Nanocellulose 3-D structures for regenerative medicine (CELL-3D)

  • Funding: Romanian National Authority for Scientific Research and Innovation, CNCS - UEFISCDI
  • Programme: P4 Basic and Frontier Research - Exploratory Research Projects PN-III-P4-ID-PCE-2016-0431
  • Project number: PN-III-P4-ID-PCE-2016-0431
  • Research domain - Engineering Sciences
  • Project title: Nanocellulose 3-D structures for regenerative medicine (CELL-3D)
  • Financing contract: 148/2017
  • Total funding of the contract: 850.000 RON
  • Funding from the State Budget: 850.000 RON
  • Project duration: 30 months
  • Period of project implementation: 12/07/2017 - 31/12/2019
  • Contractor: National Institute for Research & Development in Chemistry and Petrochemistry - ICECHIM, Bucharest
  • Project Manager: Panaitescu Denis Mihaela - panaitescu@icechim.ro

   

Project summary

12/07/2017 - 31/12/2019

 

Regenerative medicine and tissue engineering may offer new hope of life in case of loss or malfunction of a tissue or organs. One of the key elements in tissue engineering is the three-dimensional (3D) scaffold which provides structural support for cell attachment, proliferation and differentiation. Biopolymer scaffolds are preferred in tissue engineering for avoiding the issues related to donor immune rejection and pathogen transfer. Cellulose, the most abundant natural polymer on earth, is an almost inexhaustible source of valuable materials and has gained high interest in the form of nanocellulose (NC). NC stands out for its high crystallinity and mechanical strength, good biocompatibility, high water-holding capacity and large surface area and it has been intensively studied for medical applications because it does not cause toxic or allergic side effects in contact with living tissue. 3D scaffolds should possess a network of interconnected pores to permit cell migration, diffusion of nutrients and clearance of wastes, they must be able to support cell adhesion and promote cell growth and they must have balanced stiffness-toughness properties to support and transfer of loads. To meet all these requirements in porous 3D scaffolding, CELL-3D has as main objective to design new 3D nanocellulose structures that can be applied in regenerative medicine.

Phase I - 2017

In the first phase, porous nanocellulose structures have been obtained starting from several nanocellulose sources: (i) nanocellulose obtained from the mechanical disintegration of bacterial cellulose membranes, (ii) nanocellulose obtained by acid hydrolysis of microcrystalline cellulose, (iii) nanocellulose from agro-industrial waste (plum seeds shells), (iv) nanocellulose obtained by tempo oxidation and mechanical defibrillation and (v) nanocellulose obtained by defibrillation and plasma functionalization. Nanodimension of nanocellulose samples was highlighted by AFM, SEM and TEM. To obtain porous structures with controlled properties nanocellulose samples were functionalized by silanes and crosslinked using non-toxic crosslinking agents with different efficiency. In another approach, ionic agents have been used to obtain nanocellulose hydrogels and cryogels. These methods led to 3D structures with controlled pore sise and interconnected micro- and nano-pores. The porous 3D structures were characterized to observe the influence of the treatments on morphology, structure, thermal and mechanical properties and to detect the influence of the treatments on these characteristics. The methodology to obtain functionalized nanocellulose was elaborated. Moreover, the scientific report on the correlation between the process parameters, composition and the thermal and mechanical properties as well as morphology of 3D functionalized nanocellulose structures was released. High resolution computerized tomography revealed a porous structure with over 80% open porosity and a high connectivity in the case of functionalized and cross-linked porous structures.

