30th Annual Conference of the European Society for Biomaterials together with the 26th Annual Conference of the German Society for Biomaterials (DGBM)
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Ceramics and Calciumphosphates

   
Shortcut: PS1-02
Date: Tuesday, 10 September, 2019, 2:45 p.m.
Room: Hall 1 / Exhibition Area
Session type: Poster

Contents

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PS1-02-36

Synthesis and characterization of new 3D Fe-doped bioceramic scaffold obtained via Sol-Gel method (#359)

N. A. Mata1, P. Ros-Tárraga2, P. Velásquez1, A. Murciano3, P. N. De Aza1

1 Universidad Miguel Hernández, Instituto de Bioingeniería, Elche, Spain
2 Universidad Católica San Antonio de Murcia, Grupo de Investigación en Regeneración y Reparación de Tejidos, Guadalupe, Spain
3 Universidad Miguel Hernández, Departamento de Materiales, Óptica y Tecnología Electrónica, Elche, Spain

Introduction

The objective of this research was to develop and characterize biomagnetic Si-Ca-P scaffolds to be used in substitution and regeneration of bone tissue. The 3D ceramic scaffolds were formed by a core of composition 1 mol% SiO2 - 25 mol% P2O5 – 68 mol% CaO – 6 mol% Li2O and was coated with layers of composition 29 mol% SiO2 – 3 mol% P2O5 – 68 mol% CaO doped with 1 - 3 mol% of iron ions. According to the literature consulted, this scaffolds is one of the first magnetic biomaterials formed with two different compositions. The incorporation of iron in the scaffold enhances the bactericidal and mineralogical properties of the bone tissue. Additionally, it provides ferromagnetic properties that can be used in medical applications such as magnetic resonance imaging, cell separation and treatment against cancer such as hyperthermia (1-2).

Experimental Methods

Scaffolds multilayers were synthesized by sol-gel and polymer replication methods. Polyurethane sponges were used as a template to make the 3D scaffold. These sponges were impregnated with the sol solution of composition 1 mol% SiO2 - 25 mol% P2O5 – 68 mol% CaO – 6 mol% Li2O and sintered at 950 °C for 8 h to obtain the resistant core of the scaffold. Subsequently, the sintered core was coated with a new sol solution of composition 29 mol% SiO2 – 3 mol% P2O5 – 68 mol% CaO doped with 1 - 3 mol% of iron ions. Finally, the scaffolds were sintered again at 950 ºC for 3 h. The characterization was made by Scanning Electron Microscopy (SEM), X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR) and mercury porosimetry techniques. The bioactivity was evaluated in vitro, the samples were immerged at different time in a simulated body fluid (SBF), which was prepared according to Kokubo.

Results and Discussion

3D scaffolds formed by a porous core coated with layers of calcium phosphate doped with iron ions were obtained through the sol-gel and polymer replication methods. Characterization by XRD demonstrated that the core of the scaffold after sintered is constituted by Ca2P2O7, Ca3(PO4)2, LiCa(PO4), SiO2 and Ca2SiO4. The majority of the core is formed by pyrophosphate ((P2O7)-4) and it is widely known that this correspond to 0.5% of all the phosphate found in the human body (3-4). Additionally, previous research has shown that the pyrophosphate is an important regulator of the process of bone mineralization (5). The presence of pyrophosphate was also evidenced through the FTIR and SEM. Finally, the bioactivity of core coated with iron-doped layers was evaluated by the ability to precipitate hydroxyapatite on the surface when submerged in SBF. Figure 1 shows the images obtained by SEM of the ceramic scaffolds doped superficially with 1% and 3% iron after 1 day of immersion in SBF. Both samples presented precipitates of hydroxyapatite, demonstrating their ability to be bioactive.

Conclusion

Multilayer 3D ceramic scaffolds with optimal porosity were developed and characterized. These scaffolds presented pyrophosphate in the composition of the core, which is an important regulator of the bone mineralization process. Moreover, the scaffolds coated with layers of calcium phosphates doped with iron in a proportion of 1% and 3%, showed bioactivity after 1 day in SBF. The scaffolds developed are an excellent alternative for bone tissue replacement with potential medical applications due to the ferromagnetic properties provided by iron.

References

  1. F. Baino, E. Fiume, M. Miola, F. Leone, B. Onida and E. Verné. Fe-doped bioactive glass-derived scaffolds produced by sol-gel foaming. Materials Letters 235 (2019) 207-211.

  2. S. Gomes, A. Kaur, J.M. Grenèche, J.M. Nedelec and G. Renaudin. Atomic scale modeling of iron-doped biphasic calcium phosphate bioceramics. Acta Biomaterialia 50 (2017) 78-88.

  3. S. Bisaz, R.G. Russell, H. Fleisch. Isolation of inorganic pyrophosphate from bovine and human teeth. Arch Oral Biol 13 (1968) 683-696.

  4. R.E. Wuthier, S. Bisaz, R.G. Russell, H. Fleisch. Relationship between pyrophosphate, amorphous calcium phosphate and other factors in the sequence of calcification in vivo. Calcif Tissue Res 10 (1972) 198-206.

  5. I.R. Orriss, T.R. Arnett and R.G. Russell. Pyrophosphate: a key inhibitor of mineralization. Current Opinion in Pharmacology 28 (2016) 57–68

Fig. 1 SEM micrograph of the scaffolds with a core and outer layers doped with: (a) 1% Fe (b) 3% Fe
Keywords: A-01 c - Ceramic biomaterials, A-03 b - 3D scaffolds for TE applications, A-08 d - Surface characterisation
PS1-02-37

Production of calcium phosphate biomaterials in a droplet-based microfluidic device (#372)

Y. Alaoui Selsouli1, H. S. Rho1, V. Galvan Chacon1, Z. Tahmasebi birgani1, P. Habibovic1

1 maastricht university, Department of Instructive Biomaterials Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht, Netherlands

Introduction

There is an increasing need for developing affordable and effective bone graft substitutes, since the gold standard for regenerating large bone defects, i.e., autologous bone, is associated with important limitations such as donor site morbidity, limited availability of the tissue and additional surgical time. Calcium phosphate (CaP)-based ceramics such as hydroxyapatite (HA) and tricalcium phosphate (TCP) are the most widely used synthetic bone graft alternatives in the clinic [1]. However, in general, their biological performance is considered inferior to that of natural bone tissue, and additional research efforts are expended to improve their bone regenerative potential [2]. In contrast to the conventional one-material-for-one-experiment methods for developing new biomaterials, here we aim to develop a method for high-throughput synthesis and screening of ceramic biomaterials. To this end, we have developed a microfluidic device in which CaP ceramics are synthesized inside water-in-oil microdroplets.

Experimental Methods

CaP ceramics were synthesized inside microdroplets generated by a flow focusing microfluidic device in which a mixture of calcium nitrate and phosphoric acid was used as water phase and mineral oil with Span® 80 was used as oil phase. Upon generation and collection of microdroplets, ammonia was added in order to increase the pH, resulting in precipitation of CaPs. The CaP precipitates were then harvested from the suspension by several washing/centrifugation steps. CaPs, similar to those generated in the microfluidic device, were produced using a conventional wet chemical technique outside the microfluidic device, with calcium nitrate and phosphoric acid as precursors and ammonia for adjusting the pH, and served as control. Varying Ca2+/PO43- ratios and pH conditions were used in both synthesis methods in order to produce different CaP phases including brushite, HA and β-TCP. In order to obtain crystalline HA and β-TCP, synthesized biomaterials were sintered at 800˚ C for two hours. The biomaterials were characterized using X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS).

Results and Discussion

Various CaP biomaterials were successfully produced in the water-in-oil-regime in the microfluidic device as well as using the wet chemical technique. Figures 1 and 2 show SEM images and EDS spectra of one of the CaP biomaterials (Ca2+/PO43-=1) produced using the wet chemical and the microfluidics methods, respectively. The conventional wet chemical synthesis, coupled with sintering treatment in case of HA and β-TCP, allowed for obtaining pure and highly crystalline CaP phases through changing the Ca2+/PO43- ratio and pH. The microfluidic setup allowed for tuning additional synthesis parameters such as flow rates, resulting in a controlled generation of microdroplets with varying sizes. Using this feature, CaP particles of varying size can be synthesized in a controlled manner. Extraction and purification of CaP particles from the water-in-oil droplets, however, proved to be challenging and some oil residues were detected on CaPs particles before sintering. Previous studies have suggested the use of different methods for removing the oil phase such as centrifugation, addition of chemical emulsifiers and filtration [3]. Further optimization of the purification steps based on these methods is currently ongoing. Beside better control during CaP synthesis, the droplet generation system in the microfluidics device provides a miniaturized platform allowing production of multiple CaP phases. In future steps, we are aiming to develop a library of inorganic bone graft substitutes using the droplet-based microfluidic device introduced here.

Conclusion

In these first development steps towards a microfluidic platform for high throughput production and screening of inorganic bone graft substitutes, CaPs were generated in a prototype droplet-based microfluidic device. In the next steps, the method will be extended to other inorganic biomaterials and optimization of synthesis parameters using design of experiments will be explored.

References

[1]H. Yuan et al. Proc Natl Acad Sci USA 107 (31) (2010) 13614-19.

[2]K. Lin et al. Acta Biomaterialia 10 (2014) 4071–102.

[3]S. Ammann et al. Energies (2018), 11(9), 2264.

Acknowledgement

The authors acknowledge financial support by Innovative Research Incentives Scheme Vidi of the  Netherlands Organization for Scientific Research (NWO). This research has been made possible with the support of the Interreg Vlaanderen/Nederland BIOMAT collaboration and of the Dutch Province of Limburg.

Figure 1: SEM image and EDS spectra of CaP (Ca/P=1) produced using the wet chemical technique
Figure 2: SEM image and EDS spectra of CaP (Ca/P=1) produced using the microfluidic technique
Keywords: A-01 c - Ceramic biomaterials, A-04 c - Organ-on-a-chip and microfluidics, A-07 d - Bone
PS1-02-38

Systemic toxicity evaluation after subcutaneous implantation of zirconium stabilized with yttrium (#517)

K. Janiczak1, B. Zawidlak-Węgrzyńska1, M. Gonsior1, P. Ścigała1, D. Gonsior1, R. Kustosz1, A. Grajoszek2, R. Wojnicz3, E. Reichman-Warmusz3

1 Foundation of Cardiac Surgery Development, Artificial Heart Laboratory, Zabrze, Poland
2 Medical University of Silesia, Center for Experimental Medicine, Katowice, Poland
3 Medical University of Silesia, Department of Histology and Embryology, Zabrze, Poland

Introduction

Biocompatibility assessment of advanced implants like heart assist devices requires a multistep analysis to confirm the safety of use. A novel rotary ventricular assist device (VAD) ReligaHeart® ROT (RH ROT) was developed [1]. Construction of implantable blood pump is a huge challenge in the aspect of long-term contact with blood. This paper presents systemic toxicity evaluation of zirconium stabilized with yttrium, as a part of the biocompatibility assessment. The investigated material is characterized by excellent physico-mechanical properties as well as biological properties evaluated in the in vitro studies and has therefore found application in the new VAD construction.

Experimental Methods

The systemic toxicity was evaluated according to PN EN ISO 10993-11 as limit test. In the assessment a higher dose of zirconium was admitted to the animals than the dose the patient would be exposed in case of  heart support with RH ROT. The investigated material was prepared in a representative process for RH ROT device ETO sterilized. The study was conducted with the utilization of 48 New Zealand White rabbits. Animals were divided in study and control groups. Implantation was carried out under infusion anaesthesia. In the study group two samples were implanted subcutaneously on the back of animals. In control group two skin incisions were mad on the back. The observation was conducted depending of the group for 4 (n=6), 12 (n=8), and 26 weeks (n=10). Animal’s body weight was controlled every 4 weeks. Before the implantation and then after the experiment before euthanasia the haematological, biochemical and coagulation blood parameters were assessed. After euthanasia a macroscopic evaluation of the implantation area and internal organs was performed. Histopathological assessment of tissues and internal organs was done.

Results and Discussion

During the observation no clinical sings of abnormalities in animal’s behaviour were found. The body weight increase was similar for all tested groups. Blood analysis did not reveal any systemic pathology. There were no inflammation (C-reactive protein negative, no leukocytosis) or organ necrosis. Animal’s organs were characterized by proper function and endurance – stable levels of total protein, creatinine, urea, total bilirubin were observed. Large fluctuations were observed in the enzyme activity. In the case of alanine aminotransferase (ALT) and gamma-glutamyltranspeptidase (GGT), the mean results significantly exceeded the reference range, however, the values ​​remained stable before implantation and before euthanasia. Blood count parameters were in reference range, so no impact on bone marrow was found. Coagulation parameters were also stable. Macroscopic evaluation of internal organs did not show any irregularities. Histopathological analysis revealed no pathological changes in internal organs or in implantation area.

Conclusion

The investigated material –zirconium stabilized with yttrium reveals no systemic toxicity in the long-term period of 26 weeks.

References

  1. R.Kustosz, et al., Archives of Metallurgy and Materials, V.60, 3/2015: p.2253–2260

Acknowledgement

The authors would like to thank NCBIR (Grant no: STRATEGMED-2/ RH-ROT/266798/15/NCBR/2015) for providing financial support to this project.

