**4. Thermal properties of brushite**

The results of the TG analysis for the brushite are reported in **Figure 5**. Brushite is considered as a water-bearing phosphate mineral [2] and its crystal structure contains compact sheets consisting of parallel chains in which Ca ions are coordinated by six phosphate ions and two oxygen atoms belonging to the water molecules [22].

**Figure 5.** *TGA of brushite: (A) cumulative mass loss, and (B) mass loss rate.*

*Brushite: Synthesis, Properties, and Biomedical Applications DOI: http://dx.doi.org/10.5772/intechopen.102007*

Brushite contains two water molecules in its lattice and adsorbed water molecules on its surface, as indicated by the presence of two sharp peaks of mass loss during heating between 80 and 220°C (**Figure 5A**) [2, 23]. Part of the chemically-bound water is released during the transformation of brushite to monetite, CaHPO4, at ~220°C [17], and later to calcium pyrophosphate, Ca2P2O7, at ~400°C [24]. Pyrophosphates are decomposed at higher temperatures of 750–800°C (**Figure 5B**) [8, 25]. The heating of pure brushite to 600°C results in a mass loss of approximately 25%wt, while the theoretical mass loss for the dehydration of brushite is 20.93%wt [1].

Dehydration of brushite over the temperature range 110–215°C takes place according to Eq. (2) and normally results in a weight loss of about 19%wt, while the formation of calcium pyrophosphate is accomplished by Eq. (3) a

$$\text{CaHPO}\_4\cdot2\text{H}\_2\text{O} \rightarrow \text{CaHPO}\_4 + 2\text{H}\_2\text{O} \tag{2}$$

$$2\text{CaHPO}\_4 \rightarrow \text{Ca}\_2\text{P}\_2\text{O}\_7 + \text{H}\_2\text{O} \tag{3}$$

**Figure 5B** shows the rate of mass loss as a function of heating temperature for brushite. More specifically, **Figure 5B** shows the dehydration peaks corresponding to the two water molecules of brushite [26, 27].

### **5. Calcium phosphate cement (CPC)**

Brushite is used to prepare the powder component, tetracalcium phosphate (TTCP), of the CPC. Brushite was calcined at 500°C and transformed into a more stable phase; calcium pyrophosphate (Ca2P2O7). Afterward, calcium pyrophosphate and calcium carbonate were mixed with a Ca/P molar ratio of 1.9 [5]. These two compounds were mixed in ethanol for 10 h. Then the resultant mixture was dried at 105°C for 24 h and then crushed. The mixture was heated at 1500°C for 4 h quenching to room temperature, see **Figure 7**. The resultant powder (TTCP) was ground into a fine powder [5]. The general equation TTCP synthesis is as follows:

$$\text{CaHPO}\_4\cdot2\text{H}\_2\text{O} + 2\text{CaCO}\_3\text{(1500°Cfor 3h)} \rightarrow \text{Ca}\_4\text{(PO}\_4\text{)}\_2\text{O} + 2\text{CO}\_2\tag{4}$$

Mannitol, sizes vary from 100 to 400 μm, was added to the TTCP with the weight ratio of 0.5. Diammonium hydrogen phosphate solution with a concentration of 33.3 wt% was mixed with TTCP-mannitol mixture, with the weight ratio of O.34 mL (solution)/g (TTCP). After mixing the CPC components for 2 min, the paste was packed in a polycarbonate mold which has an opening of 10 × 10 mm under a pressure of ~1 MPa at 37°C. The hardened samples were then demolded and immersed in Hanks' physiological solution at 37°C for 1 day [28, 29]. The composition of Hanks' physiological solution is reported in **Table 3**.

After mixing the two components of CPC, TTCP, and the hardening solution, TTCP hydrolyses through a dissolution-precipitation reactions resulting in the formation of layers of Ca-deficient hydroxyapatite (CDHA) crystals, which are similar to the mineral component of the bone from (**Figure 6**). This hydrolysis process occurs during the setting reactions, which is confirmed by XRD and SEM (**Figures 7** and **8**). These CDH layers are characterized by wide range distribution of rod-like crystals [30].

The CPC was synthesized for *in vitro* cultivation [5, 31, 32]. Mesenchymal stem cells (MSCs) were seeded on the CPC porous matrix in presence of an osteogenic


#### **Table 3.**

*Composition of Hanks' physiological solution.*

#### **Figure 6.**

*Schematic diagram of CPC preparation.*

medium for 21 days [5, 12]. As a result of *in vitro* cultivation, mineralized nodules were formed in the constructs. The seeded cells grow and their sizes increase from 5 μm to around 50 μm. The growing cells adhered to the CPC matrix and developed cytoplasmic extension as reported in **Figure 8**.

A thick layer of nano fibrous CDH crystals covers the surfaces of the CPC matrix (**Figure 7B**). The cultured CPC exhibits new connective tissues and throughout the CaP matrix (**Figure 8**). The CPC matrix contains bioactive CDH with both Ca and P, *Brushite: Synthesis, Properties, and Biomedical Applications DOI: http://dx.doi.org/10.5772/intechopen.102007*

**Figure 7.** *XRD patterns of CPC and CPC powder [5].*

**Figure 8.**

*Growth of thick Ca-deficient hydroxyapatite (CDHA) layer on the surface of the CaP matrix.*

**Figure 9.** *SEM image of the surface of CPC after MSCs culturing for 21 days.*

therefore, this matrix provides the appropriate environment for MSCs growth and osteogenic differentiation (**Figure 9**) [33].
