Caspase inhibitor attenuates the shape changes in the alveolar ridge following tooth extraction: A pilot study in rats

Abstract Objective The aim of the study was to determine whether the inhibition of apoptosis via pan‐caspase inhibitors can attenuate the changes in the alveolar ridge upon tooth extraction. Background Cells undergoing apoptosis might play a central role in the onset of alveolar bone resorption and the ensuing bone atrophy following tooth extraction. Caspases are proteases that regulate apoptotic cell death. It is, therefore, reasonable to hypothesize that blocking apoptosis with pan‐caspase inhibitors attenuates the changes in the alveolar ridge following tooth extraction. Methods In 16 inbred rats, the mandibular first (M1) and second (M2) molars of one side were extracted. Following random allocation, the rats received either a cell‐permeable pan‐caspase inhibitor or diluent. After a healing period of 10 days, changes in shape and height of the alveolar ridge were examined using geometric morphometrics and linear measurements based on micro‐computed tomography. Results Geometric morphometric analysis revealed that the pan‐caspase inhibitor prevented major shape changes of the alveolar ridge following M1 tooth extraction (P < .05). Furthermore, linear measurements confirmed that the pan‐caspase inhibitor significantly prevented the atrophy of the alveolar ridge height following M1 tooth extraction compared to the diluent controls (−0.53 mm vs −0.24 mm; P = .012). M2 tooth extraction caused no shape changes of the alveolar ridge, and thus, the pan‐caspase inhibitor group did not differ from the control group (−0.14 mm vs −0.05 mm; P = .931). Conclusions These findings suggest that the inhibition of apoptosis may attenuate shape changes of the alveolar ridge following M1 tooth extraction in rodents.

procedure termed "alveolar ridge preservation." 2 The most common technique is to fill up the extraction socket with a bone substitute material and cover it with a resorbable membrane. 2 This procedure, however, does not prevent dimensional alterations but does limit the extent to which resorption occurs. 2 As a result, there is a clear demand to identify new strategies to prevent the resorption of the alveolar ridge. In this context, a better understanding of the underlying molecular and cellular mechanisms that cause alveolar bone resorption may provide a scientific basis to develop such strategies.
Tooth extraction, similar to other injuries, may induce apoptosis, a sequence of molecular events controlling cell death. 3 Apoptotic cells are present in the periodontal ligament following tooth extraction in rats. [4][5][6] Although apoptosis is a rare event in osteocytes after atraumatic tooth extraction as compared to osteotomies 4 or following orthodontic tooth movement, 7 these apoptotic cells may release signals that ultimately trigger and coordinate tissue repair. This is of particular importance since dying osteocytes elicit the formation of osteoclasts and consequently bone resorption. [6][7][8] Furthermore, it appears that the invasiveness of the surgical procedure determines the degree of osteocytes' apoptosis and consequently alveolar bone resorption.
This suggests that apoptosis may play a major role in the subsequent catabolic changes of the alveolar ridge following tooth extraction.
Such a hypothesis prompted us to investigate whether a pharmacologic therapy based on a cell-permeable inhibitor of caspases can prevent catabolic changes after tooth extraction. Caspases belong to a family of proteases that regulate apoptotic cell death. 8,9 Inhibition of apoptosis using a pan-caspase inhibitor reduces the particle-induced osteolysis in mice 8 and prevents the trabecular bone loss caused by unloading. 9 Moreover, the increase in bone resorption does not occur in ovariectomized mice treated with a pan-caspase inhibitor. 10 There is thus a potential of pan-caspase inhibitors to attenuate catabolic changes of alveolar bone following tooth extraction. The aim of the present study was, therefore, to determine whether the use of a pan-caspase inhibitor can attenuate structural catabolic changes in the alveolar ridge after tooth extraction in rats.

| Animals and surgery
Ethical approval for this study was granted by the Federal Ministry for Science, Research and Economy (GZ BMWFW-66.009/0020-WF/V/3b/2017), and research was conducted according to the ARRIVE guidelines. Protocols, handling, and care of the mice conformed to the Austrian federal law for animal protection. Sample size calculation was based on a previous study. 11 In brief, we expected a 15% effect of the difference between the groups. Considering a power 80%, a type I error rate of 5%, and six degrees of freedom, seven rats per group were necessary and one extra rat per group was added in the event of an unexpected loss. Consequently, a total of 16 inbred male rats, 4 weeks of age, underwent tooth extraction of the right mandibular first molar (M1) and the left mandibular second molar (M2). Anesthesia and analgesia were given i.p. injecting ketamine (100 mg/kg), xylazine (5 mg/kg), and piritramide (3 mg/kg).
Enrofloxacin (Baytril 2.5% 15 mg/kg/d) was provided in 5% dextrose water against infections. Following tooth extraction, the animals were randomly allocated to receive for 10 consecutive days either a subcutaneous cell-permeable pan-caspase inhibi- euthanasia was performed on day 11 and with an overdose of pentobarbital (300 mg/kg i.c.).

