Histological and bone mineral density analyses of the mandibular lamellar/oblique fracture healing in beagles.

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Tingting Xin, Jun Li, Qian Xu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2556512/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective : The goal of this study was to determine whether there were any differences in the healing processes for linear fractures and lamellar/oblique mandibular fractures. Materials and methods : Using a random number generator, two groups of six beagle dogs each were created from a group of twelve.The experimental group was the lamellar/oblique fracture of the mandible, while the control group was the traditional linear fracture. The first premolar and the first molar were where the fracture occurred. Two 4-hole bone plates were used to repair the fracture line. Results : X-ray:the experimental group's fracture space was fuzzier and eventually disappeared between the two groups. Histology: the Collagen Volume Fraction in the experimental group was lower than that in the control group three months after surgery. The experimental group had more bone cells than the control group did one month following the surgery.Both groups' trabecular arrangements became more regular as the healing process progressed.Bone mineral density: the experimental group's bone mineral density was higher than the control group's one month following surgery. Four-point bending test:the experimental group's fracture energy was higher than the control group's one month following the surgery. The experimental group's stiffness was substantially greater than the control group's three months after the procedure. The maximal loading of the experimental group was higher than that of the control group at one and three months following the operation. Conclusion : In comparison to traditional linear fractures, lamellar/oblique fractures of the mandible have higher bone mineral density, are more rigid, and heal more quickly. Mandibular fractures Lamellar/oblique fractures Histology Bone mineral density Biomechanics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 1. Introduction Mandibular fractures are the second-most common fracture of the skull and face bones and a frequent source of traumatic morbidity [1] .Every year, more and more jaw fractures are brought on by auto accidents. The most frequent fracture in the oral and maxillofacial region occurs in the mandible, which is prominently positioned and more prone to fracture than the zygomatic and maxillary bones [2] .The most frequent fracture in the oral and maxillofacial region is this one.36%–80% of all facial fractures are mandibular fractures [3, 4] . It’s complication occurs 7% to 29% of the time [5] .It can result in malocclusion, temporomandibular joint disorder, salivary fistula, infection, abnormal masticatory function, and facial deformity if the fracture heals poorly, all of which have a significant negative impact on the patients' quality of life [6] .Life quality is negatively impacted. The process of mending a fracture resembles the regeneration of primary bone tissue and does not involve the formation of scar tissue [7] . Long limb bones grow from the mesoderm, whereas craniofacial bones develop from the cranial neural crest, making them different from long limb bones in terms of their developmental methods and embryonic origins [8] .Since the mandible becomes osteogenic through intramembranous ossification [9] , hematoma and inflammation form at the trauma site early in the fracture, and various cytokines and immunogenic factors are released to attract bone marrow mesenchymal stem cells to the fracture site [10] , the mechanism of healing may also differ. The MSCs in the bone marrow develop into osteoblasts, which release type I collagen, osteocalcin, as well as other substances [11] .The osteoblasts become osteocytes and undergo extensive cross-linking of their dendritic protrusions, which results in the formation of a network of cells that are closely connected to the neurovasculature [12] . Mandibular fractures are mostly divided into the following categories [3] :1. Anatomical division into the median joint, the region around the chin foramen, the mandibular angle, the mandibular branch, the condyle, and fractures of the coronoid.2. Classification of three levels of damage in accordance with Kazanjian and Converse's definition of the link between the dentition and fracture line. 3.Mandibular angle fracture categorization in relation to muscle activity. 4.The degree of damage according to the F-F4 rating method for mandibular fracture severity. 5.Local condylar fracture classification. 6.The position of the fracture gap on imaging is used to modify the Dingman and Natvig classification [1] . Based on the degree of alveolar ridge atrophy, the degree of dental or periodontal trauma, and the amount of bone loss, the AOCMF divides mandibular fractures other than condylar fractures into three levels [13] . In the introduction of tension screw fixation,Prof. Zhang Yi of Peking University [14] developed the classification of mandibular lamellar/oblique fractures. Mandibular linear fractures and lamellar/oblique fractures are frequently treated similarly, and this is due to the existing classification system. In a lamellar/oblique fracture of the jaw, the buccal and lingual cortices are divided, generating two thin lamellar plates on the buccal and lingual sides, and unlike in linear fractures, the lingual side is unable to relocate the fracture end directly under direct vision. Consequently, these two distinct fracture types could result in various healing processes. There are no more precise categorization or treatment guidelines for lamellar/oblique fractures, nevertheless. This investigation examined if the healing processes for linear and laminar/oblique mandibular fractures varied. 