U N I V E R S I T Y O F T R E N T O

M a s t e r d e g r e e i n M a t e r i a l s a n d P r o d u c t i o n E n g i n e e r i n g

C o u r s e o f G l a s s E n g i n e e r i n gA . Y . 2 0 1 9 - 2 0 , p r o f . V . M . S G L A V O

L AT E S T B I O G L A S S D E V E L O P M E N T S

I N B I O M E D I C A L A P P L I C AT I O N S

Daniele BugnottiAnna FamàGiacomo MoriAlessandro Pedranz

Students:

28th April 2020

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OUTLINE

INTRODUCTION

BIOACTIVE GLASS

Glass-activated regeneration of volumetric muscle loss

• 3D-printed PCL/bioglass for bone tissue regeneration

• 45S5 Bioglass doped withniobium for repairing critical-sized bone defect

OTHER APPLICATIONS

FUTURE OUTCOMES

Volumetric muscle loss and state of the art

Structure, bioactivity and role of Boron

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VOLUMETRIC MUSCLE LOSS (VML)

Large amount of muscular tissue can be removed or destroyed after traumatic events.In these cases the innate repair mechanism is not enough to regenerate the tissue so external action is required.

Two main approaches:

• Development of engineered skeletal muscle constructs in vitro

• Development of scaffolds to regenerate damaged skeletal muscle tissue in vivo

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Materials designed to guide the regeneration of the muscle can be classified in:

• Cellular Delivery of myoblasts• Acellular Delivery of growth factors

Limitations

• Insufficient vascularization• Low recruitment of cells → very slow recovery

• No uniform cell alignment

Examples

PGASynthetic polymers PLA

PCL

Decellularized scaffolds

AlginateNatural polymers Collagen

Fibrin

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Bioactive glass can support angiogenesis and stimulate gene expression for muscle regeneration without incorporating cells and growth factors, making them a good candidate for this application.

Need of new scaffolds able to enhance SC recruitment to the defect size↓

Total endogenous regeneration

Satellite cells (SCs) = skeletal muscle progenitor cells that play a critical role in skeletal muscle repair and contribute to newly formed muscle fibers

HOW THESE SCAFFOLDS CAN BE IMPROVED?

BIOACTIVE GLASS STRUCTURE

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GLASS SiO2 B2O3 P2O5 Al2O3 Na2O K2O CaO MgO

45S5 45.0 - 6.0 - 24.5 - 24.5 -

13-93B3 - 56.7 3.4 - 5.5 11.1 18.4 4.6

8A3B - 50.7 3.2 10.8 4.9 9.9 16.4 4.1Table 1. Wt% composition of some bioactive glasses

Ø Lots of network modifiers: low NC (2-3), open structureØ P2O5: orthophosphate surrounded by cations (Na+, Ca2+)

Ø Phase separation: «Phosphate rich isles»

Ø PROBLEM: Crystallization

More compact, «less bioactive»

B2O3 + alkali mixed effect + Mg

MD simulation of glass structures by increasingsilica content (from left to right)

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HOW IS GLASS BIOACTIVE?

A bioactive material is able to “elicit a specific biological response at theinterface of the material which results in the formation of a bondbetween the tissues and the material”Ø Glass dissolution: constant ions

release

Ions tune «specific biological response»:• Ca, Sr, P, Si: osteoblast, bone regrowth• B: fibroblast• Ag, Zn: antibacterial

Ø Formation of a substituted apatite layer(nm size, highly substituted)

Remodelling of mineralized human tissues!Osteoblast adherent to the apatite layer of a bioactive glass

7

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TISSUE REGENERATION?

Ø If BG are able to stimulate angiogenesis (vascularization)

Ø Extension of BG applications outside bone regrowth

Ions are good! But high amount means cytotoxicity: Controlled ion release rate

Ø Natural tissue regeneration requires blood vessels! (bone vs cartilage)

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Synthetic biomedical devices based on bioactive glasses of different degradation rates can be used to treat VML

First report on an inorganic material used in skeletal muscle regeneration through in vitro and in vivo models

Bioactive glasses Compositions (wt%) Degradation rates SBF @ 37 °C for 30 days

Silicate 45S5 45.0 SiO2 , 24.5 Na2O, 24.5 CaO,6.0 P2O5

2.4%

Borate 13-93B3 56.7 B2O3 , 5.5 Na2O, 11.1 K2O, 4.6 MgO, 18.4 CaO, 3.4 P2O5

30.4%

Aluminoborate 8A3B 50.7 B2O3 , 10.8 Al2O3 , 4.9 Na2O, 9.9 K2O, 4.1 MgO, 16.4 CaO, 3.2 P2O5

5.3%

Are bioactive glasses capable of regenerating VML without growth factors or stem cells?

