**3. Ca-aluminate – general description and property profile**

Ca-aluminates comprise double oxides of CaO and Al2O3. Several intermediate phases exist and these are - using the cement chemistry abbreviation system - C3A, C12A7, CA, CA2 and CA6, where C=CaO and A=Al2O3. See Fig. 1 above. Table 5 presents typical property data.


The interval is primarily related to the c/w ratio used, and the highest values are achieved with c/w ratio close to that of complete hydration with no excess of water

Table 5. Mean property data of dental Ca-aluminate based materials (Kraft 2002, Lööf 2008, Lööf et al 2004,2005, Hermansson et al 2008)

Due to reduced porosity based on the huge water uptake ability, the Ca-aluminate material exhibit the highest strength among the chemically bonded ceramics. The inherent flexural strength is above 100 MP based on measurement of the fracture toughness, which is about 0.7 - 0.8 MPam1/2. The actual flexural strength is controlled by external defects introduced during handling and injection of the material. The thermal and electrical properties of Caaluminate based materials are close to those of hard tissue, the reason being that Caaluminate hydrates chemically belong to the same group as Ca-phosphates, the hard tissue

Nanostructural Chemically Bonded Ca-Aluminate Based Bioceramics 53

24 h. The test starts after 24 h hydration. The test probe accuracy was 0.01 mm. Values below

Determination of Ca and Al in the solution during the hydration process of the Caaluminate based material was performed using atomic absorption spectrometry (Liu et al 2002). Standard solutions of different concentrations of Ca and Al were prepared according to the manual. Samples were prepared with a size of 10mm x 2mm height using a wet-press method, corresponding to a surface area of 224 mm2. The test pieces were placed in plastic bottles in inorganic saliva solution of pH 7. The amount of liquid was 10 ml in each bottle. The temperature selected was 37 oC. The inorganic saliva solution contained calcium chloride, magnesium chloride, sodium chloride, a phosphate buffer, hydro-carbonate and citric acid. The Ca-content in the saliva solution corresponded to 68 ppm. 1 ml solution was removed at 1, 7 and 28 days for analyses, and saliva was exchanged at 1, 7 and 28 days after every measurement. For the 28 days test additional samples were also taken 1 h after new

Measurement of pH development during hydration of the material was conducted using a standard pH-meter. Samples were prepared according to the procedure for atomic absorption described above. The pH-testing was conducted in two separate ways. First the samples were immersed in saliva solution (pH =7) at 37 oC, and pH was measured continually over the whole experiment period (Test 1). 1 ml solution was removed at 1, 7, 14 and 28 days for pH measurement. The second type of measurement comprised immersion in 10 ml saliva solution at 37 oC, where the saliva was exchanged at 1, 7, 14 and 28 days. pH was measured at the time of observation and also after one hour in new saliva (data within

Specimens at different setting stages were subjected to cytotoxicity testing by using primary cultures of human oral fibroblasts. A tissue culture insert retaining tested materials was assembled into a 12-well plate above the fibroblast monolayers. The cytotoxicity was determined by MTT reduction assay after various curing times. Specimens were set and hydrated at 37 oC for different periods of time, i.e. 0, 5, 30, 60 min, 24 h and 1 week and were then placed on tissue culture netwell for a cytotoxicity test. Both acute (1 and 24 h) and long

Summarized below are the results from several biocompatibility and bioactivity studies (Engqvist 2004, 2005, Faris 2006) where Ca-aluminate is used as a biomaterial in orthopaedic and dental applications. *In vitro* bioactivity studies show apatite formation on the surface of the Ca-aluminate materials exposed to phosphate buffer solution, an example shown in

No height reduction at all was observed for two tested Ca-aluminate materials. Thus, according to the acid corrosion test, Ca-aluminate materials are judged as stable materials. The total absence of material loss, measured as height reduction in the acid corrosion test, is related to the general basic nature of the material, with possibility of neutralization of the

term (1 week) in vitro toxicity tests were conducted with MTT assay.

**3.1.1 Biocompatibility including bioactivity of Ca-aluminate bioceramics** 

acid in the contact zone – especially in the earlier stage of the hydration process.

0.05 mm per 24 h solution impinging are judged as acid resistant.

solution was added.

brackets), Test 2.

**Biocompatibility evaluation** 

Figure 2 below.

**Corrosion resistance** 

of bone. Another important property related to Ca-aluminate materials is the possibility to control the dimensional change during hardening. In contrast to the shrinkage behaviour of many polymer-based biomaterials, the Ca-aluminates exhibit a small expansion, 0.1-0.3 linear-% (Kraft 2002).

### **3.1 Biocompatibility including bioactivity**

#### **Definitions used**

The terms biocompatibility and bioactivity are used in different ways by different categories of scientists. Below are presented the definitions used in this paper, mainly agreeing with the definitions discussed in (Williams, 1987) Biocompatibility: "The ability of a material to perform with an appropriate host response in a specific application".

