Effect of β-calcium sulphate hemihydrate on mandible healing in dog ( radiographical assessment using Image-J Program )

This study was conducted to estimate the bony tissue response to β-calcium sulphate hemihydrate (CSH) as a bone substitute via radiographic assessment using ImageJ software. The extraction sockets in dog mandible were the regions of interest (ROI). Twenty adult (12-24 months), local breed dogs were included in the experiment. All had a complete set of permanent dentition. They were randomly allocated into four groups, each containing 5 animals. Bilateral lower third premolars have been extracted. The right socket was filled with β-calcium sulphate hemihydrate, whereas no material was placed to fill that in the left side to serve as a control. Tissue response in extraction sockets was evaluated using two postoperative intra-oral periapical radiographs for each tooth socket, the first immediately after extraction and the second at the end of each study interval (i.e., after 2 weeks, 4 weeks, 8 weeks, and 12 weeks period for group I, II, III, and IV, respectively). The radiographs were converted from conventional to digital by X-ray scanner, then examined by ImageJ software. Radiographic assessment included the evaluation of differences in extraction sockets densities, bone resorption %, bone formation %, and density of the newly formed bone. The results showed significant differences between the left (control) and right (experimental) sides in all study periods in relation to differences in extraction sockets densities. Meantime, significant differences were noticed between right and left sides during a 12 week period in relation to bone resorption and bone formation %. Concerning density of the newly formed bone, significant differences were noticed during 8 week and 12 week period.In conclusion, the use of β-calcium sulphate hemihydrate as a bone substitute significantly reduced bone resorption and increased the rate of new bone formation. In addition, the density of the newly formed bone in the right (experimental) side was greater than that noticed in the left (control) side.


Introduction
Tooth extraction may encourage extensive dimensional changes of the alveolar ridge subsequent to tooth loss, leading to considerable changes in the structure and morphology of the alveolar bone.The percentage of these changes during the first year after tooth loss is almost 10 times greater than that during later years (1).The problem that dentists face is how to undertake tooth extractions without retreating ridge dimensions (2).In dentistry nowadays, the most inventive and rousing treatment modality for substituting missing teeth is the endosseous dental implant (3).That is why bone conservation after extraction plays an important role in achieving proper results in subsequent prosthetic and implant treatments (1).Consequently, bone grafting procedures to augment the alveolar ridge following tooth extraction are frequently performed in modern dentistry (3).Several graft materials have been proposed, although autogenous bone graft has been considered the gold standard.But, the main disadvantages are a limited amount of graft material, the need of an additional surgical site, increased donor-site morbidity, and the need to use general anesthesia for the extraoral bone harvesting (4)(5)(6).As an alternative to autogenous bone graft, numerous materials have been successfully employed.Collagen, ceramics, bioglasses, polymers, xenografts, allografts, alloplasts, and synthetic hydroxyapatites are among the materials encompassed in this category (7).Of great benefit to clinicians would be a material that is completely resorbable, safe, inexpensive.The ability able to maintain space, and serve as a reservoir for calcium ions (8).Interestingly, calcium sulphate is one of the first materials reconnoitered as a substitute for bone grafting in many fields of medicine (from dentistry to orthopedics).It possesses an extended history of safe use for over a century (9)(10)(11)(12).The first reported case in the modern era where calcium sulphate was used to treat cavities in bone is from 1852 by Mathysen (a Dutch army surgeon) who assimilated plaster into a bandageable form (the form with which we are familiar today).Nowadays, calcium sulphate and its derivatives continue to be the object of research and interest in dentistry and orthopedics (13).In order to determine whether a newly developed βcalcium sulphate hemihydrate bone filling material conforms to the requirements of biocompatibility, mechanical stability and safety, it must undergo rigorous testing both in vitro and in vivo.Results from in vitro studies can be difficult to extrapolate to the in vivo situation.Recently, analysis of bone texture on radiographs became a common way to investigate bone microarchitecture (14,15).Several researchers stated that in vivo radiographs are typical analytical methods for testing the biocompatibility of such materials.However, conventional radiography presents some limitations due to low sensibility and high inter-examiner disagreement (16).X-ray scanner is used to convert conventional dental radiographs into digital images and save them into computer (17,18),the scanned digital radiographic image can then be displayed by ImageJ program.It is a scientific image processing, freely available java-based publicdomain and analysis program (19)(20)(21).According to our knowledge, no previous study on the healing procedure in the extraction socket depending on the radiographic analysis using ImageJ program was found in the literatures.So, the aim was to study the bony response to β-calcium sulphate hemihydrate as a bone substitute prepared from Iraqi gypsum rocks (22) via radiographic assessment (using ImageJ software) of the material implanted into the socket immediately following extraction of dog mandibular 3 rd premolar.