Keywords: A-01 c - Ceramic biomaterials, A-07 f - Cardiovascular incl. heart valve, A-08 a - Biocompatibility
PS1-02-39

LONG TERM IN VIVO BIOCOMATIBILITY STUDY OF ZIRCONIUM OXIDE-YTTRIUM STABILIZED ZrO2 · Y2O3 IN THE ASPECT OF LOCAL EFFECTS AFTER IMPLANTATION (#562)

B. Zawidlak-Węgrzyńska1, M. Gonsior1, K. Janiczak1, P. Ścigała1, D. Gonsior1, R. Kustosz1, A. Grajoszek3, R. Wojnicz2, E. Raichman-Warmusz2

1 Professor Zbigniew Religa Foundation of Cardiac Surgery Development, Zabrze, Poland
2 Medical University of Silesia, Department of Histology and Cell Pathology, Zabrze, Poland
3 Center for Experimental Medicine, Medical University of Silesia, Katowice, Poland

Introduction

Biocompatibility is one of the main requirements required in the preclinical evaluation of medical devices. In particular biocompatibility evaluation is essential for all materials that can be used in implantable medical devices in 3rd class of risk. In the clinical prototype of Polish implantable rotary blood pump ReligaHeart® ROT (RH ROT), the motor divider is made from ceramic composite, ZrO2 · Y2O3, material with high hardness, in order to improve device wear resistance. The aim of the study was to evaluate in vivo the tissue reaction after zirconium oxide-yttrium stabilized ZrO2 · Y2O3 implantation.

Experimental Methods

The test was carried out according to the standardized assays described in ISO 10993-6. A total of 48 New Zealand white both sexes rabbits weighing from 3000 to 3500 grams were used. Two ceramic implants (discs of 5 mm diameter 1,5 mm thickness) were aseptically inserted subcutaneously on the animal’s back. As a negative control Ti6Al7Nb titanium alloy was used. The animals were observed for: 4, 12 and 26 weeks. After animals euthanasia, a macroscopic evaluation of the implantation area was performed and the biomaterial implants were removed. The tissues were fixed with 4% formalin, embedded in paraffin, and stained with hematoxylin-eosin and Trichrome Masson for histological studies.

Results and Discussion

A micro-section of the implants showed that the investigated material implanted for the different periods: 4, 12 and 26 weeks, caused no response-mild fibrosis. There was no muscle degeneration, nor necrosis, nor any other significant change observed.

Conclusion

The zirconium oxide-yttrium stabilized ZrO2 · Y2O3 implanted in dorsal rabbit muscle did not induce any adverse tissue reactions in the long-term period of 26 weeks.

Acknowledgement

The authors would like to thank NCBIR (Grant no: STRATEGMED-2/ RH-ROT/266798/15/NCBR/2015) for providing financial support to this project.

Keywords: A-01 c - Ceramic biomaterials, A-08 a - Biocompatibility, A-07 f - Cardiovascular incl. heart valve
PS1-02-40

Enhanced Stability of Hollow Calcium Carbonate Microspheres via Polymorphic Control for Orthopedic Applications (#992)

C. M. Oral1, 2, D. Kapusuz3, B. Ercan1, 2, 4

1 Middle East Technical University, Metallurgical and Materials Engineering, Ankara, Turkey
2 Middle East Technical University, BIOMATEN, Center of Excellence in Biomaterials and Tissue Engineering, Ankara, Turkey
3 Gaziantep University, Metallurgical and Materials Engineering, Gaziantep, Turkey
4 Middle East Technical University, Biomedical Engineering Program, Ankara, Turkey

Introduction

Calcium carbonate (CaCO3) is a widely occurring mineral on earth due to its presence in exoskeletons of marine creatures [1]. Non-toxic nature, dispersability in aqueous media and suitable mechanical characteristics make CaCO3 a suitable candidate for orthopedic applications [2]. CaCO3 exists in three distinct polymorphs having different stabilities in aqueous environments [1]. Calcite is the most stable form of CaCO3, followed by aragonite in terms of stability [1,2]. Vaterite is the least stable polymorph, and thus it can easily transform to aragonite or calcite depending on the environment [1,2]. Having this said, among the various CaCO3 particle morphologies, hollow spherical CaCO3 particles attracted significant interest in orthopedics due to their low bulk density, high specific surface area and the ability to use its hollow inner core as a drug reservoir [3]. However, most of the hollow spherical CaCO3 particles consisted of vaterite, which leads to decreased therapeutic effect due to undesirable polymorphic transformations upon implantation. In this study, stable CaCO3 microspheres consisting of aragonite and calcite polymorphs were synthesized via N2 bubbling method for the first time in literature. Cytocompatibility and polymorphic stability tests were conducted on the hollow aragonite and calcite microspheres to investigate the particles in vitro conditions. These experiments were also repeated using vaterite microspheres as a control group.

Experimental Methods

Calcium acetate (Ca(C2H3O2)2) and sodium bicarbonate (NaHCO3) solutions were used as precursors to precipitate CaCO3 particles. To obtain an inner hollow core, N2 gas and sodium dodecyl sulfate (SDS) was incorporated to the precursor solutions at different temperatures. Vaterite microspheres were synthesized by adjusting precursor solution pH to 9. Cytotoxicity experiments were completed with osteoblasts (ATCC CRL-11372) using a cell culture media supplemented with various CaCO3 concentrations up to 5 days of culture. Stability of synthesized microspheres were investigated by exposing them to DMEM at 37 °C for 5 days.

Results and Discussion

The structural and morphological differences between CaCO3 microspheres were shown in Fig. 1. XRD analysis revealed that these particles consisted of calcite (Fig. 1a), aragonite (Fig. 1b) and vaterite (Fig. 1c) polymorphs. Though the bulk morphology of the synthesized CaCO3 particles were all spherical, their constituent particles were platelet, needle-like and spherical for calcite, aragonite and vaterite microspheres, respectively. The differences in the constituent particles originated from their crystal structures, which further dictated their stabilities in aqueous environments. Polymorph identification of the powders were also supported with FTIR, HR-TEM and Rietveld analysis. Specifically, HR-TEM micrographs revealed d-spacing values of 0.305, 0.345 and 0.330 nm corresponded to calcite (104), aragonite (111) and vaterite (112) planes, respectively. Hollow inner cores of the CaCO3 microspheres were imaged with TEM. BET analysis exhibited porous nature of the hollow microspheres, which would allow the use of hollow inner core as a reservoir for drug release. In vitro cytotoxicity tests showed that CaCO3 microspheres did not exhibit any toxic effect at corresponding calcium ion concentrations present in the serum. However, as the CaCO3 particle concentration increased, vaterite microspheres showed slight toxicity, while hollow aragonite and calcite microspheres, synthesized for the first time in this research, promoted osteoblast cellular functions. The reason behind the increased toxicity of vaterite microspheres could be correlated with unstability of vaterite in aqueous environments. While dissolution of particles was observed for vaterite microspheres, calcite and aragonite microspheres preserved their morphology in DMEM.

Conclusion

Hollow calcite and aragonite microspheres were obtained by supplying N2 bubbles and SDS to the precipitation system. Detailed characterization experiments highlighted porous nature of these microspheres, where the hollow core can be used as a reservoir for drug delivery. In vitro cytotoxicity tests revealed differences between stable calcite and aragonite microspheres compared to unstable vaterite microspheres. It can be concluded that hollow calcite and aragonite microspheres can be promising candidates for orthopedic applications.

References

[1] Tas, (2015). Applied Surface Science, 330, 262-269.

[2] Boyjoo, et al., (2014). Journal of Materials Chemistry A, 2, 14270-14288.

[3] Mizuno, et al., (2011). Advanced Powder Technology, 20, 89-93.

Acknowledgement

We would like to thank Middle East Technical University Research Funds (BAP-03-08-2016-009) and Scientific and Technological Research Council of Turkey (117M754) for providing financial support.

SEM micrographs of microspheres

Fig. 1. SEM micrographs of a) calcite, b) aragonite and c) vaterite microspheres. Scale bars are 2 µm.

Keywords: A-01 c - Ceramic biomaterials, A-08 b - Biodegradation, A-06 a - Biomaterials for drug delivery
PS1-02-41

Characterization and biocompatibility of inversely 3D-printed β-Tricalcium Phosphate Scaffolds (#1000)

M. Seidenstuecker1, S. Lange1, S. Esslinger2, R. Gadow2, A. Bernstein1

1 Medical Center - Albert-Ludwigs-University of Freiburg, G.E.R.N. Tissue Replacement, Regeneration & Neogenesis, Department of Orthopedics and Trauma Surgery, Freiburg, Baden-Württemberg, Germany
2 University Stuttgart, Institute for Manufacturing Technologies of Ceramic Components and Composites (IMTCCC), Stuttgart, Baden-Württemberg, Germany

Introduction

Bone disorders and defects continue to increase in our society. The reason for this is the increasing average age of the population [1]. According to the Federal Statistical Office, 191,272 endoprostheses were implanted in the knee joint in Germany in 2017 [2]. The development of 3D printing and the associated tissue engineering (TE) opened up new possibilities for bone replacement [3]. There are different types of 3D printing. These include the so-called Fused Deposition Modeling (FDM) process, which was described by S. Scott Crump [4] in the late 1980s and has since become indispensable in the research and development of 3D printing. The aim of a printed replacement material is to replace healthy tissue. This means that over time the material dissolves and is replaced by the corresponding tissue at the implanted site. The aim of the project was to characterise inverse 3D printed β-TCP scaffolds and to investigate the cell growth behaviour within the scaffolds.

Experimental Methods

For the physical properties, fracture tests were performed to determine the maximum failure load of the samples. The aim was to test untreated specimens and treated specimens previously placed in a Simulated Body Fluid (SBF) [5] solution for 28 days. The SBF solution should simulate the body fluids. Furthermore, the inner pore structures of the ceramic should be examined to explain possible effects on the stability of the samples.

For biocompatibility testing, three different in vitro experiments should be performed: A proliferation test, a cytotoxicity test and a live/dead staining test. The cells used were osteoblast-like MG-63 cells (ATCC CRL 1427), which are a cell line of an osteosarcoma.

A WST assay was used to provide information on the proliferation rate of MG-63 cells on the ceramic scaffolds. An LDH assay was used to measure the cytotoxicity of the material and a live/dead staining was used to investigate the growth behaviour of the cells on the samples and in the samples. The aim was to determine whether the cells grow into scaffolds with an inverse structure or only adhere to the surface.

Results and Discussion

The scaffolds with a strand spacing of 500 µm show the highest compressive strength (190 ± 12 N) both untreated and treated with SBF (105 ± 72 N). The SBF basically reduces the stability of the samples. The pore structure within the ceramic does not play a decisive role for stability. Here, the strand spacing itself and the powder properties of the samples are decisive factors for stability. The fact that β-TCP is a biocompatible material could be confirmed by the experiments. No LDH activity of the cells was measured, which means that no cytotoxicity of the material can be detected. In addition, the proliferation rate of all three sizes increases steadily over the test days until saturation. The cells are largely adhered to or within the scaffolds and have not migrated through the samples. The cells show increased growth not only on the outer surface, but also on the inner surface of the samples. This means that the inverse pressure method is suitable for ingrowth of the cells.

Conclusion

The experiments on which this work is based have shown that the FDM method with subsequent slip casting and sintering is well suited for the production of scaffolds for bone replacement.

References

1.         Bose, S., M. Roy, and A. Bandyopadhyay, Recent advances in bone tissue engineering scaffolds. Trends in Biotechnology, 2012. 30(10): p. 546-554.

2.         Deutschland, S.B. Operationen und Prozeduren der vollstationären Patientinnen und Patienten in Krankenhäusern. 2017; Available from: https://www.destatis.de/DE/Publikationen/Thematisch/Gesundheit/Krankenhaeuser/OperationenProzeduren5231401177014.pdf;jsessionid=803B9A7B0D5EC566EB33C4334203A4FC.InternetLive1?__blob=publicationFile.

3.         Salgado, A., O. Coutinho, and R. Reis, Bone Tissue Engineering: State of the Art and Future Trends. 2004.

4.         Crump, S.S., Apparatus and Method For Creating Three- Dimensional Objects, in United States patent. 1992, Stratasys, Inc.: USA.

5.         Helebrant, A., L. Jonasova, and L. Sanda, The Influence Of Simulated Body Fluid Composition On Carbonated Hydroxyapatite Formation. 2001.

Keywords: A-01 c - Ceramic biomaterials, A-03 b - 3D scaffolds for TE applications, A-08 a - Biocompatibility
PS1-02-42

A Bone Substitute with High Bioactivity, strength, and Porosity for Repairing Large Bone Defects (#1046)

N. Golafshan1, M. Castilho1, M. Braham1, J. Albass1, S. Zaharievski1, J. Malda1

1 UMC Utrecht, Orthopaedics department, Utrecht, Netherlands

Introduction

Three-dimensionally (3D) printed of ceramic materials, mostly calcium phosphates (CaP), is an excellent method for the fabrication of individual bone scaffolds with complex geometries. Despite their inherent bioactive and osteoconductive properties, they present limited mechanical characteristics and handling properties. To overcome this limitation, blends of CaP with degradable synthetic polymers, like polycaprolactone (PCL), have been used. However, the inclusion of synthetic polymers reduces cell adhesion and bioactivity due to polymer masking. Further, magnesium phosphate cement (MgPC) modified with strontium (Sr), is a promising alternative to CaP cement substituted by biologically active agent, due to its biocompatibility and high osteoconductive potential. In this study, we aimed to investigate the fabrication of individual bone implants with improve ostinductive potential and mechanical properties via room temperature extrusion based 3D printing of MgSP-PCL composites. A bioactive bone scaffold, contains Strontium Magnesium phosphate (MgSrP) and polycaprolactone (PCL) was investigated in this study and the 3D structure can combines high mechanical properties such as strength and toughness with impressive bone regeneration ability. The scaffolds induce substantial bone formation and defect bridging after 6 months, as indicated by micro-computed tomography scanning.