Bioactivity (bioactive material): "A material which has been designed to induce specific biological activity". Another definition according to (Cao and Hench,1996) "A bioactive material is one that elicits a specific response at the interface of the material which results in the formation of a bond between the tissues and the material".

Thus a material cannot in itself be classified as biocompatible without being related to the specific application, for which it is intended. Bioactivity from a materials viewpoint is frequently divided into *in vitro* and *in vivo* bioactivity. The *in vitro* bioactivity of a material is however only an indication that it might be bioactive in a specific *in vivo* application. Another aspect of bioactivity is that this term can be adequate only when the biomaterial is in contact with a cellular tissue. However, often a material is claimed to be bioactive if it also reacts with body liquids forming an apatite-phase. *In vitro* bioactivity is tested in phosphate buffer systems similar to that of saliva or body liquid, and apatite formation is the claimed sign of bioactivity. A further aspect of bioactivity and also biocompatibility deals with the different curing times and temperatures at which the observation (testing) is performed. This is important to issues such as initial pH-development, cohesiveness and initial strength. Finally the biocompatibility and bioactivity can only be confirmed in clinical situations, with the actual implant/biomaterial in the designed amount or content and shape. This is especially important for injectable biomaterials which are formed (hydrated) and cured *in vivo*, and for implants where movements, even micro-movement, can influence the outcome.

#### **Standards and methods**

Relating to the definition aspects above, the acceptance of a biomaterial is a crucial issue, and to some extent the question has been solved by relating to the following toxicological endpoints indicating biocompatibility as referred in the harmonized standard ISO 10993:2003, which comprises the following sections:

Cytotoxicity (ISO10993-5), Sensitization (ISO10993-10), Irritation/Intracutaneous reactivity (ISO10993-10), Systemic toxicity (ISO10993-11), Sub-acute, sub-chronic and chronic toxicity (ISO10993-11), Genotoxicity (ISO10993-3), Implantation (ISO10993-6), Carcinogenicity (ISO10993-3) and Hemocompatibility (ISO10993-4).

This will be the main guideline when presenting the status of the biocompatibility of the CASPH-system, but was complemented by corrosion testing, elementary analysis, pHchange and additional cytotoxicity testing.

The corrosion resistance test – using a water jet impinging technique - was conducted according to EN 29917:1994/ISO 9917:1991,where removal of material is expressed as a height reduction using 0.1 M lactic acid as solution, pH 2.7 . The duration time of the test is

of bone. Another important property related to Ca-aluminate materials is the possibility to control the dimensional change during hardening. In contrast to the shrinkage behaviour of many polymer-based biomaterials, the Ca-aluminates exhibit a small expansion, 0.1-0.3

The terms biocompatibility and bioactivity are used in different ways by different categories of scientists. Below are presented the definitions used in this paper, mainly agreeing with the definitions discussed in (Williams, 1987) Biocompatibility: "The ability of a material to

Bioactivity (bioactive material): "A material which has been designed to induce specific biological activity". Another definition according to (Cao and Hench,1996) "A bioactive material is one that elicits a specific response at the interface of the material which results in

Thus a material cannot in itself be classified as biocompatible without being related to the specific application, for which it is intended. Bioactivity from a materials viewpoint is frequently divided into *in vitro* and *in vivo* bioactivity. The *in vitro* bioactivity of a material is however only an indication that it might be bioactive in a specific *in vivo* application. Another aspect of bioactivity is that this term can be adequate only when the biomaterial is in contact with a cellular tissue. However, often a material is claimed to be bioactive if it also reacts with body liquids forming an apatite-phase. *In vitro* bioactivity is tested in phosphate buffer systems similar to that of saliva or body liquid, and apatite formation is the claimed sign of bioactivity. A further aspect of bioactivity and also biocompatibility deals with the different curing times and temperatures at which the observation (testing) is performed. This is important to issues such as initial pH-development, cohesiveness and initial strength. Finally the biocompatibility and bioactivity can only be confirmed in clinical situations, with the actual implant/biomaterial in the designed amount or content and shape. This is especially important for injectable biomaterials which are formed (hydrated) and cured *in vivo*, and for implants where movements, even micro-movement, can influence the outcome.

Relating to the definition aspects above, the acceptance of a biomaterial is a crucial issue, and to some extent the question has been solved by relating to the following toxicological endpoints indicating biocompatibility as referred in the harmonized standard ISO

Cytotoxicity (ISO10993-5), Sensitization (ISO10993-10), Irritation/Intracutaneous reactivity (ISO10993-10), Systemic toxicity (ISO10993-11), Sub-acute, sub-chronic and chronic toxicity (ISO10993-11), Genotoxicity (ISO10993-3), Implantation (ISO10993-6), Carcinogenicity

This will be the main guideline when presenting the status of the biocompatibility of the CASPH-system, but was complemented by corrosion testing, elementary analysis, pH-

The corrosion resistance test – using a water jet impinging technique - was conducted according to EN 29917:1994/ISO 9917:1991,where removal of material is expressed as a height reduction using 0.1 M lactic acid as solution, pH 2.7 . The duration time of the test is

perform with an appropriate host response in a specific application".

the formation of a bond between the tissues and the material".

linear-% (Kraft 2002).

