CALCIUM PHOSPHATE BASED BIO-MATERIALS APPLICABLE IN ORTHOPEDIC AND DENTAL MEDICINE D. Rabadjieva, S. Tepavitcharova, K. Sezanova, R. Gergulova, M. Gabrashanska, R. Aleksandrova Institute of General and Inorganic Chemistry Institute of Experimental Morphology, Pathology and Anthropology Bulgarian Academy of Sciences
Calcium phosphate biomaterials for orthopedic and dental medicine Biological apatite Nano-sized Poorly crystallized Non-stoichiometric hydroxyapatite Ca 5 (PO 4 ) 3 OH Including Na, K, Mg, Cl and CO 3 Not toxic Calcium Biocompatible orthophosphates
Calcium phosphate biomaterials for orthopedic and dental medicine DEVELOPMENT RELATIONSHIPS of biomaterials with SYNTHESIS – STRUCTURE – improved physicochemical INTERFACE TRANSITIONS – MECHANICAL PROPERTIES – characteristics, biological BIOLOGICAL PROPERTIES adaptivity and activity
To develop a method for preparation of calcium orthophosphate fine powders with composition and particle size close to the biological apatite To modify the composition improving biological properties of the materials To use these fine powders to prepare • Composite materials with biodegradable polymer matrices • Cements or pastes To assess new materials by in vitro and in vivo studies
Method for preparation of calcium orthophosphate fine powders IDEA Biomimetic system - SBF (Na + , K + , Mg 2+ , Ca 2+ , Cl - , SO 4 2- , HCO 3 - , HPO 4 2- ) Organic molecules – amino acids, hydrogels, surfactants Ion modification - Mg 2+ and Zn 2+ APPROACH Thermodynamic modeling for prediction the system behavior, for experimental design and assessment the experimental results
Method for preparation of calcium orthophosphate fine powders SBF-CaCl 2 -K 2 HPO 4 -KOH SBF-CaCl 2 -K 2 HPO 4 -KOH SBF-CaCl 2 -MgCl 2 -K 2 HPO 4 -KOH SBF-CaCl 2 -ZnCl 2 -K 2 HPO 4 -KOH Calculated saturation indices (SI) of the salts that might be precipitated in the biomimetic systems
Method for preparation of calcium orthophosphate fine powders SBFc- Cam SBFc- Pm ZnCl 2 SBFc- Cam CaCl 2 +MgCl 2 K 2 HPO 4 Drop-wise precipitation Under ammonia/glycine buffer pH 8, room temperature Intensive stirring
Method for preparation of calcium orthophosphate fine powders
Method for preparation of calcium orthophosphate fine powders PRECURSORS Compositions of the initial precipitated solid phase Mg(Zn)/( Mg+Zn+Ca ) = 0, 1, 2, 3, 5, 10, 13, 16 mol % Ca/P = 1.3 – 1.4 Na + = 0.02-0.08 mmol/g; K + = 0.01 – 0.02 mmol/g Cl - < 0.05 mmol/g Mg 2+ = 0.03 – 0.05 mmol/g XRD powder data IR spectra 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 2 -th e ta -S c a le
Method for preparation of calcium orthophosphate fine powders PRECURSORS Posner clusters - Ca 9 (PO 4 ) 6 .nH 2 O SBF CO 3 2- , Cl - , SO 4 2- , PO 4 3- Na + , K + , Mg 2+ , Zn 2+ , Ca 2+ PO 4 3- r Сa2+ – 1.00 Å r Na+ - 0.95 Å r Zn2+ - 0.74 Å r K+ – 1.33 Å r Mg2+ - 0.65 Å Calcium vacancies partially isomorphic fill Ca vacancies substitution Са/Р<1.5 of Ca Result Ca w Mg x Na y K z (PO 4 ) 6-v (CO 3 ) v
Method for preparation of calcium orthophosphate fine powders PRECURSORS RELATIONS Prepared in Prepared in Prepared in glycine buffer ammonia buffer presence of and hydrogel of surfactant PEG 7 xanthan gum
Method for preparation of calcium orthophosphate fine powders PRECURSORS RELATIONS Synthesis (Ca+Mg+ Zn/(Ca+ Mg/(Ca+ SSA, m 2 /g Zn)/P Mg+Zn) Mg+Zn) Initial 3.00 14.00 Ammonia buffer 1.59 2.95 4.65 34 Glycine buffer 1.54 2.85 8.50 28 Glycine buffer in a 1.59 2.90 8.23 30 presents PEG-7 Glycine buffer in a 1.40 1.74 13.68 32 presents Xanthan gum
