Chemical Preparation of Beta Calcium Sulfate Hemihydrate Granules with a Special Particle Size as Bone Graft Material

As bone lesions occur in dentoalveolar system in human due to trauma, illness, and surgery, the application of the appropriate material(s) is mandatory in these situations (1,2).

Various techniques that use different types of grafts such as autologous bone graft, allograft, alloplast, and xenograft were proposed. The most success in the field of bone graft is obtained from autografts; as autograft is a living tissue with intact cells and no immune reaction. However, it has two disadvantages (3): first, it is obtained from the secondary region of the body that can make problems due to surgery in the donor region (4) and second, the limitation that exists in the amount of graft material.

Using allograft materials would avoid performing of second surgery. However, the concern about this kind of material is the possibility of infection and disease (5,6).

Allograft materials that are routinely used are demineralized freeze-dried bone, freeze-dried bone allograft, and bovine deproteinized bone (7). In comparison with autograft materials, allograft materials have fewer capacities for osteogenesis, higher rate of absorption, immune response and also more concerns about disease transmission (8). These problems led to the expansion and creation of alternative materials (9). Calcium sulphate has the longest clinical use in comparison with other materials that are currently available (10).

Calcium sulfate used in bone defects is well-tolerated, gets rapidly and completely absorbed without leaving any obvious inflammatory response. It is made of materials which are cheap and can be easily found in abundance (11, 12). Calcium sulfate is tissue compatible and when used as a binder, it expedites the healing process and avoids loss of the grafting material (13).

On the other hand, the main limitations of calcium sulfate as bone filler are poor handling characteristics, poor mechanical properties and its quick absorption (14). Therefore, in order to avoid or reduce these limitations, this study was focused on changing calcium sulfate into the granulated form with a size of 500 to 700 microns and higher hardness. So the aim of current study was to combine granulated calcium sulfate with a new binder in order to improve its mechanical and handling characteristics and then to evaluate it clinically and histologically.

Materials and Methods

Preparation of Calcium Sulphate

At first calcium sulfate anhydrous crystals were mixed with enough water and a special binder to form a paste (this binder was prepared and designed in the Department of Chemical Engineering of Ferdowsi University of Mashhad). After several minutes, the paste dehydrated and hardened. The solidified paste was ground into the desired particle size range 500-750 microns. Then the dehydrated calcium sulfate was heated in order to lose water in a process known as calcinations. Due to the presence of binder in the mixture the intensity of heating and the final temperature is critical therefore; it should be heated slowly with a specified ramp. The resulted product was calcium sulfate hemi-hydrate. Finally, this product was sterilized using gamma ray.

Hardness Test

The hardness of the specimen was measured by the Rockwell test. The depth of the indentor penetration into the specimen surface was measured. The indentor may be either a hardened CaCo₃ ball with the diameter of ¼ inch or a spherical diamond cone with 120 angles.

PH Evaluation

After dissolving caco₃ in water, the PH was measured with digital PH meter.

Surgical Procedures

Six New-Zealand rabbits with similar weight (2.5-2.8 kg) and size were participated in this study. Protocols were approved by the ethical committee for animal study at Mashhad Dental University.

The animals were anesthetized preoperatively with an intramuscular injection of ketamine (40mg/kg Rotexamedica, Trittau, Germany) and xylazine (6-8 mg/kg Alfsan, Woerden, Holland). In addition, 1ml of local anesthetic of lidocaine without vasoconstrictor (Daroupakhsh, Tehran, Iran) was injected.

Parietal bone was chosen as the surgical site; surgical area was shaved and disinfected with povidone-iodine (Najo, Tehran, Iran).

A full-thickness incision was performed to expose the parietal bone, a slow-speed dental drill equipped with a carbide round bur was used to create one 6-mmwide defect in bone under irrigation with a large amount of normal saline. The defect was filled with calcium sulphate the surgical site was sutured with stainless-still monofilament wire 3-0 (Supa Medical Devices, Tehran, Iran).

All rabbits were given water and a standard rabbit food; no postoperative complications or death occurred. Rabbits were sacrificed at months 3, 6, 9, 12, 14, 16 and a total of six defects were retrieved.

Histological Processing

All specimens were decalcified in EDTA for two months. The process of decalcification was controlled radiographically and after decalcification, all of them were fixated in paraffin. The specimens were sectioned with the micrometer set at 5 mm.

The parameters used for evaluation were: inflammatory infiltration with probable abscess formation or necrosis and new bone formation with bone typing.