**Definitions used** 

**Standards and methods** 

10993:2003, which comprises the following sections:

(ISO10993-3) and Hemocompatibility (ISO10993-4).

change and additional cytotoxicity testing.

**3.1 Biocompatibility including bioactivity** 

24 h. The test starts after 24 h hydration. The test probe accuracy was 0.01 mm. Values below

0.05 mm per 24 h solution impinging are judged as acid resistant. Determination of Ca and Al in the solution during the hydration process of the Caaluminate based material was performed using atomic absorption spectrometry (Liu et al 2002). Standard solutions of different concentrations of Ca and Al were prepared according to the manual. Samples were prepared with a size of 10mm x 2mm height using a wet-press method, corresponding to a surface area of 224 mm2. The test pieces were placed in plastic bottles in inorganic saliva solution of pH 7. The amount of liquid was 10 ml in each bottle. The temperature selected was 37 oC. The inorganic saliva solution contained calcium

chloride, magnesium chloride, sodium chloride, a phosphate buffer, hydro-carbonate and citric acid. The Ca-content in the saliva solution corresponded to 68 ppm. 1 ml solution was removed at 1, 7 and 28 days for analyses, and saliva was exchanged at 1, 7 and 28 days after every measurement. For the 28 days test additional samples were also taken 1 h after new solution was added.

Measurement of pH development during hydration of the material was conducted using a standard pH-meter. Samples were prepared according to the procedure for atomic absorption described above. The pH-testing was conducted in two separate ways. First the samples were immersed in saliva solution (pH =7) at 37 oC, and pH was measured continually over the whole experiment period (Test 1). 1 ml solution was removed at 1, 7, 14 and 28 days for pH measurement. The second type of measurement comprised immersion in 10 ml saliva solution at 37 oC, where the saliva was exchanged at 1, 7, 14 and 28 days. pH was measured at the time of observation and also after one hour in new saliva (data within brackets), Test 2.

Specimens at different setting stages were subjected to cytotoxicity testing by using primary cultures of human oral fibroblasts. A tissue culture insert retaining tested materials was assembled into a 12-well plate above the fibroblast monolayers. The cytotoxicity was determined by MTT reduction assay after various curing times. Specimens were set and hydrated at 37 oC for different periods of time, i.e. 0, 5, 30, 60 min, 24 h and 1 week and were then placed on tissue culture netwell for a cytotoxicity test. Both acute (1 and 24 h) and long term (1 week) in vitro toxicity tests were conducted with MTT assay.

## **3.1.1 Biocompatibility including bioactivity of Ca-aluminate bioceramics**

#### **Biocompatibility evaluation**

Summarized below are the results from several biocompatibility and bioactivity studies (Engqvist 2004, 2005, Faris 2006) where Ca-aluminate is used as a biomaterial in orthopaedic and dental applications. *In vitro* bioactivity studies show apatite formation on the surface of the Ca-aluminate materials exposed to phosphate buffer solution, an example shown in Figure 2 below.

#### **Corrosion resistance**

No height reduction at all was observed for two tested Ca-aluminate materials. Thus, according to the acid corrosion test, Ca-aluminate materials are judged as stable materials. The total absence of material loss, measured as height reduction in the acid corrosion test, is related to the general basic nature of the material, with possibility of neutralization of the acid in the contact zone – especially in the earlier stage of the hydration process.

Nanostructural Chemically Bonded Ca-Aluminate Based Bioceramics 55

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

Test No. At start 1h 24 hrs 7 days 14 days 28 days 1 10.5 10.3 10.7 10.3 9.9 9.8 2 10.5 10.2 10.2 (7.7) 9.9 (7.8) 9.5 (8.1) 9.2 (8.1)

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

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

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

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

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

during the hydration process. In the

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

**Change in pH during hydration** 

the hydration.

pH 9 (Jefferies et al 2009). **Cytotoxicity testing** 

dissolution time in saliva is approx. 8.

character of the material and the formation of OH-

can be considered as fully cured, i.e. stable.

material for one week (Liu et al 2002).

Fig. 2. Cross section of the apatite-containing surface layer formed, SEM (Engqvist et al, 2004)

#### **Ion release measured by atomic absorption spectrometry**

The results of Ca and Al determination in the solution during the hydration process of the Ca-aluminate based material are presented in Table 6.


Table 6. Ca and Al dissolution during hardening of the Ca-aluminate material, (The 1h testing at 28 days within brackets)

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-aluminatehydrate phases.

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 any safety concerns at all.
