Authors –S Anoop [1], Amol Chaudhari [2], Roshani Padole [3], Viswa Chandra [4], D.C Sharma [5], V. Sthevaan [6], P. Boopathi [7]

Affiliations – 

• [1], [3], [5], [6] & [7) : Palamur Biosciences Pvt. Ltd.  Karvina, Madigattla Village, Bhoothpur Mandal, Mahabubnagar- 509382, Telangana State, India. Anoop@palamurbio.com
• [2] SynThera Biomedical Pvt. Ltd. 100, NCL Innovation Park, Dr. Homi Bhabha Road, Pashan,   Pune – 411008, Maharashtra, India.
• [4] SVS Dental College Hyderabad Road, Appana Palli,  Mahbubnagar - 509002, Telangana State, India.

Introduction:

The test item PoroSynTM is a bioactive glass (BG). BGs release soluble calcium, phosphorus, and sodium ions that lead to cellular responses at the interface of the glass and bone. This interaction induces osteoconduction and osteoinduction and results in the formation of bone without forming fibrous tissue or promoting inflammation or toxicity. The high biocompatibility and reactivity of these glasses has been identified as their main advantage for their application in periodontal repair and bone augmentation.

To study naturally occurring gingivitis and periodontitis, dogs are among the most well-established models [1]. The Beagle dog is commonly used because of its extremely cooperative nature and its size [2]. In dogs, periodontal tissues and the size of the teeth are, in general, similar to those in humans. However, dogs lack lateral jaw movements and occlusal contacts at the premolars. The subgingival bacterial species are predominantly anaerobic gram-negative cocci and rods [3, 4]. 

Large animal models have been preferred for regenerative medicine using biomaterials, due to the reproducibility and surgical accessibility of experimental defects. Among those, dogs have been widely used for modeling the regeneration of periodontal defects with biomaterials. [2]

Decision Making of Laboratory Animal Model [2]

Dogs also served as the animal model in muco-gingival surgery by creating recessions on the canines which were treated either by palatine connective tissue grafts, or by guided tissue regeneration [5-7]. Dogs are used as models in guided osseous regeneration [8, 9] and in implant surgery [10-12]

Small animals (e. g. rats, hamsters, rabbits, ferrets), their physiopathology is not as similar as humans and so, experimental models needs to be reproducible in order to obtain relevant results. [2]

Non human primates are susceptible to certain infections, such as tuberculosis [13], and ethical consideration and regulation have to be adhered to, to avoid trafficking of protected species [2].

Occurrence of periodontal diseases in rats is less frequent than in human, although the pathology can be induced by inoculating bacteria, giving a carbohydrate-rich diet and fixing ligatures around the teeth. This experimental model is not optimal for studying the evolution of the disease over long periods using histology because of the continuous growth and migration of the teeth [14].

Rodents have only one incisor and three molars in each quadrant. Studies using rodents have shown disease development, after placement of ligatures in the gingival sulcus around the molar teeth, through an increase of biofilm accumulation [15]. This model may not reproduce all aspects of human periodontitis initiation and progression.

Materials and Methods:

Concisely, the procedure involves (including methods of asepsis)

1. Dental Extractions: for the removal of premolars
2. Bone trephining procedure: for the creation of test and control defects
3. Flap suturing for the approximation of flaps post-suturing
   

Biomaterials for Testing :

There were 2 graft materials for testing. The above procedure is only for 1 test material. Sample (Graft) Specifications:

1. PoroSynTM (particle size - 400 to 800 um mesh)/ PPG
2. Xenograft (a commercially available product) / XG

TREATMENT (Sample Placement):

SURGICAL PROCEDURE:

All surgical interventions were performed under general anesthesia in aseptic conditions.

Eight Beagle dogs were used for this study. At the baseline, all lower anterior teeth (excluding the canines) are carefully removed. After ensuring that the roots are removed, Bone defects were developed to an approximate dimension of 5X8X40 mm. Defects in either the left or right side in 4 animals are filled with PoroSynTM and defects in the remaining 4 animals are filled with XG , a commercially available xenograft material. Before filling into the defect, the bone graft materials are mixed with saline and carefully placed to fill the bone defect. No respective surgery is performed in the other jaw quadrant and the cavity was kept empty (control side). In both quadrants the flaps are adjusted to allow full coverage of the edentulous ridge and sutured.