2. Materials and methods The Experimental Animal Center of Xinjiang Medical University provided the experimental animals, and the study was completed in a facility that complied with all applicable national regulations. The experimental study protocol was approved by the Ethics Committee of the People's Hospital of the Xinjiang Uygur Autonomous Region (KY2018060613). All animals were grouped after one week of captivity in the experimental center, and the experimental procedures complied with laws and norms pertaining to animal care. 2.1 Grouping Twelve healthy adult Beagles (20-24 months old, average weight 8.0-12.0 kg, males and females) were split into two groups of six at random. The mandibular body in the control group was fractured in a traditional longitudinal linear fracture model, with the fracture line parallel to the bottom edge of the mandibular body. In the experimental group, a laminar/oblique fracture model of the mandibular body was established, with the fracture line measuring about 2 cm in length and forming an angle of about 25° to the long axis of the mandible. 2.2 Surgical establishment of animal models The skin beneath the left mandible of the Beagle was prepared, stained with methylene blue, and a 3.0-cm-long curved incision was made on the lower edge of the left parallel mandible. The skin mucosa and periosteum were incised with a 15-gauge surgical blade, and the periosteum was peeled along the bone surface to expose the thymus. Anesthesia was induced by mixing 0.6 mg/kg of Sulforaphane II (Jilin Huamu) with 0.75 mg/kg of Sutex (Zoletil 50, Vic, France) intramuscularly. The bone was chiseled downward to create a section at a 25°angle to the long axis of the mandible between the left mandibular body's first premolar and first molar.The left mandibular body's labio-buccal and lingual cortices were separated to form a lamellar shape, the medial and lateral bone plates of the mandible were gradually split downward, and the medial and lateral bone plates of the mandible were completely separated with a mandibular spreader to establish a lamellar/oblique fracture of the mandibular body. The linear fracture was created by cutting a gap perpendicular to the body of the mandible with a bone chisel, and the gap was moved from the buccal side of the mandible to the alveolar ridge of the lingual molar, creating a longitudinal linear fracture of the mandible. Normal occlusal-dental relationship was restored and the broken bone end was reset. Two sets of 4-hole titanium plates and screws were used to fix the fractured ends side-by-side in both groups. The periosteum and skin were sutured, and all animals were free of postoperative discomfort such as fever and infection, and the sutures were removed 1 week later. Specimens were collected at 2 weeks, 1 month, and 3 months postoperatively, respectively. 2.3 Statistical analysis Data were analyzed using SPSS software, t-test analysis was used for comparison between two groups, one-way analysis of variance (ANOVA) was used for comparison between groups, and histograms were made using GraphPad Prism5. The results were expressed as mean ± standard deviation (SD), and statistical significance was set a P < 0.05. 3. Result 3.1 X-ray findings Evaluation of the development of bone scabs and monitoring the healing of fracture lines. Clear fracture lines and less obvious bone scab formation could be seen in both the control and experimental groups two weeks after surgery, and there were no appreciable differences between the two groups. one month following surgery, the control group's lower border of the mandible still had obvious fracture lines visible, however, the fracture lines at the osteotomy alveolar ridge were blurred and there was some bone scab formation. The fracture ends were gradually approached, the fracture lines blurred, and there was a noticeable rise in bone scabs in the experimental group, although there were still gaps at the fracture ends. The distance between the fracture ends of both groups was lower than before three months following surgery. In the experimental group, the fracture gap was essentially healed and the bone scab was more substantial than in the control group, which had a blurred fracture gap and no discernible fracture line at the lower edge of the jaw.