Phase II - 2018

            In the second phase, new functionalization-crosslinking methods were developed and their efficiency was evaluated based on the thoroughly characterization of the thermal and mechanical properties as well as morphological features of the obtained 3D cellulose structures. A three-level organization in the 3D structures was detected by SEM investigation. A 5-fold increase in the specific compression and a minor decrease in thermal stability were observed depending on the cross-linking system. Acrylic copolymers were grafted on nanocellulose structures leading to a high improvement of properties: the increase of the contact angle from less than 30° to 84° indicating hydrophobic properties, reduced water absorption, increased onset degradation temperature with over 60 °C, a uniform polymer coating and a porous structure with high porosity. 3D composite structures based on nanocellulose and hydrophobic biopolymers, poly (3-hydroxybutyrate) (PHB), poly (3-hydroxybutyrate co-3-hydroxyvalerate) (PHBV) and poly (3-hydroxyoctanoate) were obtained by (i) impregnating functionalized nanocellulose structures with solutions of various biopolymers and (ii) mixing cellulosic nanofibres with these polymer solutions. Depending on the working conditions, the thermal stability of the composite structures increases by 100 °C compared to the untreated 3D cellulose structure. An extended porous network structure with pore sizes of less than 2 µm was observed on the surface of nanocellulose - PHBV nanocomposites modified by immiscible biopolymers. The report on the mechanical properties of the 3D crosslinked cellulose structures and the methodology for obtaining 3D structures from nanocellulose/hydrophilic polymers and from nanocellulose/hydrophobic biopolymers were released.

Phase III - 2019

            In the third phase, 3D porous structures were obtained from nanocellulose and biopolymers by (i) extrusion of nanocellulose mixtures with hydrophilic biopolymers and lyophilization, (ii) extrusion of nanocellulose mixtures with hydrophobic biopolymers and (iii) extrusion of mixtures of hydrophobic biopolymers, nanocellulose using blowing agents. The analysis of the correlations between the type of biopolymer, the process parameters and the characteristics of the nanocellulose and composite 3D structures allowed the selection of the representative porous structures for tissue engineering. The 3D nanocellulose structure model was developed. The potential cytotoxicity of surface-modified and cross-linked NC foams were evaluated using murine fibroblasts L929. In vitro analyzes showed that functionalized and cross-linked foams are not cytotoxic to L929 fibroblast cells, regardless of chemical treatment, which is very important for tissue engineering. The pro-inflammatory effect was also evaluated on the same 3D nanocellulose structures. NO and TNF-α were measured using the RAW 264.7 murine macrophage cell line to evaluate the influence of the chemical modifications on the activation of macrophage secretory function. TNF-α concentration was very low in the case of functionalized and cross-linked NC foams, similar to that of the negative control.

The results obtained in this project were disseminated by 8 ISI papers, 5 of which were published and three being in the evaluation and 2 non-ISI papers. The results were also disseminated in 21 international conferences, one being an invited lecture and two oral presentations.

» Project summary

CELL-3D has as main objective to design new 3D nanocellulose structures for tissue engineering. The new 3D structures should meet the required chemical, mechanical and structural properties needed in regenerative medicine. To address the overall objective of this project, several specific objectives will be considered: (1) understanding the influence of the surface modification of nanocellulose on the 3D structures, (2) understanding the influence of composition and processing parameters on the properties of 3D structures; (3) characterization of the properties and morphology of nanocellulose 3D structures in different environments. 

The 3D nanocellulose structures will be obtained by new methods based on the understanding of the relationship between structure, processing parameters and properties of nanocellulose 3D scaffolds. The results will be disseminated by articles and communications.

Panaitescu Denis

Vuluga Zina

Corobea Mihai

Frone Adriana

Chiulan Ioana

Nicolae Cristian

Gabor Raluca

Ficai Anton

Trusca Roxana

Ghiurea Marius

Bogdan Trica

Ninciuleanu Claudia

  1. Synthesis and characterization of 3D structures starting from different types of nanocellulose (NC) using surface functionalization. Understanding the mechanisms of 3D structures formation;
  2. Synthesis and characterization of 3D structures from nanocellulose - polymer composites. Analysis of process conditions in relation to nanocomposites structure and properties;
  3. Flexible 3D structures obtained by new techniques. Optimizing the process and elaboration of the model.

