Bioactive glasses-based biomaterials with potential in soft tissue engineering (#1063)
1 Tampere University, Faculty of Medicine and Health Technoloy, Tampere, Finland
Silicate-bioactive glasses have been extensively studied for bone tissue engineering. The “traditional” bioactive glasses (i.e. 45S5 and S53P4) have found space in clinics: from bone filler to ocular implants. The benefit of using bioactive glasses, lies in the ability to tailor the composition to control the dissolution/reaction rate or thermal properties (fiber drawing, scaffold sintering). More importantly, the ease to incorporate ions of therapeutic interest has led to remarkable discoveries. Indeed, control release of strontium, zinc, magnesium, silver, copper or boron, just to cite a few, has enable to process materials with excellent osteogenesis, angiogenesis or antimicrobial properties . However, while bioactive glasses, in bone related applications, was promising, the difficulties in shaping the grafts into the exact patient’s defect has, to some extent, limited their clinical use. Nevertheless, it appears clear that the unique properties of bioactive glasses are still of interest. Indeed, researchers are constantly studying their potential use in composites or hybrid biomaterials .
While many questions remain to be answered to better grasp the full potential of bioactive glasses in hard tissue regeneration, some researchers already devoted their energies to explore new avenues for such uniquely tailorable material. For instance, researchers have explored the potential of bioactive glasses in soft tissue regeneration. For example, tubular phosphate glass fibres have been found to give a promising alternative to end-to-end suturing in facial nerve reconstruction . More recently, borate bioactive glass nanofibers (MirragenTM) has proved to be excellent in treating long-term venous stasis ulcer in diabetic patients who were irresponsive to conventional treatments .
These new results are opening new horizons for the use of bioactive glasses in clinics.
RESULTS AND DISCUSSION:
In the recent years our work has focused on designing materials that, not only could be applicable to hard tissue engineering but also soft tissue. In order to develop innovative construct; able to not only support, but also trigger, soft tissue regeneration a survey study where bioactive glass (13-93) extract was performed to culture various cell types (adipose stem cells, lung fibroblasts, or uroepithelial cells). The hASCs and the WI fibroblasts remained viable in the extracts. Li and Sr appeared to have positive impact on the cell’s proliferation. However, the high Ca concentration inhibits the proliferation of UE cells.
Tubular structures (PLA/bioactive glasses) were efficiently produced with a controlled porosity (>50%) and pore size ranging from 100 to 400 µm (Fig 1). The glass (13-93) dispersion was found to be homogeneous within the structure. The glass loading (25-35 wt%) was consistent with the target loading. The presence of bioactive glass allowed to prevent acidification of the environment while contributing to the faster degradation of the polymer matrix. Hybrid biomaterials (wood based nanofibrils and bioactive glasses or gelatin/GPTMS/bioactive glasses) were also developed into inks for bioprinting. In this study we demonstrated the possibility to develop an ink that can be printed into the shape of a mandible (Fig.2) while maintaining cell viability. Composites and hybrids could have potential for nerve regeneration, urethra or trachea repair.
Finally, while ions can trigger signalling pathways leading to osteogenesis or angiogenesis, in recent years the use of light to direct tissue healing as gained increasing interest. As such, biophotonic bioactive glasses were develop. While their characterization is only starting, the ability to design material with persistent luminescence in wavelength ranging from UV to NIR has high potential in tissue engineering. Indeed, it was shown by Liu et al. that micro-patterned light emission can enable to orientate cells and create anisotropic cell sheets, thus mimicking the complex structure of soft tissue such as muscles, artery, and nervous system 
Bioactive glasses have been widely studied in hard tissue reconstruction. However, their potential in soft tissue is nowadays being investigating. Bioactive glass, when combined with natural or synthetic polymeric matrices, enables to design materials for a wide range of application (from restoring the nervous system to healing urethra defects). The benefit of ions released from the bioactive glass is undeniable. Combining controlled ion release, biophotonic and tissue engineering may open the door to significant breakthrough in soft tissue regeneration.