A 6-month period of plaque control, consisting of daily cleaning of exposed implant surfaces was initiated. Clinical examination including assessment of plaque and soft tissue inflammation was performed at the end of this period from the implant sites. After 6-month all the animals were euthanized. The mandibles were removed and placed in a fixative solution (CaCO3 buffered formalin solution). The tissue decalcification was done in nitric acid, dehydrated in serial steps of ethanol and embedded in paraffin wax. Mesio-distal sections of the central portion of the mandible were produced with a microtome and sections were stained with Hematoxylin and Eosin for histological analysis. The sections were observed under light microscope.

Result: 

Histopathology

In general the evaluation, there is no inflammatory reaction was observed for the evaluation period. Lamellar bone, bone marrow and traces of woven bone were observed in bone regeneration area (BRA). Bone remodeling was observed in that host bone was in contact with the bone grafts typically in the bone regeneration area of all the sections. 

During the healing period resorption of the bone graft particles was observed. The degradation of bone graft particles was compared after 6 months of healing period. In general, the PPG particles are very rarely observed. In the bone regeneration in the porous structure of PPG particles were observed. In the pores of the particles, the regenerated mature bone could be observed. On the other hand the XG particles were not observed at all, due to their complete degradation only their cavities were observed.

Figure 3: Histology evaluation of mandible as an effect of bone grafting [(A) PPG and (B) XG].

Conclusion and Discussion

In this study, the results of bone regeneration potential of PPG were evaluated in vivo in Beagle dog mandible model.

6 months after the graft placement, histological observed that islands or a thin continuous layer of woven bone lined a portion of the implant surface coronal to the buccal bone crest. Scattered osteoclasts were found in the corresponding locations of the lingual bone wall the internal portion of the socket region was occupied by bone marrow but included trabeculae of mineralized tissue made up of woven bone and lamellar bone [16] [17] .

Creation of artificial bony defects and sacrifice after 6 months of surgery in beagle dog, showed new bone formation extending to the coronal part of the defect, the new bone was deposited evenly around the graft material, the bone mineral was integrated fully with the new bone, and newly formed bone exhibited a trabecular pattern [18]. 

It is essential to maintain the soft tissue around the implant in a healthy condition to preserve the marginal bone, and approximately 2 mm of junctional epithelium and 1 - 1.5 mm of firm connective tissue form a biological width that serves as a protective membrane that protects the marginal bone from the invasion of microorganisms [19-20]. The attachment tissue between the junctional epithelium and the collagenous bundle parallel to the implant surface forms a biological seal [21]. Very good soft tissue attachment was observed in 6 months after dental implant insertion in dog [22]. Root resorption and connective tissue attachment to root surface observed [23].

Periodontitis follows gingivitis; periodontal pockets develop with a dense cellular infiltrate consisting mainly of plasma cells and lymphocytes. The osteoclastic resorption of alveolar bone results in deep, narrow lesions extending vertically around a single root, leaving the interdental space intact [24]. The dog is an experimental model in periodontal regeneration. For decades, dog studies have been considered as the major in-vivo testing system for new regenerative devices or techniques. Some of the earliest evidence on the efficacy of guided tissue regeneration came from dog studies [25]. The dog model was also used to assess the impact of biomechanical relationships in the oral cavity within the context of regenerative procedures [26]. Dogs served as excellent models for studying the impact of periodontal inflammation and plaque control on regeneration and new attachment formation [26, 27, 28]. Implant stability was tested in peri-implantitis models in dogs [29, 30] and novel technologies, such as laser treatment, were studied [31], with significant findings suggesting that the inflammatory process and the resulting alveolar bone destruction in peri-implantitis in dogs are similar to those in humans and can be successfully treated. These studies also initiated a debate on defect types and material composition, as well as on the surface characteristics of implants [32, 33]

The occurrence of periodontal diseases in dogs is high, increases with ageing and thus, the etiopathology is closely related to humans [34]. Although there are certain differences in the inflammatory response as well as in the bacterial population, dogs are widely used in gingivitis and periodontitis research. Calculus deposits lead to gingivitis [35] but contrary to humans, the sub-connective tissue remains almost normal in dogs. The conversion from gingivitis to periodontitis is not systematic but could be experimentally induced by fixing ligatures around the teeth [36, 37]. Gingivitis in dogs may be accelerated by an appropriate soft, minced diet, promoting the accumulation of supragingival plaque and calculus [38, 39]. Periodontitis in dogs occurs on the basis of pre-existing gingivitis. There is a formation of periodontal pockets lined with typical pocket epithelium. In the connective tissue, the dense cellular infiltrate consists mainly of plasma cells and lymphocytes. The osteoclastic resorption of alveolar bone may result in deep, narrow lesions extending vertically around a single root leaving the interdental space intact [40, 24]. 

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