Experimental Methods

To fabricate 3D bone scaffolds, the MgSP-PCL (Magnesium Strontium phosphate), MgP-PCL (Magnesium phosphate), and HA-PCL (Hydroxyapatite) solutions were prepared with the ratio of 70-30 weight ratio, dissolved in a trisolvent mix (DCM: 2-Bu: DBP mixed in a 10:2:1 weight ratio) and their processing compatibility systematically investigated according to key process parameters (pressure, feeding rate, needle size). Cylindrical scaffolds were printed with an diameter of 10 mm, height 10 mm, strand spacing 100 mm and 2 layers of no spacing at the interface side, 22G nozzle) by using a pneumatic-driven bioprinter (regenHU, Villaz-St-Pierre, Switzerland) (Fig. 1A). Structural characterisation, X-ray diffraction, ALP activity and calcium deposition, and in-vivo evaluation were performed during this study.

Results and Discussion

According to SEM images of the ceramics powder, the size of HA and MgP were 2.45±0.57 µm and 3.58±1.91 µm, respectively which is similar to the particle size of the MgSP (2.33±0.52 µm, Fig.1B). The X-ray diffraction in Fig. 1C demonstrated that resulting ceramics consisted of pure MgSP, MgP, and HA with approved crystallinity. According to Fig.1E, the reconstructed Micro-CT image revealed a comprehensive overview of the microstructures of the scaffold and the SEM image (Fig. 1B) shows the cross-sectional morphology of the scaffold. Together, the Micro-CT and SEM results demonstrated the porous structure of the scaffolds, suggesting that the scaffold has very highly porous architecture. The controlled released ions or degraded particles from the scaffolds are supposed to adjust the local microenvironment, which determines the response and behavior of host cells. For all the scaffolds during 21 days, no remarkable changes of the concentrations or burst release of ions were observed with time prolongation, and followed by a gradually increasing release and the concentrations of ions released from the scaffolds were close to the optimal from day 1 to 21 (Fig. 2A). Moreover, according to Fig.2B, Alkaline phosphatase (ALP) activity initially is the same after 7 days of culturing because the cells proliferated and spread on the surface of the scaffolds but increased significantly after 14 and 21 days as the cells underwent differentiation. The extracellular matrix (ECM) mineralization of equine MSCs with various scaffolds was tested by alizarin red staining during 38 days of culturing. These complementary techniques revealed the deposition of calcium and phosphate into the extracellular matrix by the equine MSCs (Kim et al., 2018). For all cases, more intense colors were observed on the scaffolds with ceramics particles than on PCL as control in both media (Fig. 2C). Furthermore, the MgSP-PCL scaffolds induce substantial bone formation and defect bridging after 6 months, as indicated by micro-computed tomography scanning (Fig. 2D). We analyzed the trabecular density (BV/TV) and it is apparent that the bone regeneration with the implant is significantly higher than the empty defect.

Conclusion

The ability to produce cost effective and highly bioactive bone scaffolds are able to induce bone healing in large bone defects. It will be remarkable potential to augment or substitute current surgical approaches for bone repair using autologous or allogeneic bone grafts.

References

[1]Bellucci, D., Cannillo, V., Anesi, A., Salvatori, R., Chiarini, L., Manfredini, T., & Zaffe, D. (2018). 

[2]Kanter, B., Vikman, A., Bruckner, T., Schamel, M., Gbureck, U., & Ignatius, A. (2018). 

[3]Kim, J. Y., Ahn, G., Kim, C., Lee, J. S., Lee, I. G., An, S. H., … Shim, J. H. (2018). 

Figure 2

A) Ion release from 3D printed HA-PCL, MgP-PCL, and MgSP-PCL scaffolds during soaking in water. B) The ALP activity levels were measured and normalized to DNA content. C) Micro-CT images from one of the ponies after 6 months implantation.

Figure 1.

 A) Fabrication of cylindrical scaffolds. B) SEM images of the various ceramics powder. C) X-ray diffraction patterns of pure ceramics. D) representative SEM images from the 3D printed scaffolds. E) Micro-CT scanning of the scaffolds containing MgSP. F) The porosity of the large printed constructs contains various ceramics particle (p˃0.05).

Keywords: A-01 c - Ceramic biomaterials, A-03 b - 3D scaffolds for TE applications, A-05 e - 3D bioprinting/biofabrication
PS1-02-43

Wear Behaviour of Zirconia Containing Dispersion Ceramics Combined with Accelerated Ageing (#708)

T. Oberbach1, M. Al- Hajjar2, L. Gremmilard3, K. Hans1, J. Chevalier3, L. Jennings2

1 Mathys Orthopaedie GmbH, Bioceramics, Moersdorf, Thuringia, Germany
2 University of Leeds, Institute of Medical and Biological Engineering, School of Mechanical Engineering, Leeds, United Kingdom
3 University of Lyon, INSA Lyon, CNRS, MATEIS UMR 5510, Villeurbanne Cx, France

Introduction

Ceramic-on-ceramic bearings in total hip replacement have been used for more than 45 years. The have shown their potential for young and active patients. In this study, a methodology was devised to assess the in vitro wear behaviour of Zirconia Toughened Alumina Ceramics (ZTA) and Alumina Toughened Zirconia Ceramics (ATZ) under adverse edge loading conditions in hip simulator combined with accelerated ageing in an autoclave under hydrothermal conditions.

Experimental Methods

Femoral heads and acetabular liners of hip prostheses were made of two materials: Alumina Toughened Zirconia (ATZ) and Zirconia Toughened Alumina Ceramic (ZTA). Two material combinations were tested in this study: ATZ-on-ATZ and ZTA-on-ZTA.

A total of six bearing couples were studied on the Leeds Mark II Physiological Anatomical hip joint simulator.

The study was run for a total of eight million cycles. The first two million cycles were run using standard gait conditions and the subsequent 6 million cycles were run under edge loading conditions due to dynamic separation between the femoral head and the acetabular cup.

All femoral heads and acetabular cups were hydrothermally aged during the wear study after every million cycles of testing. It lasted 2 hours at 134°C after each million cycles. Hydrothermal ageing was achieved using accelerated ageing protocol in an autoclave (Sanoclav LA-MCS, Wolf, Germany) in water vapour.

The volume monoclinic fraction was determined using XRD and Garvie and Nicholson’s equation modified by Toraya.

Scanning Electron Microscopy observations were conducted on pristine, worn and aged surfaces on the heads after various testing times, using a Supra 55 VP microscope (Zeiss, Germany), at low acceleration voltage (1 to 2 kV) so as to avoid coating the observed surfaces.

The wear was measured gravimetrically using a balance (XP205, Mettler-Toledo) at an interval of one million cycles. A coordinate measuring machine (Legex 322, Mitutoyo, Japan) was used to reconstruct the surface of the femoral head and acetabular cup. RedLux software (RedLux, UK) was used to visualise the size, shape and penetration depth of the wear areas.

Results and Discussion

The wear rates of both aged materials, ATZ-on-ATZ and ZTA-on-ZTA, under standard conditions were very low, i.e. <0.01 mm3/million cycles. There was no measureable change in wear rate due to ageing under standard conditions.

The wear rates increased when edge loading conditions driven by separation was introduced to the gait cycle. The mean wear rate of aged ZTA-on-ZTA after six million cycles of testing under edge loading conditions was 0.19 ± 0.47 mm3/million cycles. The mean wear rate of aged ATZ-on-ATZ was 0.07 ± 0.05 mm3/million cycles.

There was no visible damage on the surfaces of the femoral head and acetabular cup after testing under standard conditions. In contrast, under edge loading conditions, a stripe-like wear area was observed on the femoral head with corresponding wear on the rim of the acetabular liner. The penetration depths on the femoral heads and acetabular liners of the ZTA-on-ZTA bearings were higher than that of the ATZ-on-ATZ bearings after 6 million cycles of testing under edge loading conditions.

No significant ageing occurred in the ZTA material. The monoclinic fractions remained very low over both the wear stripe and the unworn surface. Their variations were within the error margin of XRD.

Ageing of ATZ heads was significant. On the wear stripe, each autoclave step increased the monoclinic fraction. However, each one million cycles of wear simulation decreased the monoclinic fraction significantly.

SEM observations of the worn ATZ and ZTA surfaces showed that the first 2 million cycles (without edge loading) did not significantly damage the surfaces. Microstructural damage was first observed after edge loading. In ATZ damage was located in a small wear stripe 15 µm wide. In ZTA, damage was first located in a much more diffuse area around 50 µm wide.

SEM observations further showed that the damage was mainly located in the alumina grains but not in the monoclinic-zirconia grains for ATZ. In both cases ZTA and ATZ, the phase under the highest compressive residual stresses seemed to be the most prone to microstructural damage.

Conclusion

In this study, a method was devised by which the performance of composite ceramic materials was assessed under a combination of edge loading gait conditions and hydrothermal ageing.

The damage of ceramic components increased by the symbiotic effect of ageing, wear and shocks but remained at a very low level for both ceramic materials. It was shown that the performance of ATZ-on-ATZ materials in vitro may be superior to ZTA-on-ZTA materials despite the higher zirconia content in the ATZ materials.

Ceramic composites show an extremely low wear rate, even under worst case conditions, and provides an interesting option to meet the demands of younger more active patients.

Keywords: A-01 c - Ceramic biomaterials, A-08 a - Biocompatibility, A-07 d - Bone
PS1-02-44

Inhalable Calcium Phosphate Nanoparticles for Cardiac Drug Delivery (#102)

L. Degli Esposti1, F. Carella1, A. Adamiano1, P. Carullo2, A. Tampieri1, M. Miragoli3, 2, D. Catalucci2, M. Iafisco1

1 National Research Council, Institute of Science and Technology for Ceramics, Faenza, Italy
2 National Research Council, Institute of Genetics and Biomedical Research, Milano, Italy
3 University of Parma, Department of Medicine and Surgery, Parma, Italy

Introduction

Cardiovascular diseases (CVDs) are a worldwide growing problem that cause 17.3 million annual premature deaths1. This situation prompts to identify new therapeutic compounds as well as to develop efficient drug-delivery systems for the treatment of CVDs. Among therapeutic compounds, microRNAs (miRs) and peptides are of great interest, since they are key regulators of cardiac dysfunction and protein activity2,3. However, the approaches that employ therapeutic miRs or peptides are still not optimal, since in vivo delivery has resulted so far to be inadequate. Nanoparticles (NPs) delivery platforms hold great promise to overcome such limitations, providing a strategy for efficient drug-delivery approaches. Therefore the aim of the present work was the generation of effective nanoparticles formulation for the delivery of novel therapeutic drugs into cardiac tissue. Calcium phosphate nanoparticles (CaP NPs) have been selected, since they possess superior biocompatibility and biodegradability compared to other inorganic nanoparticles, and they are able to bind a plethora of therapeutic agents4.

Experimental Methods

A straightforward one-pot CaP NPs synthesis protocol using citrate as stabilizing agent has been adopted. Two aqueous solutions containing respectively CaCl2 (100 mM) + Na3Citrate (400 mM) and Na2HPO4 (120 mM) were mixed (1:1 v/v) and the pH was adjusted to 8.5; when drug conjugation was performed aqueous solution of miR (100 μg/ml) or peptide (500 μg/ml) was added. The CaP suspension was kept in a water bath at 37°C for 5 min. Afterward, the CaP NPs suspension was dialyzed for 6 h. CaP NPs were characterized by TEM, DLS and ζ-potential in order to measure nanoparticle morphology, size distribution and surface charge, respectively. The biocompatibility and the mechanism of internalization of CaP NPs in cardiac cells were studied on murine HL-1 cells. The test of the ability of CaP NPs to reach the heart was made on CD1 mice, administering CaP NPs by gavage, intraperitoneal, and intravenous injection as well as by intratracheal nebulization. The test of the therapeutic efficiency was made via inhalation of peptide-loaded CaP NPs in a mouse model of streptozotocin induced diabetic cardiomyopathy.