The author would like to acknowledge all the PhD students and Post-Doctoral researcher that took part of the various projects. The funding bodies, i.e., the Academy of Finland, The Jane and Aatos Erkko Foundation and EU-H2020 are acknowledged for their financial support.
 Hoppe, A. et al., Biomaterials, 32:2757, 2011
 Houaoui, A. et al., Materials Science and Engineering C, 107:110340, 2020
 Gilchrist, T. et al., J Plast Surg, 51:231, 1998.
 Jung, S. et al., Wound Repair Regen, 19:A30, 2011
 Liu, C. et al., ACS appl. Mater. Interfaces, 9 :36513, 2017
µCT 3D reconstruction of a PLA/13-93 composite scaffold foamed by ScCO2
Ptinting of a gel containing bioactive glass particles as well as cells, into a mandible shape
Keywords: Bioactive glasses, Cells, ion release
Applications of inorganic materials in soft tissue regeneration: progress and challenges (#1317)
A. R. Boccaccini1
1 University of Erlangen-Nuremberg, Institute of Biomaterials, Erlangen, Germany
In 2021 the biomaterials community celebrates 50 years of bioactive glass (BG), the first man-made material capable to bond to tissues, discovered by the late Prof. Larry L. Hench . It is well-known that the traditional applications of BGs have been in bone substitution, for example as bone defect filler, small bone and dental implants and as bioactive coatings for orthopedic and dental applications. Moreover BGs emerged as attractive inorganic materials for bone tissue engineering (TE) given their bone bonding ability and the effect of BG dissolution products on osteogenesis. More recently, BGs have started to be considered in soft TE and wound healing. Such TE applications exploit the biochemical interactions occurring at the interface between BG surfaces and the biological environment, which lead to the (controlled) release of biologically active ions to activate specific cellular pathways . As an example, the effect of selected metallic ions on vascular endothelial growth factor release from stem cells has been demonstrated to indicate the angiogenic potential of BGs.
For applications in soft TE, BGs must be "softened" which is achieved by smart combination of BGs, usually in particulate or fibre form, with suitable biodegradable polymers. Processing techniques for biopolymer scaffolds such as freeze-drying, electrospinning and 3D printing, are commonly applied to develop BG containing scaffolds with suitable mechanical properties, porosity and degradation behaviour for soft tissue repair applications. Typical examples of such composites for a variety of soft TE applications, including wound healing, nerve and muscle regeneration will be discussed.
In the second part of the presentation, applications of BGs (e.g. as mesoporous nanoparticles) in the field of 3D bioprinting (biofabrication), which have been emerging in the last few years, will be introduced. Such applications expand the scope of applications of BGs in TE. In this context, the progress in the development and characterization of cell laden hydrogel-BG scaffolds by 3D bioprinting will be discussed. Examples of such applications will be presented highlighting the novel developments of hydrogel-bioactive glass composites as innovative multimaterial bioinks for cell encapsulation and for biofabrication of TE scaffolds of increasing complexity . The author’s views on the challenges and opportunities for further research in the field will be presented.
 Hench, L. L., et al., Bonding mechanisms at the interface of ceramic prosthetic materials, J. Biomed. Mater. Res. 5 (1971) 117–141.
 Hoppe, A., et al., A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics, Biomaterials 32 (2011) 2757-2774.
 Heid, S., Boccaccini, A. R., Advancing bioinks for 3D bioprinting using reactive fillers: A review, Acta Biomater. 113 (2020) 1-22.