 

ISI papers

  1. 1.     Adriana N. Frone, Denis M. Panaitescu, Ioana Chiulan, Cristian A. Nicolae, Angela Casarica, Raluca A. Gabor, Roxana Trusca, Celina M. Damian, Violeta Purcar, Elvira Alexandrescu, Paul O. Stanescu, Surface treatment of bacterial cellulose in mild, eco-friendly conditions, Coatings 2018, 8, 221
  2. Sorin Vizireanu, Denis Mihaela Panaitescu, Cristian Andi Nicolae, Adriana Nicoleta Frone, Ioana Chiulan, Maria Daniela Ionita, Veronica Satulu, Lavinia Gabriela Carpen, Simona Petrescu, Ruxandra Birjega, Gheorghe Dinescu, Cellulose defibrillation and functionalization by plasma in liquid treatment, Scientific Reports 2018, 8, 15473
  3. Denis Mihaela Panaitescu, Adriana Nicoleta Frone, Ioana Chiulan, Cristian Andi Nicolae, Roxana Trusca, Marius Ghiurea, Raluca Augusta Gabor, Mona Mihailescu, Angela Casarica, Irina Lupescu, Role of bacterial cellulose and poly(3-hydroxyhexanoate-co-3-hydroxyoctanoate) in poly(3-hydroxybutyrate) blends and composites, Cellulose 2018, 25, 5569–5591
  4. Denis Mihaela Panaitescu, Eusebiu Rosini Ionita, Cristian Andi Nicolae, Raluca Augusta Gabor, Maria Daniela Ionita, Roxana Trusca, Brindusa-Elena Lixandru, Irina Codita, Gheorghe Dinescu, Poly(3-hydroxybutyrate) modified by nanocellulose and plasma treatment for packaging applications, Polymers 2018, 10, 1249
  5. Adriana Nicoleta Frone, Denis Mihaela Panaitescu, Ioana Chiulan, Augusta Raluca Gabor, Cristian Andi Nicolae, Madalina Oprea, Marius Ghiurea, Dan Gavrilescu, Adrian Catalin Puitel, Thermal and mechanical behavior of biodegradable polyester films containing cellulose nanofibers, 2019, Journal of Thermal Analysis and Calorimetry, (2019) 138: 2387-2398; DOI: 10.1007/s10973-019-08218-4.
  6. Adriana Nicoleta Frone, Denis Mihaela Panaitescu, Cristian Andi Nicolae, Augusta Raluca Gabor, Roxana Trusca, Angela Casarica, Paul Octavian Stanescu, Dora Domnica Baciu, Aurora Salageanu, Bacterial cellulose sponges obtained with green cross-linkers for tissue engineering, submitted to Materials Science and Engineering C
  7. Denis Mihaela Panaitescu, Cristian Andi Nicolae, Augusta Raluca Gabor, Roxana Trusca, Thermal and mechanical properties of poly(3-hydroxybutyrate) reinforced with cellulose fibers from wood waste, submitted to Industrial Crops and Products
  8. Madalina Oprea, Denis Mihaela Panaitescu, Cristian Andi Nicolae, Augusta Raluca Gabor, Adriana Nicoleta Frone, Valentin Raditoiu, Roxana Trusca, Angela Casarica, Nanocomposites from functionalized bacterial cellulose and poly(3-hydroxybutyrate-co-3-hydroxyvalerate), submitted to Polymer Degradation and Stability

 

Non-ISI papers

  1. I. Chiulan, A. N. Frone, D. M. Panaitescu, Calin Brandabur, Recent advances in 3D printing of aliphatic polyesters, Bioengineering 2018, 5(1) 2
  2. Elena Ruxandra Radu, Ioana Chiulan, Cristian Andi Nicolae, Raluca Augusta Gabor, Valentin Raditoiu , Monica Florentina Raduly, Denis Mihaela Panaitescu, Preparation and characterization of porous cellulose–polymer foams for biomedical applications, Proceedings 2019, 29(1), 8

 