Results and Discussion

Nanoparticle morphological characterization showed that CaP NPs are round shaped particles of about 50 nm in diameter (Fig. 1). DLS analysis evinced that the nanoparticles have a small hydrodynamic diameter and possess a strongly negative surface charge, indicating that the negatively-charged citrate molecules covered the surface of NPs. CaP NPs were proven to be highly biocompatible and showed no significant cytotoxicity effects on cardiac HL-1 cells5. In addition, CaP NPs were proven to not alter cardiac cells functional properties. In order to test the efficacy of drug loaded CaP NPs, cardiac HL-1 cells were exposed to CaP NPs carrying the therapeutic miR-133. qRT-PCR analyses revealed a time-dependent increase in the levels of intracellular delivered miRNA, confirming the effective loading of miR into CaP NPs as well as their cellular uptake. Additionally, a cell-based luciferase assay confirmed that the administered miR-133 efficiently repressed a miR-133-specific target5. Subsequently CaP NPs were tested in vivo, evaluating the heart uptake of fluorescent-labelled nanoparticles administered by several routes. Parenteral administration and inhalation resulted in rapid delivery of CaP NPs to the myocardium, with inhalation being the most efficient delivery method (Fig. 2, left)6. Moreover, with the inhalation route, the myocardial accumulation was paralleled by a gradual reduction in signal from the lungs, confirming the passage of CaP NPs across the pulmonary barrier (Fig. 2, right). To prove the efficacy of drug-loaded CaP NPs formulation in synergy with the inhalation administration we have treated a mouse model of streptozotocin induced diabetic cardiomyopathy. Inhalation of therapeutic peptide loaded CaP NPs led to a complete recovery of cardiac function in diabetic mice, with the restoration of protein-related contractile properties6.

Conclusion

Our results have proven that CaP NPs can easily bind miRs and peptides, that are molecules with enormous therapeutic potentials. In particular, we have demonstrated that miR- or peptide- loaded CaP NPs are biocompatible and non-toxic for the highly sensitive myocardial tissues. We have proved in vivo in mice that CaP NPs efficiently reach the heart, especially with the non-invasive inhalation route. Finally, we have demonstrated the successful delivery of a peptide payload to the heart by CaP NPs, that exerts a therapeutic effect and restores cardiac functionality in a diabetic cardiomyopathy mouse model. In conclusion, the combination of innovative therapeutic agents such as miRs and peptides with CaP NPs and with the non-invasive inhalation route provides a unique and highly efficient approach to treat CVDs.

References

  1. WHO. Cardiovascular diseases.  www.who.int/cardiovascular_diseases/en/
  2. Poller W, Hajjar R, Schultheiss HP, Fechner H Cardiovasc. Res. 86(3) (2010) 353–364
  3. F. Rusconi et al. Circulation 134 (2016) 534–546
  4. Degli Esposti L, Carella F, Adamiano A, Tampieri A,  Iafisco M Drug development and industrial pharmacy, 44(8) (2018) 1223-1238
  5. Di Mauro V, Iafisco M, Salvarani N, Vacchiano M, Carullo P, Ramírez-Rodríguez G-B, Catalucci D Nanomedicine 11(8) (2016) 891-906.
  6. Miragoli M et al. Science translational medicine 10(424) (2018) eaan6205.

Acknowledgement

The authors acknowledge the H2020-NMBP-2016 720834 European project CUPIDO (www.cupidoproject.eu).

Figure 1
TEM micrograph of CaP NPs. Inset: corresponding SAED pattern. Reprinted with permission from Ref.5
Figure 2
Left: quantification of fluorescence signals from heart tissue of mice treated with Cy7-CaP NPs via gavage, intraperitoneal (ip), intravenous (iv), and inhalation administration. Right: time-course quantification of fluorescence signals from heart and lung tissue of mice treated with Cy7-CaP NPs via inhalation administration. Reprinted with permisison from Ref.6
Keywords: A-01 d - Calcium phosphates, A-06 a - Biomaterials for drug delivery, A-07 f - Cardiovascular incl. heart valve
PS1-02-45

Synthesis and characterization of OCP with different preparation condition (#181)

T. Hayashi1, G. Tanabe1, M. Ando1, T. Kumei1, K. Tsuchiya2, R. Hamai2, O. Suzuki2

1 Japan Fine Ceramics Co, LTD, Development, Sendai, Japan
2 Tohoku University Graduate School of Dentistry, Division of Craniofacial Function Engineering, Sendai, Japan

Introduction

     Autologous bone has been used for repairing bone defects in patients as the first choice due to its bone regenerative property. However, there are quantitative restrictions on the use of autogenous bone, and there is concern that another obstacle may occur at the site from which the autologous bone is collected.

     Thus, artificial bones and their materials for substituting for the autologous   bone have been studied. Calcium phosphate ceramics such as hydroxyapatite(Ca10(PO4)6(OH)2, HA)and β-tricalcium phosphate (Ca3(PO4)2, β-TCP) have been applied as the bone substitute materials in the field of orthopedic and oral surgeries [1].

     In recent years, it has been reported that octacalcium phosphate (Ca8H2 (PO4)6 ·5H2O, OCP), which is a precursor of HA, has superior characteristics as bone regeneration material, showing osteoconductivity and biodegradability compared to HA and β-TCP, because OCP promotes osteoblastic differentiation and osteoclast formation [2, 3].  On the other hand, previous studies indicate that OCP with non-stoichiometric composition was easily obtained [4]. It has been known that synthesis method and treatment of OCP affect molar ratio of Ca/P in OCP. Especially, Suzuki et al reported that the ratio of hydrogen phosphate in all phosphorus (HPO4/P ratio) of OCP was present as labile form which can be changed reversibly by pH in solution [4].

     Previously, Miyatake et al revealed that the chemical composition of OCP influenced its osteoconductivity and biodegradability in bone defect [5]. Based on these studies described above, we hypothesized that HPO4/P ratio of OCP could be related to its chemical characteristics which may affect the bone regeneration by OCP.

     In this study, we prepared OCP batches having different HPO4/P ratio and investigated their dissolution behavior under a physiological condition in vitro.

Experimental Methods

Synthesis of OCP with different HPO4 / P ratio

     OCP was prepared by the wet synthesis method using a system for continuous synthesis which can easily control conditions of the synthesis compared to the batch type preparation. The synthesis of OCP was carried out by controlling the conditions (pH, flow rate, etc.) in the reaction field. The different types of the prepared OCP were abbreviated as OCP-A and OCP-B.

     The obtained compounds were identified by analyses of X-ray diffraction (XRD) and HPO4/P ratio. The XRD pattern of the crystals was recorded using step scanning at 0.02 deg intervals from 3.0 to 60.0 deg with Cu  X-rays on a diffractometer at 40 kV and 15 mA. HPO4/ P ratio of them was determined indirectly based on the chemical analysis by heat-induced pyrophosphate method [4, 6]. The powdery OCPs were heated above 500°C overnight. The formed pyrophosphate was hydrolyzed in an acidic solution (perchloric acid solution) under boiling. The phosphate concentrations of both the hydrolyzed and non-hydrolyzed samples were determined colorimetrically using a phosphorous analytical test agent.

 

Analysis of difference in dissolution behavior

     In order to compare the dissolution behavior of OCP with different HPO4/P ratio, the two types of OCPs were immersed in tris(hydroxymethyl)aminomethane (Tris)-HCl buffer for 1, 7 and 14 days. After the immersion, OCPs were washed with pure water and then lyophilized. Subsequently, they were characterized by XRD and subjected for the chemical analysis to measure HPO4/P ratio. Moreover, the morphology of them was observed using a transmission electron microscope (TEM).

Results and Discussion

Characterization of OCP synthesized by the continuous synthesis method

     As a result of the chemical analysis, it was confirmed that two types of OCPs having different HPO4/P ratio were obtained by controlling the conditions of the reaction field during the synthesis. The HPO4/P ratio of OCP-A was larger than that of OCP-B, while XRD analysis confirmed that both crystals had the structure of OCP.

 

Analysis of dissolution behavior

     The behavior of change in the HPO4/P ratio was different between OCP-A and OCP-B during the immersion in Tris-HCl Buffer, which was measured by the chemical analysis [4, 6]. In addition, XRD patterns of the OCPs indicate that the hydrolysis of OCP-B tended to advance earlier than OCP-A in the Tris-HCl buffer. TEM observation also showed that both OCP underwent morphological changes after immersion in the buffer.

 

Consequently, it was suggested that the chemical composition of OCP is related to the change in dissolution behavior of OCP in Tris-HCl buffer.

Conclusion

     The present study suggests that OCPs with different ion balance could be synthesized by controlling the reaction field. The results of this study also suggested that the balance of ions characterized by HPO4/P ratio could be involved in the dissolution behavior of OCP.

References

1. R.Z. LeGeros. Chem Rev 108:4742-4753, 2008

2. O. Suzuki. Jpn Dent Sci Rev 49:58-71, 2013

3. T. Sato et al. Acta Biomater 2019 in press. doi: 10.1016/j.actbio.2019.03.001

4. O. Suzuki et al. J Dent Res 74:1764-1769, 1995

5. N. Miyatake et al. Biomaterials 30:1005-1014, 2009

6. A. Gee, V.R. Deitz. Anal Chem 25:1320-1324, 1953

Acknowledgement

This study was carried out by a joint research program between Japan Fine Ceramics Co, LTD and Tohoku University.

Keywords: A-01 d - Calcium phosphates, A-01 c - Ceramic biomaterials, A-07 d - Bone
PS1-02-46

Bone regeneration using interconnected pores β-tricalcium phosphate (β-TCP) block made by the setting reaction of β-TCP granules (#216)

K. Ishikawa1, K. Hayashi1, T. S. Putri1

1 Kyushu University, Department of Biomaterials, Fukuoka, Japan

Introduction

Interconnected porous β-tricalcium phosphate (β-TCP) block may be useful as artificial bone substitutes. Although various porous β-TCP blocks are proposed further improvement is awaited due to insufficient interconnectivity or imbalance of mechanical strength and porosity. In this study, interconnected porous β-TCP blocks are fabricated by setting reaction of β-TCP granules. And its potential value was evaluated by physical measurement and histologically using rabbits.

Experimental Methods

Porous β-TCP blocks were fabricated by the setting reaction of β-TCP granules (200 to 300 µm) using a HNO3 solution followed by sintering at 1100°C. XRD, µ-CT, SEM and universal testing machine were used for measurement of their physical properties. For histological evaluation, porous β-TCP blocks and dense β-TCP blocks used as control were implanted in bone defect at medial femoral condyle of rabbits. After 4 weeks, H-E stained specimens were observed using all-in-one fluorescence microscope.

Results and Discussion

When β-TCP granules were mixed with 5mol/L HNO3, dicalcium phosphate dihydrate (DCPD) is formed and bridged β-TCP granules one another. Following heat treatment at 1100°C, interconnected dual porous pure β-TCP block was fabricated as shown in Fig 1. It has fully interconnected porous structure with pore size of 40 to 160 µm. The interconnection was achieved by the setting reaction of the granules and micro porous structure was obtained by the micropore of the β-TCP granules. Diametral tensile strength (DTS) and porosity of the porous β-TCP block were 1.4 ± 0.2 MPa and 58.1 ± 1.7%, respectively. The DTS value is enough for clinical use and balance between mechanical strength and porosity seems reasonable.

Four weeks after implantation, both porous and dense β-TCP block bonded with the existing bone and no inflammatory response was observed. In the case of dense β-TCP block, only 0.2 ± 0.1% of the β-TCP was resorbed, and amount of the formed new bone was limited (0.1 ± 0.1 %). And no no cells or tissues were observed interior of the dense β-TCP block. In the case of porous β-TCP, 9.2 ± 3.1% of the β-TCP was resorbed, and amount of new bone was 18.9 ± 5.5%. Moreover, osteoblasts, osteoclasts, osteocytes, red blood cells, bone and fibrous tissues were observed within the porous β-TCP block. Presence of osteoblasts, osteoclasts, and osteocytes indicate active bone remodeling process interior of the porous β-TCP block. Presence of accumulated red blood cells indicates capillary vessel or Haversian canal-like structure formation. Clear difference between porous and dense β-TCP blocks demonstrated the importance of the interconnected porous structure.

Conclusion

Porous β-TCP blocks fabricated by the setting reaction of β-TCP granules presented interconnected dual porous structure, as well as balanced porosity (58.1 ± 1.7%) and mechanical strength (DTS: 1.4 ± 0.2 MPa). Resorption of β-TCP (9.2 ± 3.1%) and new bone formation (18.9 ± 5.5%) after 4 weeks implantation exhibit a good potential value to be good artificial bone substitutes.

Acknowledgement

This research was supported, in part, by AMED under Grant Number JP18im0502004.

Interconnected pores β-TCP block
Photo (a), μ-CT (b), and SEM (c,d) images of interconnected pores β-TCP block
Histological images
H-E stained picture of dense (a, c, e) and porous (b, d, f) β-TCP blocks after four weeks implantation.

NB: New bone; HB: Host bone; FT: Fibrous Tissue; S: Sample; RBC: Red blood cells; Blue arrowheads: Osteoblasts; Red arrow: Osteoclasts; Yellow arrow: Osteocyte

Keywords: A-01 d - Calcium phosphates, A-07 d - Bone , A-01 c - Ceramic biomaterials
PS1-02-47

Novel electrical characterization of hydroxyapatite coatings on titanium: Influence of the layer thickness and manufacturing temperature (#332)

D. Romero-Guzmán1, 2, 3, V. Luque-Agudo1, 2, 3, A. M. Gallardo-Moreno1, 2, 3, M. L. González-Martín1, 2, 3

1 University of Extremadura, Department of Applied Physics, Badajoz, Spain
2 Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Badajoz, Spain
3 University Institute of Biosanitary Research of Extremadura (iNube), Badajoz, Spain

Introduction

Titanium and its alloys are within the most widely used metallic biomaterials in implants due to their good mechanical properties and high corrosion resistance. On the other hand, hydroxyapatite (HAp, Ca10(PO4)6(OH)2) is considered the best ceramic biomaterial in the field of medicine1. This last material is used to coat metallic materials, such as titanium, due to the similarity in the chemical composition and its excellent biocompatibility with bone tissue.