( Figure 1) 3.2 Histological observations The amount of new bone, trabecular formation, size and shape of the osteocytes in the bone traps, and the direction of collagen fiber alignment were observed. Masson staining, which stains the collagen fibers blue to show the maturation process of newly formed bone while staining red the mineralized bone, was used to detect the amount of new bone [15] .By utilizing Image Pro Plus to calculate the quantity of osteocytes and the proportion of collagen fibers to the surface area of the whole tissue (Collagen Volume Fraction (CVF)), bone mineralization formation was evaluated semi-quantitatively [16] .The analysis included microscopic images at a magnification of 100x, Masson staining with high color resolution, the same HSI mode for all images, representation of the blue-stained collagen fiber regions, calculation of the CVF, and statistical analysis of all results. The same microscopic 100x pictures were chosen for HE-stained slices in order to manually count the osteocytes. (Figure 2) At two weeks after surgery, both control and experimental groups showed blue-red stained areas in Masson staining, with more proliferated blue collagen fibrous tissue. The mean collagen fibrous area ratio was 0.2820±093 in the control group and 0.2370±039 in the experimental group, with the irregular interwoven arrangement of fibers. Osteoblasts were also visible in the fibrous tissue, scattered throughout the bone traps. HE In both groups, staining revealed dense connective tissue and osteoblasts on the surface of the bone trabeculae; osteocytes, new capillaries, and Haver's system bone were uniformly distributed within the trabeculae, and the trabeculae were grouped irregularly. At one month following surgery, Masson staining revealed more new bone in the experimental group compared to the control group, with collagen fibers arranged in a parallel pattern, and more fibrous tissue in the control group compared to the experimental group, albeit with an uneven pattern. In the experimental group, the mean collagen fiber area ratio was 0.1390±019, compared to 0.2000±074 in the control group. Osteocytes in bone traps were visible within the trabeculae, and the osteocytes in the experimental group had smaller cytostomes with a flat oval shape compared to the control group. Bone trabeculae were more regularly arranged in HE staining than at 2 weeks, connective tissue was reduced than before, and osteocytes were visible within the trabeculae. In Masson staining at 3 months after surgery, the experimental group had collagen fibers arranged parallel to one another, with obvious bone scab formation and woven bone forming lamellar bone, whereas the control group still had more fibrous tissue, primarily woven bone. The mean collagen fiber area ratio was 0.4290±006 in the control group and 0.1140±090 in the experimental group. There was no connective tissue visible and the bone trabeculae were properly structured. They also contained numerous osteocytes and osteoblasts. In both groups, the level of bone scab reconstruction was comparable(Figure3,Figure4). At 2 weeks and 1 month postoperatively, there was no statistically significant difference between the two groups when comparing the CVF data, however at 3 months postoperatively, there was a statistically significant difference between the control and experimental groups (P<0.05)(Figure5).With passing time, there was no statistically significant difference in CVF between the two groups (Figure6). Osteocyte counts in the control and experimental groups were 137.5±23.3, 121.5±31.8, 88.0±2.8, 108.0±1.4, 44.5±17.6, and 37.5±16.2, respectively, after 2 weeks, 1 month, and 3 months following surgery(Figure7) .Statistical analysis of osteocyte counts showed a statistical difference between the two groups at 1 month postoperatively (P< 0.05). When compared longitudinally, osteocytes in the control group were statistically different (P< 0.05) when compared at 2 weeks and 3 months (Figure8). 3.3 Bone mineral density results To assess bone scab growth and determine whether there was a difference in bone mineral content between the two groups, BMD was measured using dual-energy X-ray absorptiometry and coupled with imaging.The mean BMD of the experimental group two weeks after surgery was 0.898±0.231 and that of the control group was 0.532±0.182, no statistically significant difference was seen between the two groups (P=0.221). The mean BMD of the experimental group at 1 month postoperatively was 0.765±0.076 and that of the control group was 0.142±0.024, which was statistically different between the two groups (P<0.05). The mean BMD at 3 months postoperatively was 0.444±0.086 in the experimental group and 0.250±0.074 in the control group, with no statistically significant difference between the two groups (P=0.139)(Figure9).BMD values gradually increased with the extension of the fracture healing period in both groups, and no statistical difference was observed(Figure10). 3.4 Biomechanical measurements The fracture energy, stiffness, and maximum load of fracture specimens were assessed using a four-point bending experiment. The experimental group's mean fracture energy measures at two weeks, one month, and three months postoperatively were 61.6±4.0,89.0±3.4, and125.3±6.8,while the control group's mean values were 57.0±3.0,77.8±2.5,and110.4±6.1,respectively.The two groups were statistically different at one month postoperatively (P<0.05), at which point the fracture energy of the experimental group was higher than that of the control group. Stiffness measurements were not statistically different between the two groups at two weeks and one month postoperatively, and the stiffness of the experimental group was greater than that of the control group at three months postoperatively (P<0.05). Maximum load measurements were not statistically different at two weeks postoperatively and were higher in the experimental group than in the control group at 1 month and 3 months postoperatively (P<0.05) (Figure11). The fracture energy, stiffness and maximum