Simona CavaluProfessor

Faculty of Medicine and PharmacyUniversity of Oradea

ROMANIA

New bioceramics for hard tissue

replacement and drug/ protein delivery:

physical and biological approach

Highlights

Bioglass for controlled drug release (antibiotics)

Surface functionalization and conformational changes of

proteins adsorbed on Bioglass

Insulin microencapsulation and release from zinc/silica

microparticles

A real story…turns to history

Bioglass® 45S5, the first formulation

composed of SiO2, Na2O, CaO and P2O5,

developed by Professor Larry Hench at the

University of Florida in the late 1960s.

The 45S5 name signifies glass with 45 wt.%

of SiO2 and 5:1 ratio of CaO to P2O5. Lower

Ca/P ratios do not bond to bone.

The first successful surgical use of Bioglass

45S5 was in replacement of ossicles in

middle ear, as treatment of conductive

hearing loss.

Different Bioglasses composition reported in literature

Bioglasses are divided in two categories:

Class A:•Osteoproductive

•Osteoconductive.

•Bind with both soft tissues and

bone.The HCA layer forms within

several hours.

Class B:• Osteoconductive (such as ydroxyapatite)

• Bonding to soft tissues is not facilitated.

The HCA layer takes one to several days to

form.

Advantage/disadvantage

The primary advantage of bioactive glasses is their rapid rate ofsurface reaction which leads to fast tissue bonding.

Their primary disadvantage is mechanical weakness and lowfracture toughness due to an amorphous two-dimensional glassnetwork.

Tensile bending strength of most of the compositions is in therange of 40-60MPa which make them unsuitable for load bearingapplications.

For some applications low strength is offset by the glasses’ lowmodulus of elasticity of 30-35 GPa. This value is close to that ofcortical bone.

Compositions such as 45S5 Bioglass with high rates of

bioactivity produce rapid regeneration of trabecular

bone with an amount, architecture and bio-mechanical

quality of bone that matches that originally present in the

site.

The rapid regeneration of bone is due to a combination

of processes called osteostimulation and osteoconduction

Processing bioglasses

Melting processT= 1100-1300°C

Sol-gel route advantages: low processing

temperature and controlling textural

properties

Sol-gel-derived bioactive glasses are more bioactive

and degrade more rapidly than melt-derived glasses of

similar compositions.

This is because sol-gel glasses have a nanometer-scale textural

porosity which increases the specific surface area by two orders of

magnitude compared to a melt-derived glass .

Sol–gel processing

TEOS: tetraethoxysilane, Si(OCH2CH3)4.

Pharmaceutical applications

Design of bioglass and bioceramic drug delivery systemswhich allow the effective targeted delivery dosage ofproducts, ranging from inorganic and organic molecules ofdifferent size and properties, to be controlled .

Client bespoke formulations for specific applications,including the controlled release of pharmaceutical activecompounds, such as vitamins, antibiotics and anti-inflammatory drugs, and also the enabling of sequential, andtime and site well-ordered, delivery of multiple agents

Characterization to monitor and evaluate controlledreleased (from hours to days, even, to months)

Bioglass for controlled drug

release: our results

BIOACTIVE GLASSES FOR ANTIBIOTIC CONTROLLED

RELEASE- our results

Bioglass composition and treatment:

Specimen 1: 0.55SiO2•0.41CaO•0.04P2O5, maturated at room temperature for 70 days and heat treated at 310°C for 1 hour.

Specimen 2: 0.55SiO2•0.41CaO•0.04P2O5, air dried at 80°C for 50 min., maturated at room temperature for 70 days and then heat treated at 310°C for 1 hour.

Specimen 3:0.45SiO2•0.245CaO•0.06P2O5•0.245Na2O,

maturated at room temperature for 70 days and then heat treated at 310°C for 1 hour.

S. Cavalu & all, Journal of Molecular Structure 1040 (2013) 47–52

TTC was incorporated by immersion of the specimens in

solution C=7 mg/ml under continuing stirring 1h.

