biology, Leeds Dental School, University of Leeds, UK3 Department of Trauma and Orthopaedic Surgery, Leeds General Infirmary, UKPm15ni@leeds.ac.ukAbstract: An IR femtosecond pulsed laser was used for micropatterning of biomineralcontaining chitosan membranes, aiming to enhance bone mineralization and angiogenesis. Preand post irradiation materials have been characterized with XRD, SEM and spectroscopictechniques.OCIS codes: 310.6845, 310.6870, 320.70901.  IntroductionBone is a complex living tissue with significant metabolic and regenerative activities, which are disrupted whenthe tissue is damaged. Healing of fractured bone is a complicated process therefore; an ideal bone scaffold shouldhave: i) exceptional osteogenic potential in order to promote new bone formation; ii) load-bearing properties; iii)appropriate microstructure for promoting angiogenesis for circulation of nutrients and iv) antibacterial resistanceto avoid infections that lead to failure of the surgery. Research efforts have been focused on development of the“perfect” biomaterial, targeting to achieve all the aforementioned properties with a single material system; e.g.blocks of calcium phosphates, coated Ti-implants [1], polymers [2] etc. The aim of our research is to establish anovel approach, for the development, fabrication and implantation of bone scaffolds, based on the fundamentalsof advanced manufacturing using femtosecond lasers.In the present work we discuss the fabrication of a multi-layered scaffold which has the potential t promote bothbone mineralization and the formation of intrinsic vasculature. To achieve this we developed two types of chitosanmineral loaded samples. The Type-1 sample, aims to enhance osteogenic potential, it is loaded with calciumphosphate minerals (e.g.฀-calcium pyrophosphate) and is fabricated through freeze drying in order to achieve ahighly porous honeycomb structure. The Type-2 samples are based on membrane geometries, fabricated throughcasting of chitosan solutions loaded with CeO2 and Ce2O3 nanoparticles which are known for their excellentantibacterial and angiogenetic properties already observed in systems for in vitro and also in vivo models [3]. ACoherent Libra-S-1K (100fs) femtosecond laser (1 kHz repetition rate), at a wavelength of 800 nm, was utilisedto fabricate microchannel networks and patterns on the Type-2 membrane surfaces. Micropatterning andmicrochannels are expected to promote guided cellular growth leading eventually to the formation of vasculature.In addition, the increased capillary forces in the channels will allow for better circulation of the nutritionalcomponents which are necessary for the formation of new blood vessels. The scaffold can be constructed througha numbering up approach where several layers of Type-1 and Type-2 membranes are piled up together in order tomimic the natural structure of cancellous bone.2.  Materials & CharacterizationsCerium oxide nanoparticles were formed using a hydroxide mediated approach. A 0.3M aqueous solution ofsodium hydroxide was added dropwise to a cerium nitrate solution (1M) at 25°C. The mixture was left undercontinuous stirring for 24h until it gets a cloudy pink colour (is due to precipitation of the NPs??). After filtrationthe collected nanoparticles were washed several times with distilled water and were subsequently frozen at -80°Cfor 24h and then placed into a freeze drier for 24 hrs. X-Ray powder diffraction (Fig.1a) and FTIR have been wereused to verify the formation of the CeO2 nanoparticles.For the synthesis of both Type-1 and Type-2 materials, 6g of chitosan flakes was dissolved in a 2% v/v aqueousacetic acid solution under continuous mixing for 24h. Type-1 samples were synthesised by adding to the chitosansolution different quantities of calcium phosphate minerals (i.e. 20, 30 and 40% w/w). After freeze drying, porousmembranes have been obtained with pore sizes in the range of 42µm and 94µm (fig.1b) which are appropriate forbone regeneration (the apparent porosities are shown in fig.1c). For Type-2 membranes, CeO2 nanoparticles havebeen added to chitosan solution and the mixture left for t drying until the formation of a compact membrane.3. Femtosecond Pulse Laser & PorosityExperiments were carried out to identify the ablation threshold of Type-2 materials for the fabrication of cha nelsand micropatterns (report the ablation threshold). The initial average power of the laser was set at 40mW and thenconsecutive channels were formed by gradually increasing the power in steps of 20mW per line (maximum powerused was 220mW). As it was expected, the channel width is increased by increasing the power of the laser in arange between 30mm and 140µm. Fabrication of microchannels on the membrane surfaces were successful, as canbe seen in Figure 2a.Fig. 2. Hitachi SU8230 SEM images (a) Regular and periodic microchannels fabricated with the aid of a femtosecond lar per ting at800 nm; (b) Optical microscope image of inital osteoblast contact test on chitosan-calcium phospahte membraneInitial tests verified that both the Type 1 and Type 2 samples doesn’t not present any toxicological risks. Osteoblastcells (cell line G292) were grown on and close to our samples as shown in fig.2b. Further experiments are stillunder process in order to confirm that cell growth and proliferation occurs within the micropatterns/channels.4.  Acknowledgements: The acknowledge the financial support from the EPSRC DTP grant for NI and EU MarieCurie IIF support for AA and CM on PreFacto and BonE-GraPH projects, respectively.5.  References[1] Oh, S.-H., et al.,Growth of nano-scale hydroxyapatite using chemically treated titanium oxide nanotubes. Biomaterials, 2005.26(24): p.4938-4943.[2] Das, S., et al.,The induction of angiogenesis by cerium oxide nanoparticles through the modulation of oxygen in  intracellularenvironments. Biomaterials, 2012.33(31): p. 7746-7755.[3] Boccaccini, A.R., et al., Polymer/bioactive glass nanocomposites for biomedical applications: A review. Composites Science andTechnology, 2010. 70(13): p. 1764-76.Fig. 1. (a)  XRD analysis of cerium oxide nanoparticles; (b) Type-1 freeze dried sample containing 30 wt% calcium phosphate minerals;(c)  Calculated apparent density of freeze dried calcium phosphate-chitosan samples00.020.040.060.08ApparentDensity(g/cm3)aabcb

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Fabrication of Multi-Layered Bone Scaffolds using Femtosecond Pulsed Lasers

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