Besides the importance of bulk properties of the biomaterials, surface properties are decisive in the biocompatibility of any implant. In particular, the surface charge has great influence on any bioadhesion process happening on the surfaces of any medical device. The electrical interactions can promote osseointegration and avoid bacterial colonization. However, there are scarce information of the electrical interaction potential (zeta potential) of large metallic biomedical surfaces and, in particular, those modified with coating layers.

This research deepens in the electrical behaviour of thin hydroxyapatite coatings on pure titanium using streaming current. Initial offset values of current-pressure plots and temporal electrical stability are within the new proposed parameters to better understand the electrical response of the biomaterials.

Experimental Methods

The samples have been coated with HAp2 by a sputtering process carried out by the company Sidrabe (Rīga, LV-1073, Latvia). Two temperatures were used in the manufacturing process: 400°C and 500°C, and two sputtering time that translates into different thickness of the HAp layer: 100 and 150 nm. Samples have been named as 400°C-100nm, 400°C-150nm, 500°C-100nm and 500°C-150nm, accordingly.

The electrical analysis has been carried out by means of an Electro Kinetic Analyzer (EKA) using streaming current measurements. In each determination, the variation with time (from 0 up to 2 hours) of the initial streaming current is analysed.

Results and Discussion

Two interesting behaviours on the electrical response of HAp layers have been observed. Firstly, there is a temporal evolution in the zeta potential of samples. At initial stages of the experiments, for given value of temperature, or thickness, the highest superficial charge corresponds to the lower thickness, or temperature, respectively. Therefore, the 400°C-100nm HAp sample is the only one with an initial surface charge greater than the titanium control. Time evolution shows a decrease in the absolute value of zeta potential for the titanium control and HAp coatings made at 400°C. This effect is not observed in samples manufactured at 500°C. Surface charge of coatings prepared at the highest temperature are stable over the time. Because of the different time evolution of coatings, zeta potential of samples prepared at 500°C becomes the most negative after 120 minutes of measurement, exhibiting a higher absolute value even than titanium control.

Secondly, it is remarkable the very different response observed in the initial offset of the electrical measurements. Residual charge in the HAp layer prepared at 400°C may be related with the especially abrupt offset observed for these coatings.

Conclusion

Analysis of zeta potential changes with time provides a valuable characterization of the electrical behaviour of different HAp layers. This new procedure for the study of streaming current and zeta potential allows to a better knowledge of the properties of HAp obtained by different procedures. Electrical stability is obtained for coatings prepared at the highest temperature.

References

[1] Harun, W.S.W., Asri, R.I.M., Alias, J., Zulkifli, F.H., Kadirgama, K., Ghani, S.A.C., Shariffuddin, J.H.M. (2018). A comprehensive review of hydroxyapatite-based coatings adhesion on metallic biomaterials. Ceramics International, 44 (2), 1250–1268.

[2] Pluduma, L., Gross, K.A., Rey, C., Ubelis, A., Berzina, A. (2018). Production and characterization of oxyhydroxyapatites. Key Engineering Materials, 762, 48-53.

Acknowledgement

Financial support is acknowledged to the Spanish Junta de Extremadura and FEDER grants for the projects IB16117 and GR15089, and to the Ministerio de Economía y Competitividad for the project MAT2015-63974-C4-3-R y PCIN-2016-146.

Keywords: A-01 d - Calcium phosphates, A-01 a - Metallic biomaterials/implants, A-02 b - Coatings
PS1-02-48

Coatings Based on Organic/Non-Organic Composites on Bioinert Ceramics by Using Biomimetic Co-Precipitation (#522)

G. Desante1, N. Labude2, S. Neuss2, 3, R. Telle1, K. Schickle1

1 RWTH Aachen university, Department of ceramics and refractory materials, Aachen, North Rhine-Westphalia, Germany
2 RWTH Aachen university hospital, Institute of pathology, Aachen, North Rhine-Westphalia, Germany
3 RWTH Aachen university hospital, Helmholtz institute for biomedical engineering, Biointerface group, Aachen, North Rhine-Westphalia, Germany

Introduction

Bioinert ceramics such as alumina or zirconia have been commonly used in the field of orthopedics and dentistry due to its excellent mechanical properties, esthetic look, good biocompatibility and chemical inertness in biological environment. An activation of its bioinert surface could bring additional advantages for better implant-integration with surrounding tissues in vivo. Therefore, the aim of the present study was to develop an innovative biomimetic co-precipitation technique by using modified Simulated Body Fluid (SBF) to obtain a composite biomimetic coating made of organic and non-organic components enhancing a bioactivation/functionalization of this inert biomaterial.

Experimental Methods

Zirconia samples were biomimetically coated by immersion in two times concentrated SBF-solution prepared according to Tanahashi et al. (1994) and kept at a body temperature for 3h [1].

Bovine Serum Albumin (BSA) in 5 different concentrations (0.01, 0.1, 1, 10, 100 gL-1 and 0 gL-1 as a control) has been chosen as a standard protein to be incorporated into the CaP-coating during the precipitation process. The incorporation of BSA into the SBF solution occurred on the half of the samples directly (“direct” coating) and for the other half on samples already pre-coated with SBF (“with pre-coating”).

BSA/Alexa FluorTM 488 conjugates were applied to visualize the incorporated proteins into the surface. To evaluate a role of sedimentation of protein in the solution, the coating produced on horizontal and vertical (positioning shown in Fig.2) samples were compared. Samples were imaged by using fluorescence microscope. To determine the morphological changes on the substrate surfaces after soaking in SBF, scanning electron microscopy was applied. Carbon-content of the HAp-coating dependent on concentration of BSA in the solution were established by using EDX measurements. Moreover, the thickness of HAp-coatings could be measured by imaging of cross-section of ZrO2-substrates.

Results and Discussion

The control samples (0 gL-1 BSA) as well as samples coated in SBF-solution containing 0.01 gL-1 BSA exhibit typical coral-like crystal structures [2] with app. 100 nm long crystal-plates. In contrast to BSA-concentrations >0.1 gL-1 the crystal structure appears to be altered or protein-overlayed (Fig.1).

The incorporation of protein within the HAp-coatings was visualized by using fluorescence microscopy to detect BSA/Alexa-FluorTM-488 conjugates, which gives a green signal (Fig.2). The intensity of green signal is stronger with increasing protein concentration in the solution. Additionally, the content of carbon was measured by EDX. The results showed a logarithmic growth of carbon content in the HAp-coating with increasing BSA concentration in SBF solution by the precipitation process.

The influence of the sedimentation process on the intensity of fluorescence signals proportional to the amount of proteins in the coating could also be observed. The results were in correlation with the chemical analysis of the coated surfaces (EDX).

Analysis of the cross-section of the obtained coating on CaP-pre-coated samples showed the apatite growth for all tested samples in comparison to the pre-coated control sample. The thickness of the coating decreases with the increase of protein concentration in the solution, which is in correlation with the SEM images.

Conclusion

It could be shown, that it is possible to co-precipitate an organic/non-organic coating based on HAp and biological agent such as BSA. This method could create a new biomaterial group, which surfaces could be tailored designed according to its desires and requirements. Based on these results with a standard protein, BSA has been replaced by specific proteins like Bone Morphogenetic Protein 2 (BMP-2) as a potential osteoinductive factor and Hepatocyte Growth Factor (HGF) as a growth factor. These proteins have already evidenced a strong influence on the crystal growth and the HAp-coating morphology as well. Thus further systematic analyses and cell culture tests are still on going in order to better understand biologically efficacy or bone growth factor response of the protein incorporated into the CaP-coating.

References

[1] Tanahashi M., Yao T., Kokubo T., Minoda M., Miyamoto T., Nakamura T., Yamamuro T., Apatite coating on organic polymers by a biomimetic process, J Amer Ceram Soc, 1994, 77(11), 2805-2808.

[2] Liu Y., Layrolle P., de Bruijn J.D., van Blitterswijk C., de Groot K., Biomimetic coprecipitation of calcium phosphate and bovine serum albumin on titanium alloy, J Biomed Mater Res, 2001, 57, 327-335.

Acknowledgement

Funded by the Excellence Initiative of the German federal and state governments (Grant Nr. OPSF408)

Fig.1

SEM view of vertical samples coated with SBF containing BSA in “direct” coating (left) and after pre-coating (right) with different concentrations of BSA in the SBF solution: 0, 0.01, 0.1, 1.0, 10, 100 gL-1. The vertical positioning of the samples is also represented for each group of samples.

Fig.2
Fluorescence images of the vertical (v) and horizontal (h) pre-coated samples using precipitation from the SBF solution containing 0, 0.01 and 0.1 gL-1 BSA. Positioning of the samples - vertically or horizontally - is here represented.
Keywords: A-01 d - Calcium phosphates, A-06 c - Biomaterials for growth factor delivery, A-01 c - Ceramic biomaterials
PS1-02-49

Thermally treated carbonated amorphous calcium phosphate (#542)

J. Vecstaudza1, J. Locs1

1 Riga Technical University, Rudolfs Cimdins Riga Biomaterials Innovations and Development Centre of RTU, Institute of General Chemical Engineering, Faculty of Materials Science and Applied Chemistry, Riga, Latvia

Introduction

Amorphous calcium phosphate (ACP) is essential in formation of mineralized bone and in development of bone substitutes. ACP is highly bioactive and biocompatible with living organisms. The reactivity of ACP is mainly attributed to the hydrated layer surrounding ACP particles and its high specific surface area (SSA). Previously we have reported new synthesis technology of nanosized carbonated ACP [1]. Thermal treatment is a common processing step when calcium phosphates are considered for biomaterial development. At the moment there is no study related to thermal treatment induced changes of surface properties of carbonated amorphous calcium phosphate nanoparticles, that hold potential in bone substitute development. Here we present continuation of thermal and crystallization studies of the nanosized carbonated ACP [2] with detailed follow-up of ACP structural and specific area changes at 100-1000 °C.

Experimental Methods

Carbonated ACP was precipitated from calcium and phosphate ion containing solution by rapid rise of solutions pH up to 10 [1]. Freeze dried ACP was subjected to XRD, FT-IR, heating microscopy, DSC-TG, specific surface area (SSA) and particle size dBET analyses. Further ACP was heat treated at temperatures ranging from 100 to 1000 °C with hold times of 5 or 60 min. Phase and chemical composition (XRD and FT-IR), SSA and dBET were systematically analysed for heat treated products.

Results and Discussion

Initial characteristics of ACP. XRD and FT-IR analysis confirmed the amorphous nature of obained ACP sample. SSA of ACP was 115 m2/g and dBET was 15 nm. Heating microscopy and DSC-TG analyses showed crystallization related events in temperature range of 500-650 °C. Compaction of ACP sample was observed during the same temperature range. This clearly demonstrated that crystallization of ACP is accompanied by compaction of ACP particles [2].

Specific surface area and dBET. Heat treatment of carbonated ACP reduced its specific surface area in a manner dependent on temperature. Heat treatment up to 1000 °C reduced SSA of ACP for 14 times. Negligible reduction of SSA was observed in range of  100-300 °C and 700-1000 °C. These negligible differences at those exact temperature ranges were attributed to the unchanged phase composition at those exact temperatures and/or reaching some equilibrium: ACP at 100-300 °C and mixture of hydroxyapatite (HAp) and beta tricalcium phsopahte (β-TCP) at 700-1000 °C.

Phase and chemical composition. First crystalline phases were detected in XRD patterns at 500 °C. Increasing heat treatment temperatures decreased amount of residual ACP phase. From 500 to 600 °C poorly crystalline calcium phase or phases (e.g. calcium deficient hydroxyapatite) were present. At 650 °C two crystalline phases were identified - HAp and β-TCP, that remained at higher temperatures as well. In FT-IR spectra carbonate ions were present  up to 600 °C.

Impact of temperature hold time. Phase composition, SSA, particle size dBET, chemical groups and mesoporosity characteristics of heat treated calcium phosphate products were independent of temperature hold time (5 or 60 min).

Conclusion

This study contributes to understanding of thermally induced crystallization process of carbonated nanosized ACP from perspective of surface property change. Specific surface area of carbonated ACP was found to remain the same whether 5 or 60 min hold times were tested.

References

[1] Vecstaudza, J., Locs, J. Novel preparation route of stable amorphous calcium phosphate nanoparticles with high specific surface area (2017) J. Alloy. Compd., 700, pp. 215-222.

[2] Vecstaudza, J., Gasik, M., Locs, J. Amorphous calcium phosphate materials: Formation, structure and thermal behaviour (2019) J. Eur. Ceram. Soc., 39 (4), pp. 1642-1649.

Acknowledgement

This research is funded by the Latvian Council of Science, project Future of synthetic bone graft materials - in vivo guided biosynthesis of biomimetic hydroxyapatite, project No. lzp-2018/1-0238.