Tetracycline loading and release – SEM

Before loading

After TTC loading

Specimen 1: 0.55SiO2•0.41CaO•0.04P2O5

Tetracycline loading and release – SEM

Before loading

After TTC loading

Specimen 2: 0.55SiO2•0.41CaO•0.04P2O5

Tetracycline loading and release – SEM

Before loading

After TTC loading

Specimen 3 : 0.45SiO2•0.245CaO•0.06P2O5•0.245Na2O

BET specific surface area and mean pore volume values determined

for the bioactive glass specimens before and after tetracycline loading.

Sample Specific surface area (m2/g) Pore volume (ml/g)

Before TC loading TC loaded Before TC loading TC loaded

S1 106.8 83.1 0.43 0.34

S2 96.2 77.1 0.38 0.30

S3 98.2 79.0 0.41 0.31

The procedure of drying at 80 ºC caused a decrease of the pore size

The pore size and surface area decreased after immersion in tetracycline

solution due to tetracycline attachment.

Specimen 1, which exposed a larger surface, is able to incorporate more

tetracycline compared to the specimen 2 and 3.

TTC stability by UV/VIS spectroscopy

pH

stability

Red shift: transformation of TTC molecule from TTC0 to TTC- anion

concomitant with the transition of π to π* states . TTC- tends to attract

reactive species, such as .OH, due to the high electrical density on ring

system.

The main degradation products during photolysis in aqueous

medium

UV-VIS and EPR spectroscopy detecting free radical formation

during the samples preparation procedure.

200 300 400 500

TC3

TC2

Inte

nsity (

a.u

.)

Wavelength (nm)

TC1

3300 3320 3340 3360 3380 3400 3420

3333.6 3334.5 3335.4

Magnetic field (G)

TC3

TC2

TC1

Experimental EPR spectra of withdrawn tetracycline solutions

obtained upon filtration of each bioglass specimen. Inset: top of the

lower field spectral lines, showing quantitative differences in spin

concentration. Bottom: Experimental EPR spectra of tetracycline

hydrochloride starting solution (7 mg/mL)

265

383

UV-VIS spectra of withdrawn

tetracycline solutions obtained

upon filtration of each bioglass

specimen

EPR experimental spectra of the bioglasss

specimens upon TTC loading, filtration and drying

procedure.

EPR simulations of liquid spectra

The values of g and A magnetic tensors components obtained from EPR spectra simulations for

tetracycline withdrawn solutions:

τTC1< τTC2< τTC3

τ (ps) Axx (mT) Ayy(mT) Azz(mT) gxx gyy gzz

TC1 30.2 23.50 7.36 21.52 2.00475 2.00418 2.00212

TC2 34.3 23.71 7.38 21.26 2.00473 2.00392 2.00217

TC3 49.4 23.53 7.292 21.61 2.00429 2.00410 2.00285

EPR simulations of TC immobilized on porous bioactive

structure

τTC1< τTC2< τTC3

Differential Pulsed Voltammetry (DPV) -tetracycline release

profile in SBF

0.0 0.2 0.4 0.6 0.8

0

1

2

3

4

I (

A)

E (V)

2h

5h

24h

48h

96h

0.0 0.2 0.4 0.6 0.8

-1

0

1

2

3

I (

A)

E(V)

2h

5h

24h

48h

96h

0.0 0.2 0.4 0.6 0.8

0

1

2

3

E (V)

I (

A)

2h

5h

24h

48h

96h

Specimen 1 Specimen 2 Specimen 3

Time

(h)

Specimen 1 Specimen 2 Specimen 3

I (μA) C(μM) I (μA) C (μM) I (μA) C(μM)

2 3.50 0.851 3.28 0.712 2.59 0.295

5 3.27 0.703 3.11 0.614 2.32 0.121

24 2.95 0.511 2.73 0.379 2.14 0.071

48 1.99 0.025 0.25 0.040 0.19 0.008

96 1.56 0.014 0.15 0.020 0.10 0.004

Tetracycline hydrochloride may act as a chemical spin trap.