PHASE TRANSITIONS Low temperature - precursors maturation Calculated saturation indices (SI) Phase *Precipita МАP SAP tion SBFc SBFr SBFcg SBFc SBFr SBFcg Mg3(PO4)2:8H2O 0.2 -2.84 -2.38 -2.43 -6.58 -3.88 -5.57 CaCO3 1.46 -0.6 0 -0.82 -2.87 -1.75 -2.74 CaHPO4:2H2O 2.7 -0.75 -0.67 -0.6 -1.25 -1.78 -1.39 Ca3(PO4)2(am2) 9.94 0 0 0 -4.18 -4.45 -4.25 Ca4H(PO4)3:2.5H2O 13.69 -4.22 -5.01 -4.44 Ca9MgH(PO4)7 16.43 -5.41 -5.49 -5.4 Ca5(PO4)3(OH) 24.06 0 0 0
PHASE TRANSITIONS Low temperature - precursors maturation SBFc (HCO3 – 4 mmol/l) SBFr (HCO3 – 27 mmol/l) SBFg (~SBFc + Glycine) 37 o C, pH 7.3; SBF Static regime; Solid/liquid ratio 4 g/l ; Duration – 1, 2, 4, 6, 24 h, 3, 10 days, 1 month; SBF solution changes - after 3-rd day
PHASE TRANSITIONS Low temperature - precursors maturation KINETIC STUDIES PO 4 3- Ca 2+ Mg 2+ SBFc SBFc 3.50 SBFr SBFr 1.8 3.25 SBFc SBFg SBFg SBFr 3.00 1.6 1.6 SBFg 2.75 1.4 1.4 2.50 1.2 2.25 1.2 2+ , mmol/l 2+ , mmol/l 2.00 3- , mmol/l 1.0 1.0 1.75 0.8 0.8 1.50 Ca Mg PO 4 0.6 1.25 0.6 1.00 0.4 0.4 0.75 0.2 0.2 0.50 0.0 0.25 0.0 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 time,h time, h time,h 3- , Ca 2+ and Mg 2+ contents in liquid Kinetic profile of PO 4 phases after different maturation times
PHASE TRANSITIONS Low temperature - precursors maturation XRD STUDIES SBFr SBFg SBFc 300 300 300 002 002 002 720h 4h 2h 1h initial 10 20 30 40 50 60 10 20 30 40 50 60 10 20 30 40 50 60 2-theta-Scale XRD powder data of solid phases after different maturation times
PHASE TRANSITIONS High temperatures Calcination at 600 o C
PHASE TRANSITIONS High temperatures Calcination at 800 and 1000 o C Mg- TCP Zn- TCP Zn13 Mg16 Zn10 Mg10 Zn 5 Mg5 Zn3 HA + Mg- TCP Zn- TCP Mg2 Zn1 HA + TCP HA + TCP Mg0 P0 20 30 40 20 30 40 2-theta-scale 2-theta-scale
PHASE TRANSITIONS High temperatures Mg/Zn-ACP mixed crystals r Mg2+, r Zn2+ < r Ca2+ unit cell distortion volume decrease Destabilization of Mg/Zn-( -TCP) Hydroxiapatite structure
PHASE TRANSITIONS High temperatures -TCP CaO n coordination polyhedrons (n=3,6,7,8) substitution substitution in CaO n n =3, 6 in CaO n n =7,8 most suitable strong distortion isomorphic substitution -TCP
Method for preparation of calcium orthophosphate fine powders Precursor Calcination precipitation Second Suspension Heating at gelling and gelling and 300 o C and liophilization liophilization washing
Method for preparation of calcium orthophosphate fine powders Effect of ion modified calcium phosphate materials on viability and proliferation of human Lep 3 cells (MTT,72h)
Method for preparation of calcium orthophosphate fine powders Zn-β-TCP, on the 14 th week following intramuscular implantation as a paste: a) Small fragments of the Zn-β-TCP samples are encapsulated by a fibrous capsule (FC) and are separated by ingrown connective tissue. There is no lymphoid hyperplasia within a regional popliteal lymph node (PLN). b) Slight foreign body reaction including giant cells (GC) and macrophages. c) Ingrown new blood vessels (Vsc) within the newly formed connective tissue. A paraffin section stained with hematoxylin eosin.
Composite materials with biodegradable polymer matrices Calcium Hydrogels phosphate Gelatin fine Xanthan gum powders Carrageenan Saccharose
Composite materials with biodegradable polymer matrices Test in model bone system and Simulated Body Fluid SEM images before and after 1 month in SBF
Composite materials with biodegradable polymer matrices Effect of composite scaffolds on the viability and proliferation of rat bone marrow cells cultivated for 6 days in the control presence of composite scaffolds
Acknowledgments This work was financially supported by the Bulgarian Ministry of Education and Science under Project DFNI T02-5/2014 .
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