Glass-activated regeneration of volumetric muscle loss

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• Ion exchange• Leaching• Dissolution

Chemical etching by H2O

• Increase of pH• Loss of weight• Precipitation of

apatite phase

Observed

• Release of Ca2+

and Si4+ or B3+

ions • Support

angiogenesis

Degradation study in SBF

d

Time (hr)

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IN VITRO studies: bioactive glass (both silicate and borate families) can support angiogenesis through ions release + stimulate gene expression for muscle regeneration

• 3 tested bioactive glasses à secretion of growth factors for muscle defect healing(CX43 and IGF-1)

• Ionic products associated with glass degradation à VEGF (vascular endothelial growth factor) secretion in mouse muscle myoblast C2C12 cells and in HUVECs (human umbilical vein endothelial cells)

Strong evidence for their ability to stimulate angiogenesis

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Al2O3-containing 8A3B glass most attractive composition:• Moderate degradation rate, higher than 45S5• Least impact on the pH of the solution while releasing

calcium ion over the studied time• Most effective in supporting in vitro angiogenesis

Improvement of durability of 8A3B

Formation of aluminum polyhedral [AlO4],

[AlO5] and [AlO6] units↓ non-bridging oxygens

in the network

↓ number of tetrahedral boron (B IV) in 8A3B

than 13-93B3Alkali rather associate

with Al than B

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IN VIVO work (in a rat model): ability of bioactive glasses in activating SCs formation and vascularization for effective healing of VML is confirmed

Nucleated muscle fibers = indication of muscle regeneration• observed in defects treated with

glass powders of 13-93B3 and 8A3B • 8A3B group à muscle fibers of much

larger size (~20 µm) than in 13-93B3 group (5-10 µm)

Presence of blood vessels for:• sufficient vascularization of

the new muscles • maintaining cell viability

during muscle tissue growthand structural organization

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IN VIVO work (in a rat model): ability of bioactive glasses in activating SCs formation and vascularization for effective healing of VML is confirmed

Bioactive glass is found to activate the production of satellite cells (SCs), muscle stem cells, without the incorporation of extra stem cells or growth factors

• Immunofluorescent images: ↑ implantation time à ↑ number of satellite cells • 8A3B-treated rats: highest number of cells

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• Degradation rate + kinetics can be varied over a wide range• Ionic products associated with glass degradation support

angiogenesis + expression of essential muscle-defect related growth factors from cells

• Without stem cells or extra growth factors, bioactive glass alone is capable of activating satellite cells formation + angiogenesis

FINDINGS

• Clinical trials to design treatment for skeletal muscle defects• Further research on muscle regeneration and ability of bioactive

glasses to:

• Trials for identifying a better performing boron release profile for promoting angiogenesis and stem cells formation

• Testing mechanical properties

FUTURE OUTCOMES

- Stimulate the formation of satellite cells- Trigger orientation and fibers alignment

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Research demonstrates the suitability of bioactive glasses in contact with tissues outside the skeletal system

Chronology of the special applications ofbioactive glasses outside the skeletal system

Non-osseous applications à adequate compliance with soft tissues à bioactive glasses as inorganic fillers in a polymeric matrix (↑ mechanical properties)

But still bone-bending ability is under continuous improvement

Hard tissue repair à 3-D porous scaffoldsthrough versatile methods such as spongereplica technique, sol–gel foaming, polymericparticle burning-out and rapid prototypingtechniques

3D-printed PCL/bioglass (BGS-7) composite scaffolds with high toughness and cell-responses for bone tissue regeneration

Problem: BGS-7 bioglass present high bioactive properties, but also high brittleness

Solution: composite material scaffold (PCL/bioglass) showed increased mechanical properties

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3D printing → highly interconnected porous mesh structure, easy manipulation of the pore size, tortuosity, morphology, porosity of the scaffold

Fabrication process of the scaffolds using a 3D printing system

(a) compression and (b) 3-point bending test for the scaffolds

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What aboutbio-properties?

Key parameters– Porosity– Roughness – Protein adsorption ability

CONCLUSIONS:

↑ %wt of bioglass↑ cellular activity (including osteogenic genes)↓ toughness (PBGS-60)

PBGS- 40 (40 wt% of bioglass) composite scaffold showed an outstanding toughness with reasonable cellular activities

Future research: in vivo tests

In vitro cellular activities of the scaffolds

Evaluation of effectiveness of 45S5 Bioglass doped withniobium for repairing critical-sized bone defect in

in vitro and in vivo models

Why Niobium?