Each of the parameters was scored from 0 to 3, with 0 = normal, 1 = mild increase, 2 = moderate increase,
3 = marked increase. Sparsely scattered neutrophils, plasma cells, macrophages, eosinophils and mast cells arranged in a random fashion were considered normal. Localization of 3-10, 11-30, or 31 or more cells in the wound tissue per × 400 magnification fields were considered as mild, moderate, and marked increased respectively.

Sparsely scattered necrotic cell debris, extravasated erythrocytes (indicative of acute hemorrhage), and hemosiderin-laden macrophages (indicative of chronic hemorrhage) were considered as a mild increase for each of these parameters. Focal dense accumulations of these components in the wound tissue were considered as a moderate increase. Extensive tissue necrosis, massive hemorrhage involving the surrounding tissue, and the presence of greater than 30 hemosiderin-laden macrophages per × 400 magnification fields were considered as marked increase for each of these parameters respectively (15).

New bone formation was considered as inter membranous bone development in which it was highly cellular with rim of active plump osteoblasts. The type of osseous was diagnosed according to histological structure.

Results

Mechanical Hardness

Hardness was measured in scale A and its value was equal 120 shore.

PH Measurement

The PH of caco3 was measured 7.2. The solubility of calcium sulphate in water at this PH is Ksp=2.23g/lit.

Clinical Observation

The rabbits were carefully monitored and examined, during the first 16 months after putting the matter. Throughout the monitoring period, any problems, including death, weight loss and clinical signs of swelling did not occur and flap adaptation and retention of wound closure was achieved in all rabbits. At the time of biopsy, the skin of all the animals was healthy. The exposed bone was carefully studied, and their skin was fully touched. The surgical procedure was well tolerated by the animals; healing was uneventful.

Histological Evaluation

After Three months: one of the rabbits was sacrificed and histologically examination showed normal trabeculation of bone containing Haversian canals and lacunar spaces contained osteocytes, as well as osteoblastic rim. Inflammatory cells and fibrous tissue existed in the sample. Chronic diffuse inflammatory cells and infiltration was seen around the collagen fibers and blood cells and the giant cells, also reaction to foreign body was observed (Figs. 1 and 2).

At sixth months: in this sample the same trabeculation containing haversian canals and lacunar spaces with osteocytes were observed, osteoblast cells existed as a marginal rim. Also, focal infiltration of chronic inflammatory cells without foreign body reaction was visible. Fibrosis was more prominent in this sample (Fig. 3).

Figure 1. The defect at 3 months. NB: normal bone; FT: fibrous tissue; IC: Inflammatory cells. 100 X and H & E staining

Figure 2. The defect at 3 months. NB: normal bone; HC: haversian canal; OR: osteoblastic rim.400 X and H & E staining

Figure 3. The defect at 6 months. FCI: focal infiltration of chronic inflammatory cells; NB: normal bone. 400 X and

H & E staining

After nine months: In 3rd sample the normal bony structure similar to the previous samples was observed; likewise, in this case no sign of inflammation could be seen. However, fibrous tissue was also observed. Bone marrow fibrosis was also present and was advanced (Fig. 4).

At twelfth month, in the 4th sample, normal bony structure was observed. Cartilaginous tissue and inflammation were not found. Reversal lines were easily seen in this sample. In some instances, severe fibrous tissue of bone marrow was also observed.

At the fourteenth month: normal bony structure and osteoblastic rim were observed. Focal infiltration of chronic inflammatory cells was found surrounding the fibrous tissue of the bone marrow and in the periphery may be done after an accidental trauma or rest of previous inflammatory response (Fig. 5).

Figure 4. The defect at 9 months. SF: severe fibrous.400 X and H& E staining

Figure 5. The defect at 14 months. FCI: focal infiltration of chronic inflammatory cells; NB: normal bone.

At the sixteenth month: the new bone can be seen clearly. Normal bone with normal trabeculation that contains Haversian canal and lacunar space with osteocytes and osteoblastic rim beside new bone that shows more cellularity than trabeculation was observed and fibrous tissue without infiltration of inflammatory cells was also found in the bone marrow space (Fig. 6).

One of the most important findings in analysis of cases was that even in the presence of infiltration; no sign of sequestrum was observed (Table 1).

Figure 6. The defect at 16 months. FT, fibrous tissue; NB, new bone.400 X and H& E staining

Table 1. Histological data in different months