Characterizations of alveolar repair after mandibular second molar extraction: an experimental study in rats

Abstract Characterizations of rat mandibular second molar extraction socket with significantly different buccal and lingual alveolar ridge width remain unclear. Objective: To observe alterations in the alveolar ridge after extraction of mandibular second molars, and to examine processes of alveolar socket healing in an experimental model of alveolar ridge absorption and preservation. Methodology: Eighteen Wistar rats were included and divided into six groups regarding healing time in the study. Bilateral mandibular second molars were extracted. The rats with tooth extraction sockets took 0, 1.5, 2, 3, 4 and 8 weeks of healing. Histological observation, tartrate-resistant acidic phosphatase (TRAP) staining, Masson’s trichrome staining, immunohistochemical staining and micro-computed tomography (micro-CT) were applied to estimate alterations in the alveolar ridge. Results: Different buccal and lingual alveolar ridge width led to different height loss. Lingual wall height (LH) decreased significantly two weeks after tooth extraction. Buccal wall height rarely reduced its higher ridge width. From two to eight weeks after extraction, bone volume (BV/TV), density (BMD), and trabecular thickness (Tb.Th) progressively increased in the alveolar socket, which gradually decreased in Tb.Sp and Tb.N. LH showed no significant change during the same period. Osteogenic marker OCN and OPN increased during bone repair from two to eight weeks. The reduced height of the lingual wall of the tooth extraction socket was rarely repaired in the later repair stage. Osteoclast activity led to absorption of the alveolar ridge of the alveolar bone wall within two weeks after operation. We observed positive expression of EMMPRIN and MMP-9 in osteoclasts that participated in the absorption of the spire region. Conclusion: Extraction of rat mandibular second molars may help the study of alveolar ridge absorption and preservation. The EMMPRIN-MMP-9 pathway may be a candidate for further study on attenuating bone resorption after tooth extraction.


Introduction
Dental implantation is an advanced and effective clinical method to repair tooth loss, and it has an excellent aesthetic restoration effect and a high survival rate. 1,2 However, alveolar ridge resorption after tooth extraction, especially the loss of height and width, has been the primary challenge of implant restoration. 3,4 Tooth extraction models of different teeth in different animals have been reported, including the extraction of rats' maxillary incisors, 5,6 maxillary first molars, 7-9 mandibular first molars 10,11 and beagle dogs' molar extraction. 12,13 Researchers have found useful data from the models. Beagle dogs need four to six months of healing after tooth extraction to show alveolar repair. Histological analysis of 1, 2, 4 and 8 weeks of healing reveals marked osteoclastic activity, which causes resorption of the crestal region of the buccal and the lingual bone wall. 12 The extraction sockets show an area of minor mineralized bone after three months. The borderline between new and pre-existing bone disappears after six months. 14 Compared to beagle dogs, rats are cheaper and easier to obtain and raise. Eight weeks of healing is usually applied to observe alveolar repair in rats. Bone healing after extraction in the rats can be divided into the following three stages: the blood clot stage, the connective tissue and new bone formation stage, and the ossification stage, all completed within 28 days. 5,15 Similarly, another study on rats has shown that the extraction socket was gradually filled in the following two weeks from the margins inwards. The socket outline is invisible 30 days after tooth extraction. 16 Rat mandibular incisor is difficult to extract because its root is long and runs through the mandible, which is a suitable experimental model to study ameloblast activity 17 and tooth eruption 18 for continuous growth. The width of the alveolar ridge of rat maxillary incisors, maxillary first molars and mandibular first molars, was similar between the buccal and lingual sides.
However, few studies report the crestal region resorption and alveolar repair in tooth extraction model with significantly different buccal and lingual width. The buccal wall of mandibular second molars was more than twice as thick as the lingual wall. Rat mandibular second molar with three roots is similar to human mandibular first molar, which is one of the most easily lost teeth in humans. 19 Rat mandibular second molar is easier to extract than rat mandibular first molar with four roots. 20 Rat mandibular incisor is not applied because its root keeps growing. The high bone density of the rat mandible better characterizes the bone to observe alveolar ridge absorption and repair using micro-computed tomography (micro-CT).
In this study, we extracted bilateral mandibular second molars to observe changes in the alveolar bone wall after tooth extraction and to examine the process of alveolar socket healing. This experimental model can help to assess if rat mandibular second molar extraction is a suitable experimental model to study alveolar ridge absorption and preservation.
Previous studies have suggested that extracellular matrix metalloproteinase inducer (EMMPRIN) and its downstream matrix metalloproteinase-9  are involved in osteoclast activation and periodontal inflammatory bone destruction. 21 EMMPRIN is essential in the maturation of dental hard tissue and the formation of an eruption pathway 22 and gets involved in osteoclastogenesis and alveolar bone resorption during orthodontic tooth relapse. 23 The expression level of EMMPRIN in the alveolar ridge resorption after tooth extraction is unclear.
In this study, the expression levels of EMMPRIN and MMP-9 in the alveolar ridge absorption were

Micro-CT detection
The mandibles containing the extraction sockets were assessed three dimensionally using a micro- were performed to determine the expression of these proteins in the alveolar bone healing. PBS was obtained as negative control.