Keywords: A-01 d - Calcium phosphates, A-01 c - Ceramic biomaterials, A-07 d - Bone
PS1-02-50

Elastic and resistant calcium phosphate cement for vertebral fracture treatment (#631)

S. S. Ramirez1, 2, C. Mellier3, J. M. Bouler2, F. Granier4, S. Marie4, F. X. Lefèvre2, J. C. Scimeca5, R. Debret6, J. Sohier1, B. Bujoli2

1 Université Lyon, INSA Lyon, UMR 5510 CNRS, MATEIS, Villeurbanne, France
2 Université de Nantes, CNRS, UMR 6230, CEISAM, UFR Sciences et Techniques, Nantes, France
3 Graftys SA, Pôle d’activités d’Aix en Provence, Aix en Provence, France
4 Colcom, Clapiers, France
5 CNRS, INSERM, Université de Nice Sophia Antipolis, iBV, Nice, France
6 CNRS, UMR 5305, Laboratory of biology tissue and therapeutic engineering LBTI, Lyon, France

Introduction

The development of a synthetic bone substitute is presented as a potential candidate for the treatment of vertebral fractures, a painful and invalidating pathology. The current treatment includes immobilization under corsets or percutaneous vertebral augmentation by injection of a poly(methylmethacrylate) PMMA resin in the vertebra to stabilize the fracture. However, there is a medical need to replace this material, since its in situ polymerization generates high temperature and PMMA is a non-resorbable and rigid material that may cause secondary fractures. Our bioinspired approach is a material composed of an inorganic apatite calcium phosphate cement (CPC) combined to an organic hydrogel that will form in situ. These composite materials are bioresorbable and, thanks to the presence of CPC, are also osteoconductive. The candidates should have appropriate rheology and cohesion for injectability and show mechanical properties close to that of bone tissue.

Experimental Methods

Two commercial formulations of CPS were used, Graftys® HBS and Graftys® HBS without hydroxypropyl methyl cellulose (HPMC), having different cohesion and setting time (12 and 9 min, respectively). The hydrogel is formed by condensation of a commercial lysine-grafted dendrimer (Colcom® DGL) and a polyethylene glycol chain with NHS end-groups (PEG-NHS) [1]. Pastes were prepared by mixing the hydrogel components with the cement powder. The concentration of hydrogel components was modulated and the effect of the initial setting time of pastes was measured using the Gillmore method. Injectability and compressive strength measurements were performed on pastes and composite cylinders respectively, using an AMETEK LS5 texture analyzer. Decalcified samples of CPC/hydrogel composites were obtained by Ethylenediaminetetraacetic acid (EDTA) treatment. Organic and inorganic distribution were evaluated by SEM observations of polished cross-section of composite samples.

Results and Discussion

A series of composite materials were successfully fabricated. The formation of a continuous 3D hydrogel network in the CPC, once hardened, was verified by decalcification of the samples (figure 1a-b, showing the organic phase). The hydrogel contributed significantly to enhance the injectability of the resulting paste, as compared to a purely inorganic CPC (HBS without HPMC), and the paste cohesion (75% versus 95% of extruded paste). A homogeneous distribution of the inorganic and organic components was observed by SEM characterization of the composites (figure 1c, showing the hydrogel network). The working time, initial setting time, compressive strength, Young´s Modulus and injectability of the composite systems, studied as a function of the DGL and PEG-NHS contents, DGL/PEG-NHS ratio, type of CPC powder, ionic strength of the liquid phase and liquid/powder ratio, resulted in significantly different mechanical properties (figure 1d -e, showing hydrogels of different elasticity). In-vitro tests performed on a selection of these composite materials using osteoblasts and osteoclasts cells.

Conclusion

A series of inorganic/organic composite cements of versatile injectable and mechanical properties were successfully prepared, opening potential applications for the treatment of vertebral fractures. Further studies are focusing on the biological evaluation of these materials.

References

[1] Pantent: Debret, R., C. Faye, J. Sohier and P. Sommer, Polypeptide derived from tropoelastin and biocompatible material comprising same. 2016 (WO2017194761).

Elastic and resistant calcium phosphate cement for vertebral fracture treatment

Figure 1. Composite material before (a) and after decalcification (b). SEM observation of a fracture plane of the obtained hydrogel network after decalcification (c). Hydrogels obtained after decalcification of composite materials showing different mechanical properties (d-e).

Keywords: A-01 d - Calcium phosphates, A-01 f - Polymeric biomaterials, A-07 d - Bone
PS1-02-51

Reinforced bone cement based on metastable α'-Ca3(PO4)2 (#753)

A. Goncharenko1, Z. Zyman1, M. Epple2, E. Onyshchenko1

1 V.N. Karazin Kharkiv National University, Physics of Solids Department, Kharkiv, Ukraine
2 University of Duisburg-Essen, Institute of Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE), Essen, North Rhine-Westphalia, Germany

Introduction

Calcium-phosphate materials, including ceramics and cements, are widely used in orthopedics, osteoplasty and maxillofacial surgery due to their chemical similarity to the mineral composition of bone.  Calcium-phosphate cements are considered to be an excellent synthetic material for filling bone cavities and defects [1].

The cement formation is a consequence of the chemical reaction between a solid component (as a powder) and a liquid phase. Usually, suitable calcium phosphate powders are obtained at high temperatures, and their particles are several micrometers large. This results in decrease of their reactivity [2]. Therefore using powders consisted of metastable phases processed at medium temperatures from amorphous calcium phosphate, ACP, with nanoscale particles should significantly improve the reactivity of cements.

Since cements are usually fragile and have low compression strength and fracture toughness, it is expected that the addition of certain reinforcing particles should significantly improve the mechanical properties of cement. It is desired that the particles would have bioactive properties similar to those of the cement [3].

Experimental Methods

Metastable αʹ-TCP powder was obtained by quenching an ACP powder heated (crystallized) at 700 °C for 1 hour. A fast variation of the nitrate synthesis was used to produce the ACP powder with a Ca/P ratio of 1.5. HA whiskers (40 µm length and 2 µm wide) were prepared by an original hydrothermal route at 235 °C and 20 atm [4].

For cement preparation with various solid/liquid ratios and amounts of HA whiskers, a certain amount of 2.5% Na2HPO4 solution was added to the powder portion and thoroughly kneaded with a spatula. The resulting paste was formed under slight pressure into cylinders and heated at 37 °C.

The setting moment of the cement was determined by the Vick method. Initial and reinforced cements were examined as prepared and after heating (5 K min-1) at 1150 °C in a muffle furnace, respectively, by XRD, TG-DTA, IR, ESEM and EDX.

Results and Discussion

At low solid/liquid ratios, the composition of the cement is almost identical to that of the initial powder and is represented only by αʹ-TCP. At a ratio of 1/0.75, besides αʹ-TCP diffractions, apatite maxima in the range of 26-32°2θ were detected. With further increasing the liquid`s amount, the intensity of the αʹ-TCP maxima decreased, and those of the apatite increased substantially.  

The cement annealed at 1150 °C for 1 hour consisted of β-TCP. It means that the formed apatite was non-stoichiometric with a Ca/P ratio near 1.5. ESEM examination has shown that the cement consisted of agglomerates of very thin lamellar crystals. The compressive strength of the set cement was about 4 MPa at the 1/1.5 solid/liquid ratio. Enrichment of the cements by various amounts of HA whiskers (1–15 wt%) improved the mechanical properties. For example, the compressive strength increased twice for the 4 wt% whisker amount.

Conclusion

The effect of powder/liquid ratio on the stoichiometry, phase composition and morphology of apatite cements based on metastable αʹ-TCP was revealed. The possibility of reinforcement of cements by HA whiskers for the ACP–αʹ-TCP cement system was firstly explored. The 1/1.5 powder/liquid ratio provides a complete transformation of αʹ-TCP into apatite. The cement set during 15 minutes and had a compressive strength about 4 MPa. The addition of 4 wt% of HA whiskers in the cement increased its compressive strength twice. The developed reinforced processingly simple cement can be used as a non-carcinogenic bone substitute for bone tissue in non-bearing areas of the human skeleton.

References

  1. Dorozhkin S V, J Funct Biomater, 4:209-311 2013
  2. Bohner M, J Mater Chem, 17:3980-3986 2017
  3. Hockin H K X et al., J Biomed Mater Res 52:107-114 2000
  4. Zyman Z Z, et al., Biomaterialien, 7:252  2006
Keywords: A-01 d - Calcium phosphates, A-01 h - Composites and nanocomposites, A-07 d - Bone
PS1-02-52

Synthesis of Amorphous Calcium Phosphate with Biomimetic Chemical Composition (#901)

J. Vecstaudza1, J. Locs1

1 , Riga Technical University, Rudolfs Cimdins Riga Biomaterials Innovations and Development Centre of RTU, Institute of General Chemical Engineering, Faculty of Materials Science and Applied Chemistry, Riga, Latvia

Introduction

There is still ongoing need for effective materials for bone regeneration that copy both structure and composition of the mineral phase of the human bone. At the very beginning of the bone mineral formation it is amorphous and besides calcium and phosphorus it contains other biologically relevant ions. Further, structurally the bone mineral is nanomaterial. Both of these important features can be combined into ionically enriched amorphous calcium phosphate (ACP). It is known that amorphous structures are more susceptible to inclusion of large quantities of various ions than crystalline ones. Therefore, the current work presents development of synthesis of novel ACP nanoparticles with biomimetic bone-like chemical composition. The elemental composition of the biomimetic ACP will be composed of Ca2+, PO43-, OH-, CO32-, Mg2+, Sr2+, Na+, K+, Cl-, F-, Zn2+ and citrate ion building blocks.

Experimental Methods

Synthesis of the biomimetic ACP is based on recently developed wet chemistry method [1]. The basis of the method is to obtain a homogenous solution of both calcium and phosphate ions by dissolution of calcium phosphate salt e.g. hydroxyapatite or beta tricalcium phosphate. Afterwards strong base is rapidly added to the transparent solution and precipitation of ACP occurs. The precipitate is filtered, washed and later dried at 80 °C. To incorporate the rest of the biomimetic elements into structure of ACP, various salts of the respective ions (MgCl2, SrCl2, sodium citrate etc.) were added before the dissolution of calcium phosphate. Quantities of the biomimetic elements were matched to the ones in human bone. Characterization of the synthesized materials was done with x-ray diffraction analysis (XRD), Fourier transform infrared spectrometry (FT-IR), Brunauer–Emmett–Teller (BET) specific surface area analysis and scanning electron microscopy coupled with energy dispersive x-ray spectrometry (EDS).

Results and Discussion

Phase composition determined by XRD showed that the modified synthesis method yields only ACP phase and no crystalline phases were detected. Addition of multiple ions to the synthesis of ACP might hinder its crystallization, however more detailed studies are needed. Further, FT-IR spectra confirmed the XRD data as absorption bands were wide and rounded that are characteristic to amorphous structures. Presence of both carbonate and citrate ions was detected by FT-IR. The specific surface area of the synthesized products was over 50 m2/g thus proving the nanostructure aspect. Previous studies [1] of the reference ACP without biomimetic ions have shown that it is possible to synthesize nanosized (<20 nm) ACP with specific surface area over 150 m2/g that is stable in air for at least one year. EDS spectra confirmed the presence of the inorganic ions in the biomimetic ACP.

Conclusion

Overall it was possible to obtain nanostructured ACP with biomimetic bone-like chemical composition with specific surface area over 50 m2/g by modified wet precipitation technology. Addition of the supplementary ions to the synthesis system of ACP was successful in terms of preserving the amorphous structure. The obtained calcium phosphate nanostructures with the biomimetic composition will provide all the necessary preconditions for successful use in bone tissue engineering.

References

1. J. Vecstaudza, J. Locs, J. Alloy. Compd., 700 (2016), 215-222.

Acknowledgement

This research is funded by the Latvian Council of Science, project Future of synthetic bone graft materials - in vivo guided biosynthesis of biomimetic hydroxyapatite, project No. lzp-2018/1-0238.

Keywords: A-01 d - Calcium phosphates, A-07 d - Bone , A-01 b - Biodegradable metals
PS1-02-53

In vivo degradation, osteoinduction and osteogenesis of biomimetic hydroxyapatite: Effect of nanostructure and carbonate substitution (#1013)

M. - P. Ginebra1, A. Barba1, 4, A. Diez-Escudero1, M. Espanol1, C. Ohman-Magi2, C. Persson2, M. C. Manzanares3, J. Franch4

1 Universitat Politecnica de Catalunya, Department of Materials Science and Metallurgy, Barcelona, Spain
2 Uppsala University, Department of Engineering Sciences, Uppsala, Sweden
3 Universitat de Barcelona, Department of Pathology and Experimental Therapeutics, Hospitalet de Llobregat, Spain
4 Universitat Autonoma de Barcelona, Small Animal surgery Department, Bellaterra, Spain

Introduction

Synchronisation between biomaterial degradation and bone formation is one of the crucial requirements for the ideal synthetic bone graft. In practice, this synchronization is the result of a complex network of interactions between the material, the physiological fluids and the cells of the osseous system, very difficult to unravel. The active degradation of synthetic bone grafts in vivo results from the dissolution of the material in the acidic environment produced by bone-resorbing cells. Therefore, bone graft degradability depends not only on composition and textural properties, like porosity and specific surface area (SSA), but also on the inflammatory reaction and the osteoclastogenesis elicited by the material once implanted. Likewise, the degradation of the material modifies the ionic composition of the extracellular fluid, this acting as a powerful chemical signalling path that trigger specific cellular responses. In an effort to shed light on this complex system, in this work the in vivo behavior of calcium deficient hydroxyapatite with different composition and nanostructure is compared, in terms of degradation, osteoinduction (i.e. ectopic bone formation) and osteogenesis. Moreover, to better understand the interplay between chemical and biological phenomena, the results were compared with the in vitro accelerated degradation of the same scaffolds.

