New bioceramics for hard tissue replacement and drug/ protein delivery: physical and biological approach  

1. Simona CavaluProfessorFaculty of Medicine and PharmacyUniversity of OradeaROMANIA
New bioceramicsfor hard tissue replacement and drug/ protein delivery: physical and biological approach

3. 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

4. A real story…turns to history
Bioglass®45S5, the first formulation composed of SiO2, Na2O, CaOand P2O5, developed by Professor Larry Hench at the University of Florida in the late 1960s.
The 45S5 name signifies glass with 45 wt.% of SiO2and 5:1 ratio of CaOto P2O5. Lower Ca/P ratios do not bond to bone.
ThefirstsuccessfulsurgicaluseofBioglass45S5wasinreplacementofossiclesinmiddleear,astreatmentofconductivehearingloss.

5. Different Bioglassescomposition reported in literature

6. Bioglassesare divided in two categories:
ClassA:
•Osteoproductive
•Osteoconductive.
•Bindwithbothsofttissuesandbone.TheHCAlayerformswithinseveralhours.
ClassB:
•Osteoconductive(such as ydroxyapatite)
•Bonding to soft tissues is not facilitated. The HCA layer takes one to several days to form.

8. Advantage/disadvantage
Theprimaryadvantageofbioactiveglassesistheirrapidrateofsurfacereactionwhichleadstofasttissuebonding.
Theirprimarydisadvantageismechanicalweaknessandlowfracturetoughnessduetoanamorphoustwo-dimensionalglassnetwork.
Tensilebendingstrengthofmostofthecompositionsisintherangeof40-60MPawhichmakethemunsuitableforloadbearingapplications.
Forsomeapplicationslowstrengthisoffsetbytheglasses’lowmodulusofelasticityof30-35GPa.Thisvalueisclosetothatofcorticalbone.

10. Compositions such as 45S5 Bioglasswith high rates of bioactivity produce rapid regeneration of trabecularbone 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 osteostimulationand osteoconduction

11. Processing bioglassesMelting process
T= 1100-1300°CSol-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 .

12. Sol–gel processing
TEOS: tetraethoxysilane, Si(OCH2CH3)4.

13. Pharmaceutical applications
Designofbioglassandbioceramicdrugdeliverysystemswhichallowtheeffectivetargeteddeliverydosageofproducts,rangingfrominorganicandorganicmoleculesofdifferentsizeandproperties,tobecontrolled.
Clientbespokeformulationsforspecificapplications, includingthecontrolledreleaseofpharmaceuticalactivecompounds,suchasvitamins,antibioticsandanti- inflammatorydrugs,andalsotheenablingofsequential,andtimeandsitewell-ordered,deliveryofmultipleagents
Characterizationtomonitorandevaluatecontrolledreleased(fromhourstodays,even,tomonths)

14. Bioglassfor controlled drug release: our results

15. BIOACTIVE GLASSES FOR ANTIBIOTIC CONTROLLED RELEASE-our results
Bioglasscomposition 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.

16. Tetracycline loading and release –SEM
Before loading
After TTC loading
Specimen 1: 0.55SiO2•0.41CaO•0.04P2O5

17. Tetracycline loading and release –SEM
Before loading
After TTC loading
Specimen 2: 0.55SiO2•0.41CaO•0.04P2O5

18. Tetracycline loading and release –SEM
Before loading
After TTC loading
Specimen 3 : 0.45SiO2•0.245CaO•0.06P2O5•0.245Na2O

19. 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
Theprocedureofdryingat80ºCcausedadecreaseoftheporesize
Theporesizeandsurfaceareadecreasedafterimmersionintetracyclinesolutionduetotetracyclineattachment.
Specimen1,whichexposedalargersurface,isabletoincorporatemoretetracyclinecomparedtothespecimen2and3.

20. TTC stability by UV/VIS spectroscopy
pH
stability
Red shift: transformation of TTC molecule from TTC0to 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.

21. The main degradation products during photolysis in aqueous medium

22. UV-VIS and EPR spectroscopy detecting free radical formation
during the samples preparation procedure.
200 300 400 500
TC3
TC2
Intensity (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

23. EPR experimental spectra of the bioglasssspecimens upon TTC loading, filtration and drying procedure.

24. EPR simulations of liquid spectra
The values of gand Amagnetic 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

25. EPR simulations of TC immobilized on porous bioactive structure
τTC1< τTC2< τTC3

26. 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

27. 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

28. Surface functionalizationand conformational changes of proteins adsorbed on Bioglass
Native structure of methemoglobinby
X-ray crystallography
Composition as classical 45S5 Bioglass:
45% SiO2, 24.5% Na2O, 24.5% CaOand 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 MHbwith 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, Biochimicaet BiophysicaActa1824 (2012) 873–881

29. 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 NaClcrystals are not covered by proteins.

