**Change in pH during hydration**

54 Biomaterials – Physics and Chemistry

Apatite phase formed

Fig. 2. Cross section of the apatite-containing surface layer formed, SEM (Engqvist et al,

Ion tested 1h, ppm 24 hrs, ppm 7 days, ppm 28 days, ppm Ca 66 64 44 50 (70) Al 11.4 9.3 9.6 8.6 (1.2) Table 6. Ca and Al dissolution during hardening of the Ca-aluminate material, (The 1h

The results of Ca and Al determination in the solution during the hydration process of the

The release of metal ions in water was below 5x10-2 ppm/(mm2 material) for aluminium and below 30x10-2 ppm/(mm2 material) for calcium, whereas somewhat higher aluminium content was measured in artificial saliva. The ion concentrations detected are generally not time-dependent during hydration. After the initial hydration time the ion concentration (molar) is determined by the solubility product of the phases formed (katoite = 5x10-26 and gibbsite = 3x10-24). Since the concentration of Ca in saliva is higher than what is obtained in the non-physiological aqueous solution (distilled water), it can be assumed that the filling material releases very limited amounts of Ca or Al once the material has hardened. The presence of Ca in saliva will decrease the solubility tendency of the calcium-aluminate-

Based on a search in the literature, the FAO/WHO Joint Expert Committee on Food Additives (JECFA) has provided a provisional figure for tolerable weekly aluminium intake of 7 mg/kg body weight. This corresponds to 1 mg/kg /day. The daily intake of aluminium via digestion/food is approximately 5 mg per day. For calcium the NIH Consensus Development Conference on Optimal Calcium Intake recommended an intake in the range 800 mg/day for young children to 1000 – 1500 mg/day for adults depending on gender and age. For many people there is a need to supply additional calcium in order to stay healthy. The ion concentrations measured and the amounts of Ca and Al released are far below the concentrations of the elements produced from food intake and should therefore not pose

**Ion release measured by atomic absorption spectrometry** 

Ca-aluminate based material are presented in Table 6.

testing at 28 days within brackets)

hydrate phases.

any safety concerns at all.

2004)

The results of the measurement of pH development during hydration of the Ca-aluminate based are shown in Table 7. The initial *in vitro*-pH is 10.5 in saliva. After 1 week, pH after 1 h dissolution time in saliva is approx. 8.


Table 7. Change of pH during initial hydration of Ca-aluminate based materials

The pH is high during the early stage of the hydration, but decreases with time and approaches neutrality. The reason for the high pH in the beginning is the general basic character of the material and the formation of OH during the hydration process. In the clinical situation saliva is produced in a dynamic way, creating an environment capable of buffering surrounding solution to neutrality. In the clinical studies performed so far no adverse reactions have been reported from a possible elevated pH during the early part of the hydration.

When Ca-aluminate material is combined with glass ionomer system the pH-system becomes initially acidic. However after 10 min the pH is above neutral, but will not exceed pH 9 (Jefferies et al 2009).

#### **Cytotoxicity testing**

The *in vitro* MTT reduction test of the experimental Ca-aluminate dental filling material in human oral fibroblast culture showed no obvious cytotoxicity. The average level of MTT reduction of the experimental dental filling material was close to 100% of the control values. The maximal variation (SD) was less than 30%. Different curing times of the test material did not seem to affect the cytotoxicity test results although one week curing produced the most stable testing results both in the short and long term tests. After a week the material can be considered as fully cured, i.e. stable.

Morphological changes were not observed in any of the test groups at different MTT reduction testing points. As shown in Figure 3, the cell culture was typically fibroblastic with a slender and elongated form in both the control group and the group exposed to the examined material. In the exposed picture B even some precipitated hydrates are seen.

Fig. 3. Morphological observation of human oral fibroblasts on an experimental Caaluminate based material. A: Normal control. B: After exposure to the experimental filling material for one week (Liu et al 2002).

Nanostructural Chemically Bonded Ca-Aluminate Based Bioceramics 57

studies Ca-aluminate material was compared to the PMMA-material CMW 1, and in the sheep study Ca-aluminate material was compared to the PMMA-material Vertebroplastic,

6-week femur Rabbit CMW-1 Minimal inflammation,

6 (12) -month femur Rabbit CMW-1 No Al- accumulation

Table 9. Implantation studies in femur rabbit, and in vertebrae sheep, details in

Cortoss

The 6 months femur study in rabbits included a 12 months subgroup. The amount of aluminium in blood and selected organs was analysed. The main target organs of the animals (kidney, lung, liver) were histopathologically investigated. Granulomatous inflammation in the cavity, pigmented macrophages and new bone formation were the treatment-related observations at 6- and12-months examination. No difference between Caaluminate material and PMMA was detected. There were no signs of aluminium

In the 12-week study, the histopathology of vertebrae obtained one week after surgery showed the most severe inflammatory reaction to the surgery in the *sham* operated vertebrae. The bone marrow in the vertebrae filled with Ca-aluminate was not reported to be infiltrated by any inflammatory cells. In vertebrae obtained 12 weeks after surgery no inflammatory reactions were reported, and no obvious differences were observed in the pathological reactions to the surgery (sham) or the filler materials. Overview of the

The analysis of serum samples showed low concentrations of aluminum in comparison to what is normal in humans. Since the concentration of aluminum did not increase after surgery and in some instances was lower after surgery than in the 0-samples, one may

Fig. 4. Histology image of an experimental Ca-aluminate material (black) in close contact

histological contact zone to the Ca-aluminate based material is shown in Figure 4.

12-week vertebrae Sheep Vertebroplastic

regard these concentrations as within the normal variation.

(Hermansson et al 2008)

accumulation in the analysed tissues.

with sheep vertebral bone.

very few inflammatory cells were present in bone, bone marrow and adipose

No inflammation, no Al-accumulation

tissues.

and to the Bis-GMA material CortossTM. The results are summarized in Table 9.

Implantation studies Species Reference material Result

The standard for cellular biocompatibility in *in vitro* testing has been stated in the International Organization for Standardization (ISO) standards documents. The standard allows for the contact testing of solid dental materials for cytotoxicity with cell lines. Due to several disadvantages of direct contact testing, indirect testing methods have been developed and compared to the direct testing assays (Tang et al 2002). Introduction of a standard cell culture device, i.e. cell culture insert or transwell, provided an opportunity for such cytotoxicity screening of dental materials with indirect contact between material specimens and cell culture monolayer. It is believed that such a testing system more closely mimics the *in vivo* exposure pattern by providing the test of the material in both its solid and dissolved phases at the same time. It has been shown that this testing system has produced the most stable results as compared to other testing systems, such as direct contact test. In a complementary cytotoxicity test using the pulp derived cell response, the experimental CAmaterial showed no sign of toxicity (Schmalz 2002).