load were significantly higher and the data were statistically different in both groups as the fracture healing time increased. (Figure12) 4. Discussions When comparing the two groups laterally, it could be seen that there were no significant differences between the two groups during the pre-healing period. At 1 month postoperatively, the osteocytes, bone density, fracture energy, and maximum load were greater in the experimental group than in the control group, and at 3 mon boths mandibular body lamellar/oblique fractures and linear fractures achieved good healing.maximum volume proportion of collagen fibers The experimental group exhibited higher bone maturity and better mechanical characteristics, as evidenced by the CVF of the experimental group being lower than that of the control group and the maximum load of the experimental group being higher than that of the control group. The fracture line gradually blurred, the osteoblasts gradually decreased, the bone matrix gradually increased, and the bone trabeculae were regularly and orderly arranged in parallel when compared longitudinally within groups. Additionally, both groups' bone density and mechanical properties gradually increased over time. At the shattered ends of the bone, more collagen fibers can be seen in the early stages of fracture healing. As healing time advances, however, there is a steady decrease in collagen fibers and an increase in bone tissue. Bone scab size, bone density, and biomechanical qualities are all directly related to fibrous tissue area and bone tissue area, which are important stage indicators of fracture healing [17] .Cell differentiation and fracture healing may be impacted by changes in the mechanical environment of the fracture end due to the fracture inclination [18] . Ramasamy [19] discovered that the mechanical function of bone in mice is significantly influenced by the orientation of collagen fibers in the anatomical loading axis, with longitudinal fibers forming a tissue with better tensile properties and maximum strain. The lamellar/oblique group in our trials had a fracture inclination angle of 25°, and the outcomes demonstrated superior mechanical qualities to the linear group, which is also consistent with the Miramini et al. study. When the mandibular fracture is correctly and steadily repositioned anatomically, studies have shown improved bone healing and fewer complications [20–21] . Restoring occlusion and ensuring bone healing have long been the main objectives of jaw fracture treatment [22] . Both groups of animals in this experiment received care in accordance with the OR/IF standardized fracture treatment approach [23] . The gap at the fracture break after fixation may be larger than in the linear fracture group because the laminar/oblique group had trouble resetting the lingual bone portion under direct view.According to Tetsuo [24] experiments on sheep with metatarsal fractures, significant micromotion (0.7 mm) results in an increase in the amount of new bone growth when the fracture gap is small (2 mm), but on the other hand, when the fracture gap is large (6 mm), the micromotion magnitude can result in a delay in the healing process. The most suitable micromovement at the fracture end is currently thought to be a 0.2 to 1.0 mm axial movement [25] . This implies that the right kind of intermittent motion between fractures will encourage the development of bone scabs and hasten the healing of fractures. A four-point bending test, which involves applying two downward forces on the skeletal scaffold equidistant from the center of the bone, was used in this experiment. This test is crucial for determining the final fracture repair because it allows a more uniform propagation of the bending moment between the two points where the force is applied [17] . The strains caused by weight-bearing activity or muscle contraction at the fracture site cause interfragmentary movement (IFM), which alters the size of the initial fracture gap and causes interfragmentary strain ε [26] .Moderate-sized IFM can promote bone healing.Axial motion, as opposed to the translational and torsional shear motion, which slows bone healing, is found by some researchers to produce better healing in mechanical simulations and finite element analysis [27] .The maximum mineralized bone crust area, four-point bending experimental stiffness, and mineralized tissue density, on the other hand, were higher in the torsional shear group than in the axial group in Bishop's experiments in a sheep tibial fracture model, suggesting that interfragmentary shear motion does not necessarily delay bone healing but instead stimulates bone crust formation and promotes fracture healing [28] .Our research also supports the notion that shear does not always prevent bone healing because the stiffness and maximum load of the lamina/bevel group were higher in the late phase of fracture healing than those of the linear group. BMD can react to the development of a bone scab and changes with the speed and depth of healing. The mandible shares several characteristics with long bones, and BMD of long bones is frequently used to evaluate changes in the balance between osteoblast bone creation and osteoclast bone resorption [29] . A number of writers have employed BMD measurements obtained from DXA in animal studies recently to quantify fracture healing and statistically examine healing variations [30-32] .Our