EPR and UV/VIS spectroscopy have shown that the specimen with a larger surface

are is able to incorporate more tetracycline.

The maximum TC amount was released after 2 h, and thereafter the release continued

slightly for 24 h, followed by a drastic diminution after 48 h.

The pores size modification and specific surface area after tetracycline loading seems

to be the main factor in tetracycline controlled released process.

Similar results were obtained for different pharmaceutical compounds:

hydrocortisone, propolis, β-cyclodextrin [ Z.R. Domingues & all, Biomaterials 25

(2004) 327–333; A. L. Andrade & all, Journal of Non-Crystalline Solids 355 (2009) 811–

816].

Observations

Surface functionalization and conformational changes of

proteins adsorbed on Bioglass

Native structure of methemoglobin by

X-ray crystallography

Composition as classical 45S5 Bioglass:

45% SiO2, 24.5% Na2O, 24.5% CaO and 6% P2O5 (in molar%).

Sol-gel route.

Aging 30 days at room temperature and heating at 310º C 1h.

Incubation 4h in protein solution ( 25 mg/ml MHb with TBS).

Glutaraldehyde (GA) solution (1 mol/L) as protein coupling agent

Particles size distribution of the milled glass

(by laser diffraction method).

V. Simon, S. Cavalu, Solid State Ionics 180 (2009) 764–769.

C. Gruian, S. Cavalu, V. Simon, Biochimica et Biophysica Acta 1824 (2012) 873–881

SEM images of BG without GA before (A) and after

immersion in protein solution (C)

An uniform layer of protein covers the BG surface; the NaCl crystals are not covered by proteins.

Methemoglobin attachment on the Bioglass surface after

functionalization with GA

SEM images of the BG with GA, before (A) and after immersion in protein solution with 10 mMNaCl (B) and 500 Mm NaCl (C).

Protein

cluster

FTIR spectroscopy

X –ray Photoelectron Spectroscopy

evidence of MetHB adsorbed on

Bioactive glass

Amide I and amide II absorption bands are sensitive to changes in protein

secondary structure.

Qualitative and quantitative structural information can be obtained by second

derivative spectrum and deconvolution.

Native MeMb

MethMb on BG

MetMb on BG-GA

Native MetMb MethMb on BG MetMb on BG-GAF

TIR

sp

ect

rosc

op

y a

nd

de

con

vo

luti

on

α helix % β sheet% β turns % Random % Side chain%

Observations

One additional peak centered at 1648 cm−1 appearsoriginating from random structure.

Also, the major bands at 1656 and 1654 cm−1, assigned to α-helix structures in the native methemoglobin are shifted tohigher wavenumbers upon adsorption. The higher bandposition corresponds to weaker hydrogen bonding, leading tomore flexible helices, as a consequence of the interactionbetween the proteins and bioactive glass.

The protein loose approximately half of the α-helicalstructure after adsorption on BG, but only 1/3 when BG isfunctionalized with GA.

XPS survey spectra

SampleElemental composition (at %)

Si Ca P Na C O N S

BG 32.6 5 2.3 1.4 5.6 53 – –

MetHb – – – – 64.8 18.4 16.7 0.1

MetHb

on BG16.9 3.8 3.7 0.6 28.2 42.4 4.3 –

BG–GA 18 2.9 1.5 – 40.5 33.6 3.4 –

MetHb

on BG–

GA

5.8 2.1 1 0.1 58.1 21.4 11.5 –

BG

MetHB lyophylized

MetHb on BG

BG with GA

MetHb on BG-GA

C 1s high resolution XPS spectra and

deconvolution

284.6 eV

290.4 eVC03

286.2 eV C-C and C-H

288.7 eV NH-CHR-CO and –NH2

N 1s and O 1s high resolution XPS spectra

(a) BG

(b) MetHb lyophilized

(c) MetHB on BG

(d) BG with GA

(e) MetHb on BG with GA.

