1.

Ladies and gentleman, it‘s a pleasure for me to present you my work on Calcium phosphate bone
cements, their synthesis, characterization and drug release properties. I preferomed this work at the
patras university in Greece where I was on Erasmus.

2. To begin with the purpose of my experiments I must ephasize few facts. As the population age
distribution shifts to the upper end, more bone related defects and diseases will play bigger role. Un
he US alone aprox. 100 k of ununits happen every year and older methos such as auto and
allotransplants of bone grafts are extremely invasive. A materiál that could fit in the defect by
injection would be very valuable. And CPC might be one of these materials.

3. So what was the aim of my work at the department of material scicences? My goal was to
synthetize a high quality a-TCP and prove its quality by X-ray diffraction. From this materiál to make a
cement paste that would prove to be injectable and washout resistant. To let this cement harden in a
PBS solution and meanwhile take samples for XRD, Raman, Compressive strength and SEM. After
these experiments were conducted a release property of this cement was examined by releasing 40
mg of ibuprofene.

4. To start with basics, I will now explain what a calcium phosphate cement is. It is a biomaterial
found in bone tissue capable of forming the hardest materiál found in human body – hydroxyapatite.
Its main advantage in comaprison with other bone substitute sis that the setting reaction desn’t
produce any heat, unlike for examle PMMA. The injectable form of cement is a cement paste made
from a-TCP and PBS.

5. Here you can see a simplified diagram of how the proces of forming hydroxypapatite goes. First
calcium carbonate reacts with calcium orthophosphate at specific conditions (1450°C) for 48 hrs. A-
tcp is formed in its ceramic form, after that it must be quenched in air and cold ceramic materiál is
ballmilled. Fine powder of A-tcp is mixed with sufficient amount of 4% w/v Na2PO4 which forms a
cement paste. This paste was then put in molds to harden in PBS solution at 37°C.

6. What are the important characteristics of CPC? Let’s begin with 2 relatively vaguely deffined
atributes. The cement paste must be injectable, thus it must be phase stable independently on the
force applied. Second – washout resistance refers to the ablity to stay compact and not dissolve or
lose it’s structure after application. Compressive strength is a capacity to withstand loads to reduce
size, which was in the following graphs then converted to pressure. X-ray diffraction is a method
where interference of x-ray with crystalline structure gives us the information of specific crystalline
modifications. Scanning electron microscopy gives us images of microstructure of the inner and outer
surface. And last but not least raman spectroscopy which you might know form our lectures gives us
qualitative information about the sample by measuring interaction of materiál with laser beam.

7. The possiblity to incorporate drugs inside a bone cement would be beneficial for healing bone
traumas while affecting inflamation, immune systém, hormones etc. Since CPC do not disintergrate,
the release of drugs is relatively slow compared to other dosage forms. The crucial point of using CPC
as drug carrier is that it must not affect the important natures such as compressive strength, HAP
formation etc.

8. Ibuprofene was chosen as our model drug. I suppose I don’t have to present his characteristics to
you.

9. So now step by step what I have done. First A-tcp was synthtized form finely ballmilled cacium
carbonate and calcium orthophosphate at 1450°C for 48 hrs. The ceramic product must be quenched
in air because the a-tcp crystalline form is only metastable and during a slow cooling more stable B-
tcp would form. And B-tcp doesn’t react with water to form HAP so willingly, so B-tcp was for us the
undesired byproduct.

10. Here you can see a graph from X-ray diffraction. Please note that the black line of our sample
corresponds with the red peaks of standard A-tcp.

11. After the ceramic a-TCP was ballmilled to ca. 1 micrometer particles, the A-tcp was used as a solid
fase for paste preparation. As the liquid phase 4% disodium phosphate was used.

12. The paste was formed to 3 balls and put in Normal saline, Ringer and PBS solution to simulate
physiological conditions. In all of them, the balls were stable and did not disintergrate. Injectability
test was also conducted where the paste retained its consistence.

13. From the same paste were also made these cylinders. We prepared 20 cylinders from CPC and 20
with 4% ibuprofen. The ibuprofene was mixed with the solid phase and then mixed with liquid as a
whole. This is a crucial moment to which I will refer once more. The cylinders were stored
throughout the whole proces at 37°C in PBS.

14. The deformation force was measured after 24 hours, 9 days and 15 days. There i sis visible a
significant increase of compressive strength from day 1 to 9 in pure cements with unsignificant
decrease at the day 15. The difference between pure CPC and CPC with ibuprofen is massive.

15. This was the X-ray diffractometer that we used which was located at a FORTH insitute in Patra.

16. On the screen you can see a difractogram which shows the same facts as the one before, you can
see that the standard of A-TCP corresponds with our sample.

17. And these graphs show us that during time there was a shift in the crystaline structure of our
samples. Nevertheless addition of ibuprofen doesn’t seem to affect this process.

18. Raman spectroscopy a traditional nondestructive analytical method was used to obtain data if
the process of conversion from a-tcp to HAP was complete after 1 day or not.

19. Here you can see that the peaks of our cement after 1 day of hardening correspond with HAP.

20. The samples that we used for comrpessive strength test were then sputtered with gold and SEM
images of them taken by the EVO MA 10 ZEISS microscope.

21. Please don’t mid the date of these pictures, they just didn’t set it in the software. Here you can
see the raw a-tcp powder, please note the fine structure with diameter of about 1 micrometer.

22. On this you can see the inner surface of pure cylinder piece. You can see the plates and needles
of hydroxyapatite.

23. The outter surface has less visible mistrostructure consisting more of tiny needles.

24. On these images one can see nice plates and needles in the inner struture of cement with
ibuprofen.

25. And here the outter surface with ibuprofen.

26. And last but not least the drug release experiment. It was conducted with 4 tablets each with 40
mg of ibuprofen in 15 ml of PBS at constant 37°C. The PBS was changed after
3,6,9,24,28,72,120,168,264,432 and 504 hrs. The ibuprofen concentrations were measured by UV
spectrometer at both highest absorbance peaks.
27. Here you can see the graphs where both show release of almost 85% of materiál in course of 21
days.

28. So the results of my work: the cement paste proved to be injectable and washout resistant in our
simulated conditions. We can therefore assume that it would tay stable in the physiological
environment as well. The purity of a-TCP was at least satisfactory, only minor peak of B-tcp was
present. Raman spectroscopy showed us that the qualitative conversion of a-tcp to HAP was
complete in 24 hours. So the increase in compressive strength was probably due to the changes in
crystalline structure and growth of plates and needles of HAP. Compressive strength of our samples
reached its maximum after 9 days and did not increase afterwards. There was a very significant
decrease between the strength of pure and cylinders with ibuprofen. Which was not due to different
microstructure or crystalline structure.

29. SEM have shown us the full conversion of a-tcp to HAP plates and needles and very fine milling of
a-TCP. In the drug release experiment my sample did prove CPC to be a promising drug carrier thanks
to its interesting release properties. Because the steady release reached 85% of ibuprofen released
after 21 days.

30. Here I have few topic for discussion. The first one is probably the most important one because
the massive decrease in compressive strength would have to be somehow solved. It might be only by
adding the API to liquid phase or to the paste itself. Measuring of compressive strength would be
more practical if it would have been done in shorter intervals.