Statistical analysis
All data are expressed as the mean ± standard deviation. Student's t tests, one-way analysis of variance, and the Newman-Keuls test were conducted using the GraphPad Prism 5 software. The differences were considered significant at p<0.05.

Results
Observation of rat mandibular second molar crestal region The root morphology of rat mandibular second molars with only three roots is similar to human mandibular molars ( Figure 1A). Mandibular second molars were completely extracted. The crestal region of the buccal bone wall is thicker than that of the lingual bone wall in mandibular second molars    The height of lingual extraction socket bone wall (black arrows) was lower than that of the adjacent crestal region (black arrows) 1.5 weeks after extraction. Osteoclasts (red arrows) participated in the marked osteoclastic activity, which caused resorption of the crestal region of the alveolar (A1-A4). Inflammatory cells and multinucleated osteoclasts (red arrows) were found around the small crestal bone islands, which had not been completely absorbed 1.5 weeks after extraction (B1-B4). AL alveolar bone, ES extraction socket. A1 and B1 (8 × magnification), A2 and B2 (20 × magnification), A3 and B4 (72 × magnification), A4 and B3 (83 × magnification) J Appl Oral Sci.

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Bone morphological parameters, including BV/TV, BMD, and Tb.Th, which were positively correlated with bone load capacity and quality, progressively from two to eight weeks. The values decrease when osteoporosis, fracture and bone resorption occur.
We observed the osteogenic marker OCN and OPN  to evaluate bone healing from two to eight weeks.
Consistent with micro-CT results, the expressions of OCN and OPN increased during bone repair after extraction. The healing process agreed with many previous reports. 5,12,15,16 We included HE and Masson's staining results of Bone absorption in the crestal region of the bone wall near the buccal side was mainly found near the extraction socket side. The width of the alveolar ridge was similar between the buccal and lingual sides in most tooth extraction models. However, rat mandibular second molar showed a significant width difference; the buccal wall was more than twice as Figure 8-Representative immunostaining of EMMPRIN and MMP-9 after tooth extraction. EMMPRIN-positive osteoclasts participated in the marked osteoclastic activity, which caused resorption of the crestal region of the alveolar (A1) (10 × magnification). EMMPRIN-positive osteoclasts (red arrows) were observed in the lacuna resorption pits (A2 and A3) (63 × and 110 × magnification). Many positive-monocytes and multinucleated cells in bone marrow cavity were found 1.5 weeks after tooth extraction (A4) (110 × magnification). MMP-9-positive osteoclasts (red arrows) and inflammatory cells (red arrows) were observed during the resorption of the crestal region of the alveolar bone wall (B1-B4). B1 (10 × magnification), B2 (28 × magnification), B3 and B4 (110 × magnification) J Appl Oral Sci. 2022;30:e20220010 10/12 thick as the lingual wall.
The lingual wall height did not increase significantly, although bone volume and trabecular thickness increased from two to eight weeks after extraction.
The reduced height of bone wall near the lingual side of the extraction socket was rarely repaired in the later repair stage. Our results also showed that alveolar ridge width was a determinant of alveolar ridge loss after tooth extraction. [27][28][29][30] The relationship between ridge width and height loss was another focus of the study.
We included tooth extraction sockets with 1.5 weeks of healing to observe osteoclastic activity that The goal of alveolar ridge site preservation is to preserve as much of the alveolar bone of the extraction wound as possible before implanting, ensuring that the implant has more bone to support it. 33,34 Studies have mostly focused on increasing bone regeneration after tooth extraction. [35][36][37][38] The repair of unilateral alveolar ridge site loss is easier than that of buccal-lingual bilateral loss. Only lingual bone wall height was reduced in the rat mandibular second molar extraction model. This study offers a new model for future study on alveolar ridge site preservation after tooth extraction.

Conclusions
1) The rat mandibular second molar extraction model suits the assessment of alveolar ridge absorption and preservation. 2) The EMMPRIN-MMP-9 pathway may be a candidate for further study on attenuating bone resorption after tooth extraction.