400 eV C-N

532 eV -OH and peptidic oxygen

( shifted to lower binding energy)

Observations

The marker bands N 1s and C 1s specific to proteins shows

an increasing intensity on GA functionalized sample.

The surface functionalization of the bioactive glass substrate

with GA provides a better protein adherence that is

considered beneficial for further interaction of biomaterial

surface with surrounding cells.

Insulin microencapsulation and

release from zinc/silica microparticles

In the presence of zinc ions, insulin dimers associate into hexamers with

greater stability.

Advantages

Microencapsulation is considered one of the best oral drug delivery approaches.

Overcome the enzimatic and physical barriers of gastro intestinal tract.

Advantages of encapsulation using inorganic silica: highly inert and stable (compared to organic polymers), amorphous silica (in contrast to crystalline silica) is not toxic being recognized by the Food and Drug Administration as safe food additive and excipient for vitamins.

Silica shell with pores typically < 10 nm

ZnO has antiseptic effect

Addition of Zn ions proved to preserve the secondary structure of some proteins.

Sol - gel route

Sol- gel 95 SiO2● 5ZnO (mol%)

20 mg Insulin addition to zinc silicate sol

(before gelation ) pH=2+

Spray- dried microcapsules

Inlet temperature T= 120 ◦C

Outlet T=75 ºC

Freeze- dried microparticles

T= -196 ºC

Dried at T= 37 ºC

X–ray diffraction and particle size analysis

XRD patterns of the insulin

entrapped in zinc-silica particles

obtained by spray drying (a) and

freeze drying (b) methods.

Particle size distribution plotted on a logarithmic scale of

the zinc-silica sprayed dried microsphere (a) and freeze

dried microparticles (b).

2.5 µm- dominant

35 µm- weakly 5µm

E. Vanea, S. Cavalu, Journal of Biomaterials Applications 28(8) 1190-1199 (2014)

SEM images

Spray –dried microspheres

Freeze –dried microparticles

FTIR spectra and deconvolution

(a) Native insulin

(b) insulin entrapped in zinc-silica microspheres (ZnSi-SD-INS)

(c) insulin entrapped in zinc-silica microparticles (ZnSi-FD-INS)

Deconvolution of FTIR Amide I absorption band of native insulin (a), insulin entrapped in ZnSi-SD-INS

microspheres (b), and insulin entrapped in microparticles (ZnSi-FD-INS) (c).

The in vitro release tests were carried out by suspending the particles in simulated

gastric fluid (pH =1.2) for 120 min, which corresponds to the gastric transit time in

the stomach and then in simulated intestinal fluid (pH=8.2) for another720 min.

Cumulative release in pH=1.2 Cumulative release in pH=8.2

Observations

Insulin encapsulation in zinc-silica microparticles following the

sol-gel route combined with freeze drying and spray drying

procedure has been demonstrated to preserve the integrity of

insulin.

Formation of insulin hexamers in the presence of zinc ions leads

to an increased stability of the insulin three-dimensional structure

during preparation, storage and release.

The release profile can be adapted by synthesis route of

microparticles.

The future of bioglasses

Bone tissue engineering- combines cells and

biodegradable 3D scaffold to repair diseased or

damaged bone tissue.

Scaffolds are needed that can act as temporary templates

for bone regeneration and actively stimulate vascularized

bone growth so that bone grafting is no longer necessary.

Bone tissue engineering

Requirements for the future:

Improvement of the mechanical performance of existing

bioactive ceramics.

Enhanced bioactivity in terms of gene activation.

Improvement in the performance of biomedical coatings in

terms of their mechanical stability and ability to deliver

biological agents.

Development smart materials capable of combining sensing

with bioactivity.

Development of improved biomimetic composites.