study demonstrates that at the middle stage of fracture union, the experimental group's BMD was higher than the control group's, and at the same time, the experimental group's callus hyperplasia was visible on X-rays. This means that the control group's bone maturity was lower than the experimental group's at this stage of fracture union. Osteocytes are the primary cells of bone metabolism, and bone reconstruction involves more than just the interaction of osteoblasts and osteoclasts [33] . Through a network of luminal tubules, osteocyte protrusions come into contact with the extracellular matrix and are responsive to a variety of mechanical and chemical stimuli [34] .In order to influence bone reconstruction, osteoclasts can transform mechanical shear stresses into biochemical signals that can be communicated with by other cells. Osteocytes trigger the production of growth factors, extracellular matrix synthesis, and the stimulation of mesenchymal cells to develop into osteoblasts in response to mechanical forces [35] .In our findings, we demonstrated that during mid-fracture healing, the lamellar/oblique group had more osteocytes, fracture energy, maximum loading force, and BMD than the linear group. This suggests that mechanical shear stress may have encouraged osteoblast osteogenesis in the lamellar/oblique fracture group, yielding a result resembling mandibular distraction osteogenesis. Schwarz [36] demonstrated that compared to resorbed fixed fractures, the fracture load of mandibular distraction osteogenesis was much higher, and the development of high-density bone mineralization was increased in the distraction osteogenesis regeneration group. This research has some restrictions: 1) The fracture line in the animal experimental model was artificially osteotomized, which makes it impossible to accurately simulate the real fracture environment. This will have an impact on the experiment's accuracy, and future mechanical loading experiments should be improved to simulate a fracture subjected to mechanical impact in a more realistic manner. 2) Because of the sample's small size and potential for error. In conclusion, we created models of lamellar/oblique and linear fractures of the mandible in Beagle dogs, assessed and compared the cellular, bone density, and biomechanical functions during the healing process of these two fracture types, both of which were converted into the new bone through osteoblast-osteoclast conversion, and observed faster bone scab formation and higher mechanical properties, such as BMD and stiffness of the bone, in lamellar/oblique fractures. This study offers evidence in favor of improving the kinds and management of mandibular fractures. Declarations Ethics approval and consent to participate The experimental study protocol was approved by the Ethics Committee of the People's Hospital of the Xinjiang Uygur Autonomous Region (KY2018060613). The experimental procedures complied with relevant guidelines and regulations pertaining to animal care. All methods are reported in accordance with ARRIVE guidelines for the reporting of animal experiments. Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests Funding This work was supported by the Natural Science Foundation of Xinjiang [Grant Number: 2018D01C099]. Authors’Contributions Qian Xu designed the study, Qian Xu and Tingting Xin analyzed the data, prepared the figures, and wrote the original draft. Jun Li critically reviewed the manuscript. All Authors read and approved the final manuscript. Acknowledgements The authors owe a debt of gratitude to Xinjiang Medical University Animal Experimental Research Center due to their support and intensive effort to our project. References Escott EJ, Branstetter BF. Incidence and characterization of unifocal mandible fractures on CT. AJNR Am J Neuroradiol. 2008. 29(5): 890-4. Ahmed A, Wu E, Sarai R, Williams R, Breeze J. 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Camal Ruggieri IN, Cícero AM, Issa J, Feldman S. Bone fracture healing: perspectives according to molecular basis. J Bone Miner Metab. 2021. 39(3): 311-331. Schwarz DA, Arman KG, Kakwan MS, Jamali AM, Elmeligy AA, Buchman SR. Regenerate healing outcomes in unilateral mandibular distraction osteogenesis using quantitative histomorphometry. Plast Reconstr Surg. 2010. 126(3): 795-805. Schwarz DA, Arman KG, Kakwan MS, Jamali AM, Buchman SR. Analysis of the biomechanical properties of the mandible after unilateral distraction osteogenesis. Plast Reconstr Surg. 2010. 126(2): 533-542. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2556512","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":175545358,"identity":"b64d3de9-7480-488b-9bac-f6b403cd6872","order_by":0,"name":"Tingting Xin","email":"","orcid":"","institution":"Xinjiang Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Xin","suffix":""},{"id":175545359,"identity":"805136b6-043b-4fc5-b144-0c330eaf9dce","order_by":1,"name":"Jun Li","email":"","orcid":"","institution":"Xinjiang Uiger Municipal People’s Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Li","suffix":""},{"id":175545360,"identity":"0fb74330-1a93-4b45-b13a-db68f670e1d4","order_by":2,"name":"Qian Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYBACefb2gw8+VPyX42dvIFKLYc+ZZMMZZ5iNJXsOEGvNDQczYc425sQNNxKI1ME4gyGNmbGNjXHmzMcbbzDU2EQT1MIu3XjsccE5HmZ+6bRiC4ZjabkNBG2ZcyDdeEaZBJvk7BwzCcaGw4S1MNxIMJPmYTPgMbh5hiQtbQkSBjd4iNQCDeQDBpI9QL8kEOMXaFQeqO9nP7zxxocaGyIchgQMJBJIUQ7RQqqOUTAKRsEoGBkAACdEQv8806mMAAAAAElFTkSuQmCC","orcid":"","institution":"Xinjiang