Experimental Methods

Calcium deficient hydroxyapatite (CDHA) foams were obtained by foaming and hydrolysis of an alpha-tricalcium phosphate (α-TCP) slurry. Two different nanostructures, fine needle-like crystals or coarse plate-like crystals (Fine-CDHA and Coarse-CDHA) were obtained by using either fine or coarse α-TCP powders respectively. Carbonated CDHA (CO3-CDHA) foams were obtained by hydrolysing α-TCP in a sodium bicarbonate solution. Sintered beta-TCP (β-TCP) and biphasic HA/β-TCP (BCP) foams were used as controls. The scaffolds were characterized in terms of composition, porosity, solubility and microstructure. The in vivo study was carried out in a standardized model of intramuscular (IM) and intraosseous (IO) implantation over 6 and 12 weeks in beagle dogs. Material resorption and bone formation was evaluated by micro-CT and SEM and histological and histomorphometrical analysis were performed. The susceptibility of the material to acidic degradation in vitro was assessed by immersing the samples in an acidic solution at 37 °C to mimic the osteoclastic environment.

Results and Discussion

Both ectopic and orthotopic bone formation, as well as scaffold degradation were drastically affected by nanocrystal morphology. Moreover, carbonate doping, which resulted in small plate-shaped nanocrystals, accelerated both the intrinsic osteoinduction and the bone healing capacity (Figure 1). Similar degradation trends were found intramuscular and intraosseously, although the absolute values were higher intramuscularly. CO3-CDHΑ Foams exhibited the highest degradation rate (60% IM and 50% IO at 12 weeks), followed by the Fine-CDHA Foams (40% IM and 42% IO at 12w), Coarse-CDHA foams exhibiting the lowest resorption (10% IM and 20% IO at 12 w). A good correlation was found between material degradation and bone formation, both ectopically and orthotopically. The materials presenting the highest osteoinduction, i.e. CDHA-CO3 and CDHA-F Foams (23 and 28% formed bone IM at 12 w, respectively), were also the ones showing the highest degradation rate. However, no clear patterns were found correlating CaP degradation and ectopic bone formation when compared with the sintered ceramics, β-TCP and BCP. Thus, BCP-Foam, presented a high amount of ectopic bone formation at 12 weeks (28% IM at 12w), despite showing the lowest degradation rate, and β-TCP, with a degradation at 12 weeks similar to Fine-CDHA showed no ectopic bone formation.

On the other other hand, the degradation in vivo was not fully consistent with the degradation results in vitro. Thus, whereas CDHA-F-Foam, with the highest SSA, showed the highest degradation rate, exceeding the more soluble β-TCP, the incorporation of carbonate in CDHA rather than increasing the in vitro degradation, significantly reduced it.

Conclusion

The size and morphology of the nanocrystals, as well as the presence of carbonate, allow tuning the osteoinductive and osteogenic potentials as well as the degradation profi le of the CDHA. Moreover, those materials with textural and compositional properties closer to the biological apatite exhibited better synchronization between material resorption and bone formation.

References

[1] Barba et al, ACS Appl. Mater. Interfaces 2019, 11:8818

Acknowledgement

MAT2015-65601-R project, from Spanish Government and European Reg. Dev. Funds; ICREA Academia award of MPG, from Generalitat de Catalunya

Figure 1.
Back-scattered SEM images of the CDHA scaffolds after 12 weeks of intramuscular implantation. [1]
Keywords: A-01 d - Calcium phosphates, A-03 b - 3D scaffolds for TE applications, A-07 d - Bone
PS1-02-54

Synthesis of Ion Modified Carbonate Apatites inspired by Biological Apatite (#1145)

B. Kołodziejska1, J. Kolmas1

1 Medical Univesity of Warsaw, FACULTY OF PHARMACY WITH LABORATORY MEDICINE DIVISION/CHAIR ANALYTICAL CHEMISTRY AND BIOMATERIALS, Warsaw, Poland

Introduction

Biological apatite is the main component of the inorganic fraction of hard tissues, such as bone tissue or mineralized dental tissues. Nanocrystals of the biological apatite are calcium orthophosphates with various foreign ions in their structure, i.e.: Mn2+, Mg2+, Cl-, Zn2+, Na+, K+, F-, CO32-, BO33-, SiO44-, etc. This unique composition causes the changes in the Ca/P molar ratio, which in the stoichiometric hydroxyapatite is 1.67.[1] Impurities in the apatite of bones, enamel or dentin affect not only the physicochemical properties, but also the biological properties of tissues. For example, silicon ions influence the process of osteogenesis by stimulating osteoblast proliferation.[2,3] The aim of the study was to develop a method of the synthesis of nanocrystalline carbonated apatites imitating the biological apatite. Our goal was to introduce foreign ions into the apatitic structure, by using different substrates. The high apatite susceptibility to ion substitutions was useful in the synthesis.[4]

Experimental Methods

The synthesis of the multi-substituted carbonate apatite was carried out using the wet method in SBF (simulated body fluid), containing ions such as: Na+, K+, Mg2+, Cl-, CO32-, SO42- and citrate. It was carried out in two ways (using different substrates). In the first method, only sources of calcium and phosphate ions were used; in the second method additional sources of magnesium ions and carbonate ions were added. The obtained precipitates were left to age at different periods of time. Finally, eight samples were produced: four without the addition of foreign ions with different aging times and four apatitic samples enriched with Mg2+ and CO32- ions with an analogous aging time. The obtained powders were characterized using the following methods: FT-IR mid-infrared and Raman spectroscopies, ICP-OES spectrometry and TEM microscopy.

Results and Discussion

The obtained results were analyzed in terms of the degree of crystallinity of powders as well as the content of individual ions in the samples. The significant changes in the crystallinity as well as in the elemental composition were observed. The differences in the content of Mg2 + ions, as well as CO32-, were evident.

Conclusion

The obtained powders were nanocrystalline, multi-substituted apatites, containing mainly magnesium and carbonates, that is characteristic for biological apatites. Their composition and crystallinity were affected by the aging period. Further physicochemical tests are required.

References

[1] S.V. Dorozhkin, Materials 2009, 2 (2), 399-498.
[2] J. Kolmas, S. Krukowski, A. Laskus, M. Jurkitewicz, Ceramics Intenational 2016, 42 (2), 2472-2487. 
[3] Q. Liu, S. Huang, J.P. Matinlinna, Z. Chen, H. Pan, Biomed. Res. Int. 2013, 2013, 1-13.
[4] J. Kolmas, A. Ślósarczyk, A. Wojtkowicz, Solid State Nucl. Magn. Reson. 2007, 32 (2), 53-58. 

Acknowledgement

FW23/N/19 - Medical University of Warsaw

Keywords: A-01 d - Calcium phosphates, A-01 c - Ceramic biomaterials, A-07 d - Bone
PS1-02-55

Synthesis of Substituted Hydroxyapatite for Application in Bone Tissue Engineering and Drug Delivery (#1181)

D. Büchner1, 2, M. Gericke2, T. Heinze2, E. Tobiasch1, S. Witzleben1, M. Schulze1

1 Bonn-Rhein-Sieg University of Applied Sciences , Departement of Natural Sciences, Rheinbach, North Rhine-Westphalia, Germany
2 Friedrich-Schiller-University Jena, Institute of Organic Chemistry and Macromolecular Chemistry, Center of Excellence of Polysaccharide Research, Jena, Thuringia, Germany

Introduction

Current approaches in bone tissue engineering focus on hybrid materials made of polymer matrices (e.g. synthetic biopolymers such as polylactic acid, collagen or polysaccharides) and inorganic constituents (e.g., hydroxyapatite, tricalcium phosphate, or bioglass). While polymers help forming light and porous biocompatible structures, the ceramic parts improve cell-attachment and mechanical properties to at least partially restore stability but should also induce the formation of new bone tissue. A straightforward approach is the fabrication of scaffolds that carry stem cells with capability to differentiate into osteoblasts, bioactive drugs (such as growth factors) which induce and support osteoblast differentiation and purinergic receptor ligands influencing the osteogenic linage commitment. [1] [2]      

Bisphosphonates are long known in the therapy of various bone associated diseases due to their osteoclast-inhibiting activity. A guided delivery and prolonged release of bisphosphonates to the site of the disease is an attractive objective to overcome the inherent drawback of their low oral bioavailability.

Experimental Methods

In this work, we present the synthesis of substituted and stoichiometric hydroxyapatites by chemical precipitation. Chemical characterization is carried out by various spectroscopic and X-ray methods (i.e. XRD, FT-IR and ICP-OES), while the product’s morphology is examined using particle size measurements and SEM.

Results and Discussion

Systematic evaluation of the reaction parameters shows a major impact of the reaction temperature on crystallite size and degree of crystallinity for the synthesis of stoichiometric hydroxyapatite, while pH and reaction time result in minor changes. Furthermore, the influence of organic templates on the crystallization and agglomeration of the products are investigated.

Conclusion

Embedding the synthesized apatites into a polysaccharide hydrogel-matrix provides a hybrid material, that resembles the physicochemical properties of natural bone, thus making it a promising candidate for bone tissue engineering and drug delivery applications.

References

[1]           P. F. Ottensmeyer, M. Witzler, M. Schulze, E. Tobiasch, Int. J. Mol. Sci. 2018, 19, 3601.

[2]           A. Leiendecker, S. Witzleben, M. Schulze, E. Tobiasch, Current Stem Cell Res. Ther. 2017, 12, 103.

Acknowledgement

Financial support was given by the BMBF project "Hybrid-KEM" (FKZ 13FH569IX6).

Keywords: A-01 d - Calcium phosphates, A-07 d - Bone , A-06 a - Biomaterials for drug delivery
PS1-02-56

Effect of two β-tricalcium phosphate bone grafting materials with varying macro- and micromorphology on bone formation and osteogenic marker expression 6 months after sinus floor augmentation in humans (#1188)

M. Rezk1, 2, A. Bednarek1, M. Stiller1, C. Knabe1

1 Philipps University , Dept. of Experimental Orofacial Medicine, Marburg, Hesse, Germany
2 Ain Shams University, Dept. of Periodontology, Cairo, Egypt

Introduction

Sinus floor augmentation (SFA) is frequently performed as a mandatory augmentation procedure prior to placing dental implants in the posterior maxillary region. Disadvantages of autologous grafts as well as the limited biodegradability of some of the commercially available bone grafting materials have prompted an ever-increasing search for adequate bone substitute materials. An ideal bone grafting material should stimulate bone formation at its surface, resorb and  be replaced by fully functional bone tissue.

Over the last two decades, the use of tricalcium phosphate (TCP) granules as alloplastic bone graft particles has received increasing attention especially for maxillary sinus augmentation procedures.

This study evaluated the effect of two highly porous TCP granules with the same overall porosity but varying macro- and micromorphology on bone formation and on osteogenic marker expression 6 months after sinus floor augmentation (SFA). This was in addition to characterizing the biodegradability.

Experimental Methods

This study comprised a series of 40 consecutive patients, in which unilateral SFA was performed. In 20 patients phase pure β-TCP polygonal particles (granule size 1000 to 2000 µm) with 65% porosity (CM)1,2 and rounded morphology were used, while in the remaining 20 patients TCP polygonal particles displaying sharp edges (granule size 700 to 1400 µm) with 60% porosity TCP (CER)3 were utilized in a combination with autogenous bone chips (10:1). At implant placement, 6 months after sinus grafting, cylindrical biopsies were sampled and processed for immunohistochemical analysis of resin embedded sections from the augmented area as described previously.2,3. Sections were stained for collagen type I (Col I), alkaline phosphatase (ALP), osteocalcin (OC) and bone sialoprotein (BSP). Subsequent to the immunohistochemical analysis, tissue sections were prepared for histomorphometry. The area fraction of newly formed bone as well as the particle area fraction were determined first, apically close to the Schneiderian membrane and second, in the center of the cylindrical biopsies.2,3

Results and Discussion

In both patient groups a statistically significant greater amount of bone formation and smaller amount of residual grating material were observed in the central area compared to the apical area. This tendency was more pronounced in the CM group (43.9% (mean) newly formed bone centrally vs. 33.3% (mean) apically) compared to the CER group (39.5% (mean) vs. 36.3% (mean) apically). Residual grafting material: CM group centrally 2.85 (mean) versus 21.1% (mean) apically; CER group 1.3 (mean) apically versus 16.0 (mean) centrally, with these differences between the CM and CER group not being statistically significant. Marked differences with respect to the osteogenic marker expression were observed when comparing both materials. Significantly higher expression of OC was noted in osteoblasts in the CM group both apically and centrally when compared to the CER group. In the CM group a higher expression of Col I was noted apically than centrally, whereas positive expression of the osteogenic markers OC and ALP were present in both areas. In the CER group a high expression of Col I and ALP in the apical and central area was indicative of bone formation which was still actively progressing 6 months after sinus floor augmentation. Furthermore, the CER particles located in the most apical layer of the biopsies were fully surrounded by fibrous tissue in combination with fibrous tissue being noticed in the pores of the particles. The trend towards a smaller amount of residual bone grafting material, i.e. higher biodegradability for the CER group compared to the CM group may be related to the slightly smaller granule size of the CER particles.