Keywords: Bioactive glasses, tissue engineering, Biofabrication
Restoring bone regeneration in diabetic (hyperglycaemic) environments with HIF mimetics and bioactive glasses (#538)
A. Rezaei1, J. Turner1, K. Shakib1, G. Jell1
1 University College London (UCL), Division of Surgery and Interventional Science, London, United Kingdom
Diabetic patients have an increased risk of fracture and non-union fracture healing1. Diabetic patients and hyperglycaemic conditions have also been shown to cause an impaired hypoxia inducible factor-1α (HIF-1α) and reduced cellular response to low oxygen conditions (hypoxia)2. Whilst the HIF pathway is undoubtedly important in angiogenic signalling and restoring the vasculature in bone repair3, the direct role of HIF-1α on osteoblast behaviour and mineralisation in both normal and hyperglycaemic conditions is unclear. There may also be differing effects between hypoxia and HIF stabilisation. Using a multidisciplinary characterisation approach (biological, ultrastructural and microstructural quantitative techniques), this study investigates the role of hypoxia (1% O2) and HIF-stabilisers (CoCl2) and dimethyloxalylglycine (DMOG), on bone formation, in normal and high glucose in vitro environments. Moreover, the effect of controlled cobalt ion release via cobalt bioactive glasses (CoBGs) on hyperglycaemic nodule formation will be studied with the aim to determine if targeting HIF pathway can promote bone regeneration in diabetic patients.
Primary osteoblasts were isolated from neonatal Sprague-Dawley rats and cultured in α-MEM supplemented with 2 mM β-glycerophosphate, 10nM dexamethasone, and 50μg/mL ascorbate. Once confluent, cells were exposed to normal (5.5mM), moderate (25mM) and high (50mM) glucose environments and kept in normoxia (20% O2) and hypoxia (1% O2) for 21 days. Cells were also treated with CoCl2 (12.5, 25 and 50µM), DMOG (250,500 and 1000µM) and CoBG conditioned medium (containing 0, 12.5 and 25µM cobalt) in the various glucose conditions. Metabolic activity, proliferation rate, alkaline phosphatase (ALP) activity and vascular endothelial growth factor (VEGF) expression. Bone nodules were characterised using Alizarin Red staining, scanning and transmission electron microscopy (SEM,TEM), Raman spectroscopy and interferometry for quantitative analysis of the nodule size.
Results and Discussion
Moderate and high glucose conditions inhibited nodule formation. Hypoxia (1% O2), inhibited nodule formation in all glucose levels. HIF stabilisers (CoCl2, DMOG and CoBG) and CoBG conditioned media did not prevent nodule formation in normal glucose, and restored nodule formation in hyperglycaemic conditions (as determined by size)(Figure 1. a&b) All HIF stabilisers enhanced HIF-1α expression and VEGF production in moderate and high glucose environments. Glucose level did not have any effect on ALP activity.
A hyperglycaemic bone model was developed that demonstrated that hyperglycaemic environments inhibit bone nodule formation, whilst the use of HIF mimetics (CoCl2, DMOG and CoBG) restored bone nodule formation in these conditions. This develops our understanding of the role of the HIF pathway in bone mineralisation and the creation of biomaterials or tissue scaffolds designed for patients with impaired bone regeneration due to an impaired HIF-1α pathway.
 Loder, RT, 2016, ‘The influence of diabetes mellitus on the healing of closed fractures’, Clinical orthopaedics and related research, 1988, 210-216.
 Thangarajah, H, et al. 2010, ‘HIF-1α dysfunction in diabetes’, Cell cycle, (9)1, 75-79.
 Semenza, GL, 2016, ‘Targeting hypoxia-inducible factor 1 to stimulate tissue vascularisation’, Investig Med, 64, 361-363.