Uiger Municipal People’s Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2023-02-06 14:44:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2556512/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2556512/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":32926786,"identity":"da8e5d76-6472-41d3-8264-7723f73574eb","added_by":"auto","created_at":"2023-02-14 16:16:27","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":45464,"visible":true,"origin":"","legend":"\u003cp\u003eA1,X-ray of the experimental group at 2 weeks.A2,X-ray of the control group at 2 weeks.B1, X-ray of the experimental group at 1 month.B2,X-ray of the control group at 1 month.C1,X-ray of the experimental group at 3 months.C2,X-ray of the control group at 3 months.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2556512/v1/b3bad4246a0ac7341c16d68e.jpg"},{"id":32926789,"identity":"d803e4d8-2b72-4862-b41a-4983bdaf5be3","added_by":"auto","created_at":"2023-02-14 16:16:27","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":119818,"visible":true,"origin":"","legend":"\u003cp\u003eA、B,Collagen fibers measurement process in Masson staining. C、D,the quantity of osteocytes in HE staining.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2556512/v1/45d4e99a8d187717d10810e9.jpg"},{"id":32926297,"identity":"a8a49fa6-6323-4c6f-b463-9cd0e74229ea","added_by":"auto","created_at":"2023-02-14 16:08:27","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":536643,"visible":true,"origin":"","legend":"\u003cp\u003eHE staining: A,control group for 2 weeks(×100). B,control group for 1 month(×100). C,control group for 3 months(×100). D, experimental group for 2 weeks(×100). E,experimental group for 1 month(×100). F, experimental group for 3 months(×100).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2556512/v1/a31ee0aacc3792e8fd5406a7.jpg"},{"id":32926787,"identity":"9db10322-c630-496b-b4ac-326a92d0c07c","added_by":"auto","created_at":"2023-02-14 16:16:27","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":130906,"visible":true,"origin":"","legend":"\u003cp\u003eMasson\u0026nbsp;staining: A,control group for 2 weeks(×100). B,control group for 1 month(×100). C,control group for 3 months(×100). D, experimental group for 2 weeks(×100). E,experimental group for 1 month(×100). F, experimental group for 3 months(×100).\u003c/p\u003e\n\u003cp\u003e★:Collagen fibers.\u003c/p\u003e\n\u003cp\u003e▲:Trabecular bone.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2556512/v1/f2c0228cb666f8b9ccd90c08.jpg"},{"id":32925630,"identity":"4ac5a5e6-6e4c-4ce1-8cf3-d7e2530f5b80","added_by":"auto","created_at":"2023-02-14 16:00:27","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":62410,"visible":true,"origin":"","legend":"\u003cp\u003eA,the quantity of CVF at 2 weeks.B,the quantity of CVF at 1 month.C,the quantity of CVF at 3 months.（*:P\u0026lt;0.05）\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2556512/v1/109666aaf2e218762e12a4ef.jpg"},{"id":32926788,"identity":"2368c3d8-c083-49c3-95ea-a6ee2b68c330","added_by":"auto","created_at":"2023-02-14 16:16:27","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":31411,"visible":true,"origin":"","legend":"\u003cp\u003eThe CVF of control and experiment groups respectively\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2556512/v1/8f945ae542fdff1f9302ac29.jpg"},{"id":32926793,"identity":"fe5d5537-b555-4591-84e5-b57ff46bc1eb","added_by":"auto","created_at":"2023-02-14 16:16:27","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":44630,"visible":true,"origin":"","legend":"\u003cp\u003eOsteocyte counts in the control and experimental groups\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2556512/v1/5a59ce3e6528bfafdc5205b8.jpg"},{"id":32927003,"identity":"52dbd468-a7ff-4179-a3fe-e02c02fa916f","added_by":"auto","created_at":"2023-02-14 16:24:27","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":31008,"visible":true,"origin":"","legend":"\u003cp\u003eThe Osteocyte counts of control and experiment groups respectively.(*:P\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2556512/v1/69e62b9173e7a6df4256f671.jpg"},{"id":32927469,"identity":"c9d6b002-ed6d-4a31-a1e1-8d5d6100555f","added_by":"auto","created_at":"2023-02-14 16:32:27","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":60461,"visible":true,"origin":"","legend":"\u003cp\u003eA,the quantity of BMD at 2 weeks.B,the quantity of BMD at 1 month.C,the quantity of BMD at 3 months.(**:P\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2556512/v1/3f765d90b4ad8fbe32473bf5.jpg"},{"id":32926300,"identity":"b642b2b1-eafa-494d-b2a7-fb39aa8e79ee","added_by":"auto","created_at":"2023-02-14 16:08:27","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":25324,"visible":true,"origin":"","legend":"\u003cp\u003eThe BMD of control and experiment groups respectively.\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2556512/v1/027f734f30c063add0fd32ba.jpg"},{"id":32925641,"identity":"e161f2f8-a49a-4ff6-b1be-367713b8dbc7","added_by":"auto","created_at":"2023-02-14 16:00:27","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":52006,"visible":true,"origin":"","legend":"\u003cp\u003eA,the frature energy of two groups at the same time.B,the stiffness of two groups at the same time.C,the maximum load of two groups at the same time.