Conclusion

Both biomaterials with identical chemical composition and a similar degree of overall porosity showed marked differences with respect to the histomorphometric and immunohistologic results in vivo. This appeared to be due to the different macro- and micromorphology of the granules. The high OC expression in osteoblasts both centrally and apically in the OC group is indicative of the excellent bioactive behaviour of this grafting material and its enhancing effect  on bone matrix mineralization. The CER particles being surrounded by fibrous tissue in the most apical layer of the biopsies may be related to the sharper edges of the CER particles compared to the more rounded morphology of the CM particles in conjunction with the relative movement between particles of the grafting material and the Schneiderian membrane during the breathing process. Both TCP grafting materials, however, supported sufficient bone formation in the augmented sinus floor for facilitating stable and reliable implant placement with residual grafting material still being present 6 months after SFA. This was in addition to matrix mineralization and bone formation still actively progressing 6 months after SFA.

References

  1. Knabe C, Adel-Khattab D, Hübner WD, Peters F, Knauf T, Peleska B, Barnewitz D, Genzel A, Kusserow R, Sterzik F, Stiller M, Müller-Mai M. “Effect of silicon-doped calcium phosphate bone grafting materials on bone regeneration and osteogenic marker expression after implantation in the ovine scapula”. J Biomed Mater Res B Appl Biomater. 2018 May 16.
  2. Knabe C, Koch Ch, Rack A, Stiller M. Effect of β-tricalcium phosphate particles with varying porosity on osteogenesis after sinus floor augmentation in humans. Biomaterials 2008;29(14):2249-2258.
  3. Knabe C, Adel-Khattab D, Kluk E, Struck R, Stiller M. Effect of a Particulate and a Putty-Like Tricalcium Phosphate-Based Bone-grafting Material on Bone Formation, Volume Stability and Osteogenic Marker Expression after Bilateral Sinus Floor Augmentation in Humans. J Funct Biomater. 201729;8(3).

Acknowledgement

The authors would like to thank the German Research Foundation for funding (KN377/5-1) and Ms. A Kopp for her excellent technical assistance.

Keywords: A-01 d - Calcium phosphates, A-01 c - Ceramic biomaterials, A-11 a - Clinical trials
PS1-02-57

Multicomponent Sr-HA and Ga-βTCP/gelatine based scaffolds for biomedical applications (#1230)

K. Szurkowska1, J. Kolmas1

1 Medical University of Warsaw, Faculty of Pharmacy with Laboratory Medicine Division, Department of Analytical Chemistry and Biomaterials, Analytical Group, Warsaw, Poland

Introduction

Calcium phosphates (CaP) play a crucial role in the regenerative medicine as cavities filling materials, scaffolds for new bone tissue, and more recently as systems delivering therapeutic substances [1]. Among CaP group, hydroxyapatite (HA) has received considerable attention because of its significant similarity to the biological apatite - the main inorganic component of biological hard tissues. Another material commonly used in bioceramics is beta tricalcium phosphate (βTCP), which is characterized by better solubility than HA. The unique capacity for the CaP group is the ability to undergo ionic substitution, which can enhance biological activity of the samples [2]. Both strontium and gallium ions enhance bone regeneration, act as antiresorptive agents and possess slight antibacterial properties. Therefore, their coexistence in the scaffold can be particularly beneficial. The addition of scaffold-binding gelatine will allow to control the degradation rate of the composite and to improve the mechanical properties of CaP samples [3]. Due to the presence of pores in the composite, proper vascularisation and delivery of the necessary nutrients to the tissue will be possible.

Experimental Methods

The synthesis of a series of calcium phosphates substituted with strontium and gallium, substituted with various amounts of ions, were carried out. A series of Sr2+ substituted hydroxyapatites (Sr-HA) was synthesized via wet precipitation method at 60 °C, left for 24 h for aging and then air-dried. A series of Ga3+ substituted beta tricalcium phosphates (Ga-βTCP) was first obtained by precipitation and then the raw precipitates were subjected to sintering at 1000 °C to allow a phase transition. In addition, pure unsubstituted CaP were synthesized as comparative materials. All the resulting powder materials were subjected to a thorough physicochemical analysis to select the ones with the most favourable parameters. Fourier Transform Infrared Spectroscopy (FT-IR), Solid State Nuclear Magnetic Resonance (ssNMR) and X-ray Powder Diffraction (PXRD) were used for structural and chemical composition assays. Transmission Electron Microscopy (TEM) was used to assess the morphology, shape and size of crystals and their tendency to agglomerate. Quantitative analysis of the introduced ions was performed by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). A preliminary evaluation of cytotoxicity in vitro was performed on BALB/c 3T3 mammalian fibroblast cell line. Porous gelatine-based scaffolds containing selected Sr-HA and Ga-βTCP samples were obtained by the lyophilization method. The resulting materials were then examined, determining the degree of porosity, morphology and ion release profiles.

Results and Discussion

Sr-HA and Ga-βTCP samples with various amounts of substituted ions were synthesized and thoroughly physicochemically examined. Moreover, all of the samples turned out cytocompatible. Multicomponent scaffolds, composed of selected Sr-HA, Ga-βTCP and gelatine were successfully obtained. Through the use of lyophilization method, it was possible to obtain a porous material that gradually releases substituted ions into the medium. Differences in the release profile of individual elements were obtained, due to the better solubility of βTCP than HA.

Conclusion

The aim of the research was to synthesize biocompatible and bioactive implant materials composed of doped calcium phosphates (CaP), which would serve as scaffolds delivering strontium and gallium ions directly into the damaged bone tissue. The determined ion release profiles are the starting point for further research. The long-term project objective is to develop scaffold that could potentially be used for local delivery of antiresorptive drugs into the injured tissue.

References

[1] M. Vallet-Regi, J.M. Gonzales-Calbet, Prog. Solid State Chem. 32 (2004) 1-31.

[2] M. Supova, Ceram. Int. 41 (2015) 9203-9231.

[3] J. Venkatesan, S.K. Kim, J. Biomed. Nanotechnol. 10 (2014) 3124-3140.

Acknowledgement

This work was supported by UMO-2016/22/E/STS/00564 grant of the National Science Centre, Poland and FW23/PM1/18 grant from the Medical University of Warsaw.

Keywords: A-01 d - Calcium phosphates, A-03 b - 3D scaffolds for TE applications, A-06 a - Biomaterials for drug delivery
PS1-02-58

Zn2+ and SeO32- modified hydroxyapatite as a potential, antitumor biomaterial for the therapy of bone cancer (#1238)

A. Laskus1, A. Zgadzaj2, J. Kolmas1

1 Medical University of Warsaw, Department of Analytical Chemistry and Biomaterials, Analytical Group, Warsaw, Poland
2 Medical University of Warsaw, Department of Environmental Health Sciences, Warsaw, Poland

Introduction

During the last few decades, when the societies have started to age, the disorders of the skeletal system have become a serious problem. Due to this, novel approaches to treat bone fractures, osteoporosis etc. have been recently developed. Considering different biomedical areas concerning the treatment of hard tissue, bone tissue engineering based on calcium phosphates (CaPs) is one of the most-developed. Among the CaPs, hydroxyapatite (HA) with the general formula Ca10(PO4)6(OH)2 should be distinguished due to its significant resemblance to the biological apatite, which builds mammalian hard tissue. On account of their excellent biocompatibility, HA-based materials are currently being used as bone fillers, coating materials or the elements of the matrices for drugs targeting bones.

One of the possible ways to create functional, novel ceramic materials based on HA, is introducing foreign ions into its crystal lattice. In this study, due to their well-known antibacterial, osteogenic and anticancer activity, Zn2+ and SeO32- were chosen as ionic dopants.

Experimental Methods

In this study, a series of hydroxyapatite containing different amounts of zinc and selenite ions were synthesized via classical, co-precipitation method. The obtained powders underwent both physicochemical and biological analysis. The techniques of FTIR, PXRD, ssNMR, TEM and ICP-OES were applied. The release kinetics of the ionic dopants was also investigated. Additionally, the cytotoxicity tests of the materials were conducted.

Results and Discussion

The physicochemical analysis confirmed that all of the obtained powders were poorly-crystalline hydroxyapatites with no other crystalline impurities in phase. The FTIR spectra, as well ICP-OES results confirmed the successful introduction of the SeO32-. The elemental analysis confirmed also the presence of the Zn2+ in the structure of HA. Based on the PXRD, FTIR and TEM studies, a clear tendency concerning the crystallinity of the powders containing Zn2+ was observed. Namely, the introduction of Zn2+ ions into the crystal structure of HA lowered its crystallinity. The interesting outcomes of the release kinetics of the ionic dopants were also obtained. Whereas in case of zinc, the level of the released element did not exceed 1 % of the total, introduced amount, the level of selenite ions reached ca. 50 % in average, which suggests that, unlike zinc ions, selenites were also partially adsorbed on the surface of HA. This, in turn, is consistent with the cytotoxicity tests, which revealed that the materials containing selenites were toxic and thus, could be used as potential, antitumor agents in bone cancer treatment. Considering well-known toxicity of selenium, the authors believe that the outcomes of the biological analysis are strongly connected with the partial adsorption of the selenites on the surface of HA.

Conclusion

In this study, a novel, HA-based biomaterial containing Zn2+ and SeO32- was synthesized. The outcomes of the cytotoxicity test suggests that the obtained powders could be used as potential, anticancer biomaterials. Due to the preliminary character of the study, it is necessary to proceed with further investigation.

Acknowledgement

The study was supported by The National Science Centre UMO-2016/22/E/STS/00564 .

Keywords: A-01 d - Calcium phosphates, A-01 c - Ceramic biomaterials, A-08 a - Biocompatibility
PS1-02-59

Dual-setting brushite-silica gel cements with high cross-linking density precursors (#775)

I. Holzmeister1, J. Groll1, U. Gbureck1

1 University Hospital of Würzburg, Department for Functional Materials in Medicine and Dentistry and Bavarian Polymer Institute, Würzburg, Bavaria, Germany

Introduction

Calcium phosphate cements (CPC) set via a dissolution–precipitation reaction and form an entangled network of crystals providing mechanical strength after setting. A further modification by adding monomers to the aqueous cement phase results in the formation of a second interpenetrating network with a strong impact on cement properties.[1] This was recently shown for cement modification with tetraethyl orthosilicate (TEOS) as precursor.[2] Here, this approach is further explored by using silica precursors with a higher density of alkoxy groups to increase network and cross-linking density in the final gel. Also modified precursors with different hydrophilic functional groups are investigated to influence the interface between silica and cement matrix. The precursors were initially hydrolyzed under acidic conditions and the combined with a brushite forming cement powder. Due to the increase in pH of the sol during cement setting leads to a simultaneous formation of cement matrix and silica hydrogel. [3]

Experimental Methods

Sol precursors were a mixture of TEOS (100 – 60%) and 0 – 40% of silica monomers with 6-12 alkoxide functionalities. Both commercially available 1,8-Bis(triethoxysilyl)octane and self-synthesized modified silica monomers with hydrophilic groups and a higher density of alkoxy groups were used (Figure 1A). Silica sols were obtained by adding the sol precursor mixture to water in a ratio of water: “Si-OEt” = 2.25 and 0.1 M HCl solution. Composite cements were produced by mixing the sol with cement raw powder, consisting of β-tricalcium phosphate (β-TCP) and monocalcium phosphate anhydrous (MCPA) in an equimolar ratio and the addition of 1 wt% citric acid. The citric acid is used as retarding agent in order to extend the setting-time of the composites.

Results and Discussion

The results demonstrated an increase of mechanical performance by using different amounts of a silica monomer (1,2-Bis(triethoxysilyl)octane) in addition to TEOS in the composite compared to the pure TEOS reference (Figure 1B). A variation of the monomers resulted in a substantial increase in the strength (Figure 1C) with an increase from 0.44 ± 0.09 MPa to 8.29 ± 1.01 MPa at low PLR of 1 g/mL. The porosity characteristics of the silica–brushite networks showed a bimodal pore size distribution in the set matrices with nanosized pores originating from the silica matrix and micrometer pores from the cement matrix (Figure 1D). The latter is thought to have a strong effect on drug release capability by retarding drug diffusion from the cement matrix.

Conclusion

It could be shown that the use of different silica monomers has a huge influence on the properties of the composites. As shown a increased strength of the composites could be observed depending of the system up to 10 times. Due to the two network in the composites, a bimodal poresize distribution could be observed.

References

[1] T. Christel et al. Dual setting α-tricalcium phosphate cements, ­­Materials in Medicine, 2013, 24(3), pp 573-581.

[2] M. Geffers et al. Dual-setting brushite–silica gel cements, Acta Biomaterialia, 2015, Volume 11, pp 467-476.

[3] I. Holzmeister et al. Artificial inorganic biohybrids: The functional combination of microorganisms and cells with inorganic materials, Acta Biomaterialia,2018, Volume 74, pp 17-35.

Figure 1

A Molecular structure of TEOS, commercially bought silica monomer(A) and synthesized silica monomers(B/C) B Compressive strength of CPC and composite reference CPC+TEOS. Increased compressive strength by adding 20/40% of silica monomer A. (PLR = 2 g/mL) C Compressive strength of CPC and composites with 5% different silica monomers (PLR = 1 g/mL) D Pore size distribution analyzed by Hg porosimetry in a range of 10-10000 nm of silica gel composite.

Keywords: A-01 d - Calcium phosphates, A-01 e - Bioglasses & silicates, A-01 h - Composites and nanocomposites