The effect of best concentrations of HIF mimetics (Co 12.5µM and DMOG 500µM) on nodule formation in low, moderate and high glucose environments. Alizarin Red calcium staining showed that non-toxic range of CoCl2 and DMOG restore bone formation in moderate and high glucose environments (scale bar is 200mm)
Keywords: Bone tissue engineering, Diabetes, Bioactive glass
Sr-containing Mesoporous Bioactive Glasses Bio-Functionalized with ICOS-Fc: an advanced tool to target osteoporotic fractures (#883)
S. L. Fiorilli1, M. Pagani1, E. Boggio2, 3, C. L. Gigliotti2, 3, C. Dianzani4, C. Pontremoli1, G. Montalbano1, U. Dianzani3, C. Vitale-Brovarone1
1 Politecnico di Torino, Department of Applied Science and Technology, Turin, Italy
2 NOVAICOS s.r.l.s, Novara, Italy
3 Università del Piemonte Orientale, Department of Health Sciences, Novara, Italy
4 Università di Torino, Department of Drug Science and Technology, Torino, Italy
Osteoporotic bone fractures represent a critical clinical issue and need personalized and specific treatments. The development of smart nano-biomaterials able to synergistically combine chemical and biological cues to impart specific therapeutic effects (i.e., pro-osteogenic, anti-clastogenic) can provide novel and effective medical solutions to target compromised bone remodeling. Recently, strontium-containing mesoporous bioactive glasses (Sr-MBGs) proved to exert a role in the activation of both osteoblast (Ob) and osteoclast (Oc) cell signaling pathways, which allows the promotion of osteoblast replication, differentiation, and survival while downregulating osteoclast activities . In this contribution, with the aim to open new perspectives for advanced treatments of osteoporotic fractures, Sr-MBGs have been bio-functionalized with ICOS-Fc, a molecule able to reversibly inhibit Oc activity. The proposed strategy (Figure 1) aims to combine in single device the intrinsic properties of MBGs with the specific effect exerted by ICOS-Fc to deliver a multifunctional platform (i.e., bioactivity, stimulation of osteoblast cells, inhibition of osteoclast activity) for the treatment of osteoporotic fractures.
The extracellular portion of human ICOS was cloned as fusion protein to the human IgG1 Fc region, generating ICOS-Fc fusion protein, according to Di Niro et al. . Sr-containing MBGs (10% mol.) were synthesized as reported in ref  and post-modified with ((3-aminopropyl)silanetriol to expose surface amino groups able to react with the carboxyl groups present on Fc residue of ICOS-Fc molecule. The Sr-MBGs after ICOS-Fc grafting were fully characterized by scanning electron microscopy, N2 adsorption-desorption analysis, FT-IR spectroscopy, thermogravimetric analysis. Flow cytometry (Attune NxT, Life Technologies, Carlsbad, CA, USA) was performed to assess the presence of ICOS-Fc molecule on MBG surface and an in house-developed ELISA-Like Assay was performed to prove the retention of ICOS-Fc functionality upon grafting and the binding stability in aqueous medium. Cell Migration Assay were carried out in the Boyden chamber (BD Biosciences, Milan, Italy) migration assay by using ICOSL positive cell lines PC-3 (prostate cancer) and U2OS (osteosarcoma). The ability of grafted ICOS-Fc to inhibit osteoclast differentiation and function was assessed by monitoring the differentiation of monocyte-derived osteoclasts (MDOCs) up to 21 days.
Results and Discussion
N2 adsorption analysis, FT-IR spectroscopy and flow-cytometry analysis proved the successful grafting of ICOS-Fc on the surface of Sr-containing MBGs, which were also proved to retain the peculiar ability to release osteogenic strontium ions and an excellent bioactivity after functionalization. An ELISA-like assay allowed to confirm that grafted ICOS-Fc molecules were able to bind ICOS-L (the ICOS binding ligand) and evidenced the stability of the surface binding to hydrolysis in aqueous environment up to 21 days. The migration assay demonstrated that, in analogy to the free form of the molecule, PC-3 and U2OS cell (ICOSL positive) migration was affected by ICOS-Fc grafted Sr-MBGs (in a dose dependent manner), at variance with HOS (ICOSL negative) cell migration, confirming the specificity of ICOS-Fc to bind ICOSL. Furthermore, grafted ICOS-Fc was able to strongly inhibit the differentiation of MDOCs, which after the treatment showed a round shape and a morphology similar to a spindle. The strong inhibitory effect was also proved by the downregulation of DC-STAMP, OSCAR, and NFATc1 expression, as evidenced by real-time PCR after 21 days.
The functionality of ICOS-Fc molecule anchored to Sr-containing MBGs was confirmed by a custom-made ELISA-like and the binding stability was assessed up to 21 days of soaking in culture medium. The inhibitory effect of grafted ICOS-Fc on cell migratory activity was demonstrated by using ICOSL positive cell lines and the ability to inhibit osteoclast differentiation and function confirmed (in analogy to the free form of the molecule) by monitoring the differentiation of MDOCs, which revealed a strong inhibitory effect, also proved by the downregulation of Oc differentiation genes.