(*:P\u0026lt;0.05**:P\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2556512/v1/ee9b59489b8257dd4abac007.jpg"},{"id":32926790,"identity":"aedc89aa-f8c7-4609-a93a-13d54f17dab3","added_by":"auto","created_at":"2023-02-14 16:16:27","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":57677,"visible":true,"origin":"","legend":"\u003cp\u003eA1、A2,The frature energy of control and experiment groups respectively.B1、B2,The stiffness of control and experiment groups respectively.C1、C2,The maximum load of control and experiment groups respectively.(*:P\u0026lt;0.05**:P\u0026lt;0.01***:P\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2556512/v1/0b89a619d4c6d3f4b33e2db8.jpg"},{"id":50080989,"identity":"89eb93a1-ad86-408f-b8b8-d436f604f857","added_by":"auto","created_at":"2024-01-24 07:42:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":861762,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2556512/v1/bad49468-04d3-4e95-9ddd-369a2eac977f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Histological and bone mineral density analyses of the mandibular lamellar/oblique fracture healing in beagles.","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMandibular fractures are the second-most common fracture of the skull and face bones and a frequent source of traumatic morbidity\u003csup\u003e[1]\u003c/sup\u003e.Every year, more and more jaw fractures are brought on by auto accidents. The most frequent fracture in the oral and maxillofacial region occurs in the mandible, which is prominently positioned and more prone to fracture than the zygomatic and maxillary bones\u003csup\u003e[2]\u003c/sup\u003e .The most frequent fracture in the oral and maxillofacial region is this one.36%\u0026ndash;80% of all facial fractures are mandibular fractures\u003csup\u003e[3, 4]\u003c/sup\u003e. It\u0026rsquo;s complication\u0026nbsp;occurs 7% to 29% of the time\u003csup\u003e[5]\u003c/sup\u003e.It can result in malocclusion, temporomandibular joint disorder, salivary fistula, infection, abnormal masticatory function, and facial deformity if the fracture heals poorly, all of which have a significant negative impact on the patients\u0026apos; quality of life\u003csup\u003e[6]\u003c/sup\u003e.Life quality is negatively impacted.\u003c/p\u003e\n\u003cp\u003eThe process of mending a fracture resembles the regeneration of primary bone tissue and does not involve the formation of scar tissue\u003csup\u003e[7]\u003c/sup\u003e. Long limb bones grow from the mesoderm, whereas craniofacial bones develop from the cranial neural crest, making them different from long limb bones in terms of their developmental methods and embryonic origins\u003csup\u003e[8]\u003c/sup\u003e.Since the mandible becomes osteogenic through intramembranous ossification\u003csup\u003e[9]\u003c/sup\u003e, hematoma and inflammation form at the trauma site early in the fracture, and various cytokines and immunogenic factors are released to attract bone marrow mesenchymal stem cells to the fracture site\u003csup\u003e[10]\u003c/sup\u003e, the mechanism of healing may also differ. The MSCs in the bone marrow develop into osteoblasts, which release type I collagen, osteocalcin, as well as other substances\u003csup\u003e[11]\u003c/sup\u003e.The osteoblasts become osteocytes and undergo extensive cross-linking of their dendritic protrusions, which results in the formation of a network of cells that are closely connected to the neurovasculature\u003csup\u003e[12]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eMandibular fractures are mostly divided into the following categories\u003csup\u003e[3]\u003c/sup\u003e:1. Anatomical division into the median joint, the region around the chin foramen, the mandibular angle, the mandibular branch, the condyle, and fractures of the coronoid.2. Classification of three levels of damage in accordance with Kazanjian and Converse\u0026apos;s definition of the link between the dentition and fracture line. 3.Mandibular angle fracture categorization in relation to muscle activity. 4.The degree of damage according to the F-F4 rating method for mandibular fracture severity. 5.Local condylar fracture classification. 6.The position of the fracture gap on imaging is used to modify the Dingman and Natvig classification\u003csup\u003e[1]\u003c/sup\u003e. Based on the degree of alveolar ridge atrophy, the degree of dental or periodontal trauma, and the amount of bone loss, the AOCMF divides mandibular fractures other than condylar fractures into three levels\u003csup\u003e[13]\u003c/sup\u003e. In the introduction of tension screw fixation,Prof. Zhang Yi of Peking University\u003csup\u003e[14]\u0026nbsp;\u003c/sup\u003edeveloped the classification of mandibular lamellar/oblique fractures.\u003c/p\u003e\n\u003cp\u003eMandibular linear fractures and lamellar/oblique fractures are frequently treated similarly, and this is due to the existing classification system. In a lamellar/oblique fracture of the jaw, the buccal and lingual cortices are divided, generating two thin lamellar plates on the buccal and lingual sides, and unlike in linear fractures, the lingual side is unable to relocate the fracture end directly under direct vision. Consequently, these two distinct fracture types could result in various healing processes. There are no more precise categorization or treatment guidelines for lamellar/oblique fractures, nevertheless.