Conferences

  1. D. M. Panaitescu, A. N. Frone, I. Chiulan, R. A. Gabor, C.A. Nicolae, M. Ghiurea, D. Florea, C. M. Ninciuleanu, Surface functionalization of nanocellulose for biomedical applications, The XIIIth International Symposium "Priorities of Chemistry for a Sustainable Development – PRIOCHEM”, Bucharest, 25 - 27 October 2017
  2. Denis Mihaela Panaitescu, Adriana Nicoleta Frone, Ioana Chiulan, Cristian Nicolae, Raluca Gabor, Elvira Alexandrescu, Angela Casarica, Design of nanocellulose aerogels by functionalization and cross-linking, 11th International conference on Advanced Nanomaterials, Aveiro, Portugal, 18-20 July 2018
  3. Adriana Nicoleta Frone, Ioana Chiulan, Raluca-Augusta Gabor, Elvira Alexandrescu, Cristian-Andi Nicolae, Denis Mihaela Panaitescu, 3D structures from cross-linked nanocellulose, 6th Portuguese Young Chemists Meeting - PYCheM2018, Setubal, 15-18 May 2018
  4. Adriana Nicoleta Frone, Denis Mihaela Panaitescu, Ioana Chiulan, Raluca Augusta Gabor, Cristian Andi Nicolae, Thermal behavior of biodegradable polyesters based membranes, 12th European Symposium on Thermal Analysis and Calorimetry, Brasov, Romania, 27-30 August 2018
  5. Denis Mihaela Panaitescu, Adriana Nicoleta Frone, Ioana Chiulan, Cristian Andi Nicolae, Augusta Raluga Gabor, Madalina Calarasu, Sergiu Stoian, Roxana Trusca, Angela Casarica, New 3D structures from nanocellulose and biopolymers, XXXVth National Conference of Chemistry, Caciulata, Romania, 2-5 October 2018
  6. Denis Mihaela Panaitescu, Adriana Nicoleta Frone, Madalina Calarasu, Sergiu Stoian, Porous structures from nanocellulose and biopolymers for biomedical application, 9th International Conference on Biopolymers and Polymer Sciences, Bucharest, Romania, 19-21 November 2018
  7. Sergiu Stoian, Adriana Nicoleta Frone, Denis Mihaela Panaitescu, Mona Mihailescu, Roxana Trusca, Cristian Andi Nicolae, Augusta Raluca Gabor, Physico-chemical characterization of surface modified cellulose, Sixth edition of the International Colloquium ‘Physics of Materials’, Bucharest, Romania, 15-16 November 2018
  8. Ioana Chiulan, Denis M. Panaitescu, Adriana Nicoleta Frone, Cristian Andi Nicolae, Raluca A. Gabor, Sorin Valentin Rădiţoiu, Elvira Alexandrescu, Bogdan Trică, Cellulose/ethylene glycol methyl ether acrylate 3D structure with controllable physical properties, 11th International conference on Advanced Nanomaterials, Aveiro, Portugal, 18-20 July 2018
  9. D. M. Panaitescu, A. N. Frone, I. Chiulan, R. A. Gabor, C.A. Nicolae, M. Ghiurea, D. Florea, C. M. Ninciuleanu, Surface functionalization of nanocellulose for biomedical applications, The XIIIth International Symposium "Priorities of Chemistry for a Sustainable Development – PRIOCHEM”, Bucharest, 25 - 27 October 2018.
  10. 10.  Denis Mihaela Panaitescu, Adriana Nicoleta Frone, Nanomaterials based on nanocellulose for biomedical applications, 4th International Congress on Biomaterials & Biosensors (BIOMATSEN), Oludeniz, Turkey, May 12-18, 2019 (invited lecture).
  11. Sergiu-Alexandru Stoian, Denis Mihaela Panaitescu, Cristian Andi Nicolae, Raluca Augusta Gabor, Paul Octavian Stanescu, Cross-linked nanocellulose foams using mono- and disaccharides, 4th International Congress on Biomaterials & Biosensors (BIOMATSEN), Oludeniz, Turkey, May 12-18, 2019.