The obtained results are very promising and pave the way to personalized clinical solutions for bone regeneration of fractures in osteoporotic patients.
This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 814410.
 Saidak, Z.; Marie, P. J. Strontium signaling: Molecular mechanisms and therapeutic implications in osteoporosis. Pharmacol. Ther. 2012, 136, 216–226, doi:10.1016/j.pharmthera.2012.07.009
 Di Niro, R.; Ziller, F.; Florian, F.; Crovella, S.; Stebel, M.; Bestagno, M.; Burrone, O.; Bradbury, A. R. M.; Secco, P.; Marzari, R.; Sblattero, D. Construction of miniantibodies for the in vivo study of human autoimmune diseases in animal models. BMC Biotechnol. 2007
 Fiorilli, S.; Molino, G.; Pontremoli, C.; Iviglia, G.; Torre, E.; Cassinelli, C.; Morra, M.; Vitale-Brovarone, C. The incorporation of strontium to improve bone-regeneration ability of mesoporous bioactive glasses. Materials (Basel). 2018, 11, 678
Schematic illustration of the effect produced by Sr-containing MBGs functionalized with ICOS-Fc on osteoblast and osteoclast cells
Keywords: nano-biomaterials, osteoporosis, ICOS-Fc biofunctionalization
3D plotted composites consisting of calcium phosphate bone cement and mesoporous bioactive glass with drug delivery function3D plotted composites consisting of calcium phosphate bone cement and mesoporous bioactive glass with drug delivery function (#475)
R. F. Richter1, T. Ahlfeld1, M. Gelinsky1, A. Lode1
1 TU Dresden, Centre for Translational Bone, Joint and Soft Tissue Research, Dresden, Saxony, Germany
Calcium phosphate bone cements (CPC) and mesoporous bioactive glasses (MBG) are two well studied biomaterial groups widely under investigation concerning their applicability to treat bone defects. Recently, a CPC-MBG composite was developed in our group which showed promising results with respect to cell response in vitro and new bone formation in vivo [1,2]. Unlike conventional powder/liquid cements, the used CPC is a hydrophobic carrier-liquid (cl) based paste; it maintains a good injectability until contact with aqueous environment that allows its application for extrusion-based additive manufacturing techniques like 3D plotting . By variation of the amount of cl, it is possible to prepare CPC-MBG composites that are suitable for 3D plotting as well and the advantage of 3D plotted scaffolds of such composites compared to bulk scaffolds regarding ion release were shown recently . The aim of the present study was to analyse the degradation behavior of different CPC-MBG composites and the influence of the single components in detail. Furthermore, due to their high specific surface area, MBG are promising candidates to be used as carrier system for proteins, growth factors or anti-inflammatory agents. This study therefore aimed to develop a method that allows to load MBG with proteins and incorporate them into CPC pastes without affecting the plottability.
CPC and strontium-modified CPC pastes were provided by INNOTERE GmbH, Germany and MBG particles (< 45 µm) were prepared as previously described . Additionally, strontium-modified MBG particles were synthesized by complete substitution of the calcium part with strontium. To study the degradation behavior, mass loss, change of porosity and ion release of 3D plotted scaffolds (Figure 1) made from different CPC-MBG combinations were evaluated and compared to those of 3D plotted scaffolds or samples made of the single components. Afterwards, a protocol for loading MBG particles with proteins was developed. The protein uptake kinetics for different types of MBG and various initial protein concentrations were studied using lysozyme as a model protein. The release of lysozyme from pure MBG and from 3D plotted CPC-MBG scaffolds was investigated and it was verified whether the released lysozyme retained its biological activity. Finally, this developed protocol was transferred to the proangiogenic growth factor VEGF (Vascular Endothelial Growth Factor) and again the release and biological activity were analysed and verified.