30. Methemoglobinattachment on the Bioglasssurface after functionalizationwith 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

31. FTIR spectroscopy
X –ray Photoelectron Spectroscopy
evidence of MetHBadsorbed on Bioactive glass

32. 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.

33. Native MeMb
MethMbon BG
MetMbon BG-GA
Native MetMb
MethMbon BG
MetMbon BG-GA
FTIR spectroscopy and deconvolution
αhelix % βsheet% βturns % Random % Side chain%

34. Observations
Oneadditionalpeakcenteredat1648cm−1appearsoriginatingfromrandomstructure.
Also,themajorbandsat1656and1654cm−1,assignedtoα- helixstructuresinthenativemethemoglobinareshiftedtohigherwavenumbersuponadsorption.Thehigherbandpositioncorrespondstoweakerhydrogenbonding,leadingtomoreflexiblehelices,asaconsequenceoftheinteractionbetweentheproteinsandbioactiveglass.
Theproteinlooseapproximatelyhalfoftheα-helicalstructureafteradsorptiononBG,butonly1/3whenBGisfunctionalizedwithGA.

35. XPS survey spectra
Sample
Elemental 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 BG
16.9
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
MetHBlyophylized
MetHbon BG
BG with GA
MetHbon BG-GA

36. C 1s high resolution XPS spectra and deconvolution
284.6 eV
290.4 eV
C03
286.2 eVC-C and C-H
288.7 eVNH-CHR-CO and –NH2

37. N 1s and O 1s high resolution XPS spectra
(a) BG
(b) MetHblyophilized
(c) MetHBon BG
(d) BG with GA
(e) MetHbon BG with GA.
400 eVC-N
532 eV-OH and peptidicoxygen ( shifted to lower binding energy)

38. Observations
The marker bands N 1s and C 1s specific to proteins shows an increasing intensity on GA functionalized sample.
The surface functionalizationof the bioactive glass substrate with GA provides a better protein adherence that is considered beneficial for further interaction of biomaterial surface with surrounding cells.

39. Insulin microencapsulation and release from zinc/silica microparticles
In the presence of zinc ions, insulin dimersassociate into hexamerswith greater stability.

40. Advantages
Microencapsulation is considered one of the best oral drug delivery approaches.
Overcome the enzimaticand 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 excipientfor vitamins.
Silica shell with pores typically < 10 nm
ZnOhas antiseptic effect
Addition of Zn ions proved to preserve the secondary structure of some proteins.

41. 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

42. X–ray diffraction and particle size analysis
XRDpatternsoftheinsulinentrappedinzinc-silicaparticlesobtainedbyspraydrying(a)andfreezedrying(b)methods.
Particlesizedistributionplottedonalogarithmicscaleofthezinc-silicasprayeddriedmicrosphere(a)andfreezedriedmicroparticles(b).
2.5 μm-dominant
35 μm-weakly
5μm
E. Vanea, S. Cavalu, Journal of Biomaterials Applications 28(8) 1190-1199 (2014)

43. SEM imagesSpray –dried microspheres Freeze –dried microparticles

44. 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)
Deconvolutionof 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).

46. 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

47. Observations
Insulinencapsulationinzinc-silicamicroparticlesfollowingthesol-gelroutecombinedwithfreezedryingandspraydryingprocedurehasbeendemonstratedtopreservetheintegrityofinsulin.
Formationofinsulinhexamersinthepresenceofzincionsleads
toanincreasedstabilityoftheinsulinthree-dimensionalstructureduringpreparation,storageandrelease.
Thereleaseprofilecanbeadaptedbysynthesisrouteofmicroparticles.

48. 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 vascularizedbone growth so that bone grafting is no longer necessary.

49. Bone tissue engineering

50. Requirements for the future:
Improvementofthemechanicalperformanceofexisting
bioactiveceramics.
Enhancedbioactivityintermsofgeneactivation.
Improvementintheperformanceofbiomedicalcoatingsin
termsoftheirmechanicalstabilityandabilitytodeliverbiologicalagents.
Developmentsmartmaterialscapableofcombiningsensing
withbioactivity.
Developmentofimprovedbiomimeticcomposites.