| Generalized Procrustes analysis and principal component analysis
Bony surfaces were generated with Amira using the half-maximum For comparing the shapes of the alveolar bone, a principal component analysis (PCA) based on eight landmarks was performed. The differences in shapes are represented by data plotted on a principal component axis. 13 Statistical significance is given when data points of groups (≥3 Individuals/group) do not overlap. The shape difference distinguishing the groups were visualized by relative warps using extreme scores (−0.25 and 0.25) on the principal component two. This analysis identified regions with the strongest shape difference which were further used for linear measurements.

| Linear measurements of alveolar bone high
We calculated the distances between the lingual 12  molars of one side were extracted, and the contralateral pristine bone sites were used as reference. When µCT images revealed remnants of teeth in the extraction alveolus, we excluded the values; according to that method, four extraction alveoli were excluded.
Lingual alveolar crest distance of M1 was size-corrected by dividing the distance by the centroid size. To further address the ridge resorption difference, we subtracted the ridge height of the pristine and extraction side to obtain a delta.  Overall, these findings suggest that caspase inhibitor attenuates the catabolic shape changes of the alveolar ridge after M1 tooth extraction.

| Caspase inhibitor reduced alveolar ridge atrophy upon tooth extraction
To further examine the effect of caspase inhibitor on bone shape, a GPA was performed. The GPA uses landmarks to superimpose the anatomical sites ( Figure 3). Superimposition of nine size-, rotation-, and translation-corrected landmarks exhibited a difference between the caspase inhibitor group and the control group ( Figure 3A). The

| D ISCUSS I ON
The present study demonstrated that pan-caspase inhibitors attenuate catabolic changes of the alveolar ridge after M1 tooth extraction. These findings can be explained by the inhibition of apoptosis that might otherwise have caused bone resorption. These biological principles are supported by a previous study showing that the pan-caspase inhibitor reduces the inflammation-induced bone resorption in a murine calvaria model. 8 Similarly, results from another study revealed that pan-caspase inhibitors were able to attenuate the hindlimb-induced bone loss in mice. 9 Thus, the present data support the hypothesis that M1 tooth extraction triggers a local cell apoptosis and the dying cells in turn generate a microenvironment that support osteoclastogenesis leading to bone loss.
Conversely, in M2 sites the alveolar ridge barely changed upon tooth extraction. This might be attributed to the M2 anatomical situation where tooth extraction may causeless mechanical stress and apoptosis, resulting in limited bone resorption.
A recent study revealed that bisphosphonates also prevent bone atrophy after the extraction of mandibular first molars in rats. 14 In that study, buccal and lingual alveolar bone atrophy was significantly reduced after 1 month of healing. Conversely, in our study we observed catabolic changes in alveolar bone at 10 days. However, it should be noted that the anatomical changes in rodent models are subtle and can hardly be localized by linear measurements. Our analyses were therefore based on geometrics morphometrics being widely recognized as robust and sophisticated diagnostic tools for determining shape changes, particularly in anthropology. [15][16][17] This type of analysis has also been used in dentistry, including orthodontics 18-20 and craniofacial surgery. 21 In addition, we have recently proposed the use of geometrics morphometrics to characterize skull deformation in sclerostin knockout mice. 11 Here, geometric morphometrics revealed that caspase inhibitors limited the deformation of the lingual alveolar ridge following M1 tooth extraction. In addition, it showed that M2 tooth extraction causes almost no catabolic changes.
We recognize that this study has a number of limitations. First, this study focused on the structural changes after tooth extraction.
In this sense, to which extent apoptotic osteocytes 4 and periodontal cells 4-6 drive bone resorption upon tooth extraction remains unclear.
Considering that both aforementioned cell types can contribute to inflammatory osteolysis of the alveolar bone, 22

| CON CLUS ION
The present study suggests that the inhibition of apoptosis via caspase inhibitors may attenuate shape changes of the alveolar ridge following M1 tooth extraction in rats. Our findings support the hypothesis that resorption of the alveolar ridge and the subsequent bone atrophy following tooth extraction is partially controlled by apoptotic mechanisms. Targeting apoptosis might become an F I G U R E 5 Atrophy differences in molar one (M1) and molar two (M2). The bone atrophy was determined by calculating the difference between the extraction sockets and the pristine corresponding sites. Significant less atrophy was observed for the caspase inhibitor group compared to the control group in M1 (A) but not in M2 (B).
interesting therapeutic approach to modulate the dimensional changes of the alveolar bone following tooth extraction.

ACK N OWLED G EM ENTS
The authors thank Patrick Heimel for acquiring the µCT data. The