Materials and methods
Twenty local breed dogs in good general health with an average age (12-24 months) and weighing (13-24 kilograms) were included in the experiment.The selected animals should have a complete set of permanent dentition.The animals were divided randomly into four groups, each containing 5 animals as shown in Table ( (B, D, F, H    the dog is more suitable as a model for human bone from a biological standpoint.In terms of mineral composition, humans and dogs do not show significant differences (28)(29)(30).In the present study, the extraction sockets in dog mandible were the regions of interest (ROI) for demonstrating the response of bone tissue to an implant material.Moreover, dog was chosen due to the anatomical and histological resemblances to those of humans.Ease of access, simplicity of procedure, less traumatic placement, the healing properties and size similarities with humans are additional factors making this model appropriate for such study as proof of efficacy or safety of the material prior to registration or human clinical trials (16,31).ImageJ program was used in the present study for radiologic assessment since it is an essential tool that fulfills most of routine image processing and analysis requirements.Another strength is the large number of automated image segmentation algorithms, again allowing the user to choose the most appropriate one, which is considered a significant advantage (32).ImageJ calculates area as a number of pixels, also it measures density in pixels depending the gray scale difference in pixel value statistics for user-defined selections (33).
The early resorption of calcium sulphate as indicated by many previous researches (7,(34)(35)(36)(37) leaves calcium phosphate lattice in the area; the presence of high concentrations of calcium ions gives more radio-opacity compared to the control side, and it encourages the subsequent ingress of osteoprogenitor cells leading to early new bone formation; that's why a highly significant differences were observed between the right (experimental) and left (control) sides in relation to differences in extraction sockets densities (Table 2, Figure 2).This was in line with other previous confirmations (38)(39)(40).Many dental and orthopedic literatures concluded that resorption of calcium sulphate is rapid and complete when compared with other implantable regenerative materials such as hydroxyapatite.It seems to be completely resorbedin 4-10weeks depending on the vascularity of the grafted site, ingress of osteoprogenitor cells and life span of the model (6,38,(40)(41)(42)(43).It was found that the presence of calcium sulphate (and the subsequent release of high concentrations of calcium ions) in the implantation siteis associated with increased concentrations of bone morpho-genetic protein (BMP)-2, BMP-7, transforming growth factor-b (TGF-b), and platelet-derived growth factor (PDGF), all of which play a role in bone regeneration.In the aggregate, these results suggest that this material does not act simply as a bio-inert filler, but it may play a more active role in osteogenesis (44).
Animal studies have shown that the dissolution of calcium sulphate hemihydrate was accompanied by formation of resorption pits due to the attachment of osteoclasts (bone resorbing cells) to calcium sulphate hemihydrateas they possess a calcium sensing receptor (CaSR), which may regulate their activity based on local calcium concentration, and subsequently lead to resorption of the material but not the surrounding bone (4,45), and this gives us a reasonable explanation for the less bone resorption in the experimental side compared to the control one in this study (Table 3, Figure 3).Meanwhile this resorption is principal to the precipitation of a calcium phosphate (CAP) lattice around the resorbed particles, to which osteoblasts (bone forming cells) attach resulting in new bone formation.This could interpret the greater bone formation percentage (Table 4, Figure 4) and the highest density of newly formed bone in this study at the experimental side (Table 5, Figure 5).Moreover, calcium ions released during dissolution of calcium sulphate will lead to local increases in calcium ion concentration, which may stimulate osteoblast proliferation and differentiation from undifferentiated mesenchymal present in the area due to the release of growth factors resulting in modulation of osteoid synthesisby a process of creeping substitution (46,47).The presentstudy concluded that the use of βcalcium sulphate hemihydrate (CSH) prepared from Iraqi gypsum rocks as a bone substitute significantly reduced bone resorption and increased the rate of new bone formation.In addition, the density of the newly formed bone in the experimental side was greater than that noticed in the control side.
Figure 3: M side R= Righ