• Effectiveness of the introduction of therapeutic ions also for bone-regeneration strategies

• Improve biological and mechanical properties of the biomaterial

• Sol-gel Nb oxide promote apatite formation

• Great biocompatibility and osteogenic differentiation of human mesenchymal stem cells

But rarely applied incorporated insilicon rich glasses…

First of all, cytotoxicity, genotoxicityand system compatibility were tested

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Live/Dead assay photomicroscopies of normal human osteoblasts (NHOsts) treated with control medium (a), 45S5 (b), BGPN2.6 (c), and ETOPOSIDE (d) for 72 hr (100×magnification)

Nb-containing glass (BGPN2.6) is not cytotoxic to Human Osteoblasts

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What about bone regeneration?

Three-dimensional (3D) reconstructions of microcomputedtomography images of rats' calvarias after 56postoperative days (a-f) and percentage of bone withinthe defect after 56 days of implant (g)

Bone regeneration showed 40% increase indefect repair compared to 45S5 bioglass

Conclusions

Biocompatibility was confirmed by the resultsof the in vivo experiments and no sign ofgenotoxicity was found. Nb-glass is a safe andefficient biomaterial to be used for bonereplacement

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Bioactive glasses: future developments

• Soft tissues applications (e.g. wound healing) à optimizing the stiffness to match the compliance of delicate collagenous tissues

• Optimization of bioactivity à 3D porous scaffolds as templates for cell alignement, proliferation and bone growth

• Technological advancement in the processing à additive manufacturing technology

• Hierarchical scaffolds with multifunctional capabilities: apatite-forming properties, controlled and local release of ions/drugs

• 3D organ printing with bioglass powder/polymer materials (bioglass support the organ growth, angiogenesis, antibacterial effects..)

REFERENCES

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• WEITAO JIA, HAORAN HU, AIZE LI, HUAYUN DENG, CARRIE L. HOGUE, JOHN C. MAURO, CHANGQING ZHANG, QIANG FU, “Glass-activated regeneration of volumetric muscle loss”, Acta Biomaterialia, v.103, p. 306–317, 2020

• JONATHAN M. GRASMAN, MICHELLE J. ZAYAS , RAYMOND L. PAGE, GEORGE D. PINS “Biomimetic scaffolds for regeneration of volumetric muscle loss in skeletal muscle injuries”, Acta Biomaterialia, 25, 2–15, 2015

• DELIA S. BRAUER, Bioactive Glasses—Structure and Properties, Angewandte Chemie Int. Ed. 54 (14), 162-177 , 2015

• P. BALASUBRAMANIAN, L. HUPA, B. JOKIC, R. DETSCH, A. GRU¨NEWALD, ALDO R. BOCCACCINI, “Angiogenic potential of boron-containing bioactive glasses: in vitro study”, J. Mater. Sci. 52, (15, 8785-8792 ), 2017

• F. BAINO, G. NOVAJRA, V. MIGUEZ-PACHECO, A. R. BOCCACCINI, C. VITALE-BROVARONE, “Bioactive glasses: Special applications outside the skeletal system”, Journal of Non-Crystalline Solids, v. 432, p. 15–30, 2016

• YONGBOK KIM, JUN YOUNG LIM, GI HOON YANG, JUN-HYUK SEO, HYUN-SEUNG RYU, GEUNHYUNG KIM, “3D-printed PCL/bioglass (BGS-7) composite scaffolds with high toughness and cell-responses for bone tissue regeneration”, Journal of Industrial and Engineering Chemistry, v.79, p. 163-171, 2019

• L. P. L. DE SOUZA , J. H. LOPES, F. V. FERREIRA, R. A. MARTIN, C. A. BERTRAN, J. A. CAMILLI, “Evaluation of effectiveness of 45S5 bioglass doped with niobium for repairing critical-sized bone defect in in vitro and in vivo models”, J. Biomed. Mater. Res., v. 108A, p. 446– 457, 2020

U N I V E R S I T Y O F T R E N T O

M a s t e r d e g r e e i n M a t e r i a l s a n d P r o d u c t i o n E n g i n e e r i n g

C o u r s e o f G l a s s E n g i n e e r i n gA . Y . 2 0 1 9 - 2 0 , p r o f . V . M . S G L A V O

L AT E S T B I O G L A S S D E V E L O P M E N T S

I N B I O M E D I C A L A P P L I C AT I O N S

Daniele BugnottiAnna FamàGiacomo MoriAlessandro Pedranz

Students:

28th April 2020