  12. 12.  Sergiu-Alexandru Stoian, Cristian Andi Nicolae, Augusta Raluca Gabor, Marius Ghiurea, Denis Mihaela Panaitescu, Effect of TiO2 on the properties of polyhydroxyalkanoates, 9th International Conference of the Chemical Societies of the South-East European Countries, Targoviste, Romania, May 8–11, 2019 (oral presentation)
  13. Madalina Oprea, Cristian-Andi Nicolae, Raluca Augusta Gabor, Roxana Trusca, Denis Mihaela Panaitescu, Biocomposites from bacterial cellulose and polyhydroxyalkanoates, 9th International Conference of the Chemical Societies of the South-East European Countries, Targoviste, Romania, May 8-11, 2019.
  14. Denis Mihaela Panaitescu, Sergiu-Alexandru Stoian, Madalina Oprea, Adriana Nicoleta Frone, Ioana Chiulan, Augusta Raluca Gabor, Cristian Andi Nicolae, Roxana Trusca, Paul Octavian Stanescu, Nanocomposites from functionalized bacterial cellulose and biopolymers, 7th International Conference on Biobased and Biodegradable Polymers (BIOPOL-2019), Stockholm, Suedia, June 16-19, 2019
  15. 15.  Denis Panaitescu, Sergiu Stoian, Sorin Vizireanu, Mona Mihailescu, Cristian Nicolae, Veronica Satulu, Gheorghe Dinescu, Influence of surface modified cellulose on the properties of biomedical polyester scaffolds, 19th International Balkan Workshop on Applied Physics and Materials Science, Constanta, Romania, July 16-19, 2019 (oral presentation)
  16. Elena Ruxandra Radu, Ioana Chiulan, Cristian Andi Nicolae, Valentin Raditoiu, Roxana Trusca, Denis Mihaela Panaitescu, Functionalized cellulose for biomedical applications, Advanced Nanomaterials Conference, Aveiro, Portugalia, July 17-19, 2019.
  17. Alexandru Sergiu Stoian, Denis Mihaela Panaitescu, Augusta Raluca Gabor, Cristian Andi Nicolae, Valentin Raditoiu, Thermal properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nanocomposites with controlled degradability, 5th Central and Eastern European Conference on Thermal Analysis and Calorimetry (CEEC-TAC5), Rome, Italy, August 27-30, 2019.
  18. Denis Panaitescu, Adriana Frone, Cristian Nicolae, Raluca Gabor, Madalina Oprea, Sergiu Stoian, Biocomposite foams based on polyhydroxyalkanoates: morphological and thermo-mechanical characterization, 5th Central and Eastern European Conference on Thermal Analysis and Calorimetry (CEEC-TAC5), Rome, Italy, August 27-30, 2019.
  19. Denis Mihaela Panaitescu, Alexandru Sergiu Stoian, Cristian Andi Nicolae, Adriana Nicoleta Frone, Augusta Raluca Gabor, Dynamic mechanical properties of polyhdroxybutirate reinforced with cellulose microfibrils, 5th Central and Eastern European Conference on Thermal Analysis and Calorimetry (CEEC-TAC5), Rome, Italy, August 27-30, 2019.
  20. I. Chiulan, D. M. Panaitescu, E. R. Radu, C. A. Nicolae, S. V. Raditoiu, S. Vizireanu, R. A. Gabor, Comprehensive Physico-chemical characterization of cellulose nanocrystals-grafted-PEG acrylates aerogels, Aveiro, Portugalia, “6th EPNOE International Polysaccharide Conference” (EPNOE-2019), October 21-25, 2019.
  21. Elena Ruxandra Radu, Ioana Chiulan, Cristian Andi Nicolae, Raluca Augusta Gabor, Valentin Raditoiu , Monica Florentina Raduly, Denis Mihaela Panaitescu, Preparation and characterization of porous cellulose–polymer foams for biomedical applications, Prioritatile Chimiei Pentru O Dezvoltare Durabila (PRIOCHEM 2019), Romania, Bucuresti, November 30-01, 2019.