Results and Discussion
Based on our previous work it was possible, by varying the amount of cl, to increase the addition of MBG to 16 wt% and maintain the extrudability of the composites. Composites with at least 8 wt% MBG showed significantly higher initial porosity and significantly greater mass loss after degradation. Regarding the ion release, again composites with a minimum addition of 8 wt% MBG showed a significantly improved release of ions, e.g. Sr2+ compared to pure strontium-modified CPC scaffolds.
Additionally, a protocol for loading MBG particles with proteins was successfully developed, that allowed the incorporation of such functionalised MBG particles into CPC pastes without impairing the plottability. The protein release from plotted scaffolds could be varied by addition of different amounts of protein-laden MBG into the composite (Figure 2) and the released lysozyme from pure MBG and from plotted composite scaffolds maintained its biological activity.
Finally, the established protocol for protein loading of MBG was successfully applied to VEGF and the release from MBG and CPC-MBG composites, while retaining the biological activity, was demonstrated.
In this study, we showed the development of a functionalised CPC-MBG composite applicable for 3D plotting that allows fabrication of patient-specific implants. Furthermore, the results of ion and protein release showed the high flexibility of this material system and suggest it as a promising material toolbox to treat bone defects.
This work was funded by the German Research Foundation (DFG) as part of the Collaborative Research Centre SFB/Transregio 79 (subproject M2).
 Wagner, A.-S., Schumacher, M., Rohnke, M., Glenske, K., Gelinsky, M., Arnhold, S., Mazurek, S., Wenisch, S. (2019). Incorporation of silicon into strontium modified calcium phosphate bone cements promotes osteoclastogenesis of human peripheral mononuclear blood cells. Biomedical Materials, 14, 025004, United Kingdom: IOP Publishing
 Kauschke, V., Schneider, M., Jauch, A., Schumacher, M., Kampschulte, M., Rohnke, M., Henss, A., Bamberg, C., Trinkaus, K., Gelinsky, M., Heiss, C., Lips, K. (2018). Effects of a Pasty Bone Cement Containing Brain-Derived Neurotrophic Factor-Functionalized Mesoporous Bioactive Glass Particles on Metaphyseal Healing in a New Murine Osteoporotic Fracture Model. International Journal of Molecular Sciences, 19, 3531, Switzerland: MDPI
 Lode, A., Meissner, K., Luo, Y., Sonntag, F., Glorius, S., Nies, B., Vater, C., Despang, F., Hanke, T., Gelinsky, M. (2012). Fabrication of porous scaffolds by three-dimensional plotting of a pasty calcium phosphate bone cement under mild conditions. Journal of Tissue Engineering and Regenerative Medicine, 8, 682–693. United Kingdom: John Wiley & Sons Ltd
 Richter, R. F., Ahlfeld, T., Gelinsky, M., & Lode, A. (2019). Development and Characterization of Composites Consisting of Calcium Phosphate Cements and Mesoporous Bioactive Glass for Extrusion-Based Fabrication. Materials, 12, 2022, Switzerland: MDPI
 Zhu, Y., Wu, C., Ramaswamy, Y., Kockrick, E., Simon, P., Kaskel, S., & Zreiqat, H. (2008). Preparation, characterization and in vitro bioactivity of mesoporous bioactive glasses (MBGs) scaffolds for bone tissue engineering. Microporous and Mesoporous Materials, 112, 494–503, Netherlands: Elsevier
Figure 1: 3D plotted scaffold
3D plotted scaffold using a CPC-MBG composite (in this case a strontium-modified CPC with the addition of 16 wt% of MBG)
Figure 2: Accumulated lysozyme release from 3D plotted scaffolds
Accumulated lysozyme release from 3D plotted scaffolds made of strontium-modified CPC (cSr) and various amounts of lysozyme-laden MBG (gCa(x)[Lyz], with x = 4, 8, 16 wt%) over 49 days (n = 6, mean ± standard deviation)
Keywords: calcium phosphate cement, mesoporous bioactive glass, drug delivery
Group discussion with all abstract presenters