\u003c/p\u003e\n\u003cp\u003eThis investigation examined if the healing processes for linear and laminar/oblique mandibular fractures varied.\u003c/p\u003e"},{"header":"2.\tMaterials and methods","content":"\u003cp\u003eThe Experimental Animal Center of Xinjiang Medical University provided the experimental animals, and the study was completed in a facility that complied with all applicable national regulations. The experimental study protocol was approved by the Ethics Committee of the People\u0026apos;s Hospital of the Xinjiang Uygur Autonomous Region (KY2018060613). All animals were grouped after one week of captivity in the experimental center, and the experimental procedures complied with laws and norms pertaining to animal care.\u003c/p\u003e\n\u003cp\u003e2.1 Grouping\u003c/p\u003e\n\u003cp\u003eTwelve healthy adult Beagles (20-24 months old, average weight 8.0-12.0 kg, males and females) were split into two groups of six at random. The mandibular body in the control group was fractured in a traditional longitudinal linear fracture model, with the fracture line parallel to the bottom edge of the mandibular body. In the experimental group, a laminar/oblique fracture model of the mandibular body was established, with the fracture line measuring about 2 cm in length and forming an angle of about 25\u0026deg; to the long axis of the mandible.\u003c/p\u003e\n\u003cp\u003e2.2 Surgical establishment of animal models\u003c/p\u003e\n\u003cp\u003eThe skin beneath the left mandible of the Beagle was prepared, stained with methylene blue, and a 3.0-cm-long curved incision was made on the lower edge of the left parallel mandible. The skin mucosa and periosteum were incised with a 15-gauge surgical blade, and the periosteum was peeled along the bone surface to expose the thymus. Anesthesia was induced by mixing 0.6 mg/kg of Sulforaphane II (Jilin Huamu) with 0.75 mg/kg of Sutex (Zoletil 50, Vic, France) intramuscularly.\u003c/p\u003e\n\u003cp\u003eThe bone was chiseled downward to create a section at a 25\u0026deg;angle to the long axis of the mandible between the left mandibular body\u0026apos;s first premolar and first molar.The left mandibular body\u0026apos;s labio-buccal and lingual cortices were separated to form a lamellar shape, the medial and lateral bone plates of the mandible were gradually split downward, and the medial and lateral bone plates of the mandible were completely separated with a mandibular spreader to establish a lamellar/oblique fracture of the mandibular body.\u003c/p\u003e\n\u003cp\u003eThe linear fracture was created by cutting a gap perpendicular to the body of the mandible with a bone chisel, and the gap was moved from the buccal side of the mandible to the alveolar ridge of the lingual molar, creating a longitudinal linear fracture of the mandible. Normal occlusal-dental relationship was restored and the broken bone end was reset. Two sets of 4-hole titanium plates and screws were used to fix the fractured ends side-by-side in both groups. The periosteum and skin were sutured, and all animals were free of postoperative discomfort such as fever and infection, and the sutures were removed 1 week later. Specimens were collected at 2 weeks, 1 month, and 3 months postoperatively, respectively.\u003c/p\u003e\n\u003cp\u003e2.3 Statistical analysis\u003c/p\u003e\n\u003cp\u003eData were analyzed using SPSS software, t-test analysis was used for comparison between two groups, one-way analysis of variance (ANOVA) was used for comparison between groups, and histograms were made using GraphPad Prism5. The results were expressed as mean \u0026plusmn; standard deviation (SD), and statistical significance was set a P \u0026lt; 0.05.\u003c/p\u003e"},{"header":"3.\tResult ","content":"\u003cp\u003e\u003cstrong\u003e3.1 X-ray findings \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEvaluation of the development of bone scabs and monitoring the healing of fracture lines. Clear fracture lines and less obvious bone scab formation could be seen in both the control and experimental groups two weeks after surgery, and there were no appreciable differences between the two groups.\u003c/p\u003e\n\u003cp\u003eone month following surgery, the control group\u0026apos;s lower border of the mandible still had obvious fracture lines visible, however,\u0026nbsp;the fracture lines at the osteotomy alveolar ridge were blurred and there was some bone scab formation. The fracture ends were gradually approached, the fracture lines blurred, and there was a noticeable rise in bone scabs in the experimental group, although there were still gaps at the fracture ends.\u003c/p\u003e\n\u003cp\u003eThe distance between the fracture ends of both groups was lower than before three months following surgery. In the experimental group, the fracture gap was essentially healed and the bone scab was more substantial than in the control group, which had a blurred fracture gap and no discernible fracture line at the lower edge of the jaw.( Figure 1)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Histological observations \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe amount of new bone, trabecular formation, size and shape of the osteocytes in the bone traps, and the direction of collagen fiber alignment were observed. Masson staining, which stains the collagen fibers blue to show the maturation process of newly formed bone while staining red the mineralized bone, was used to detect the amount of new bone\u003csup\u003e[15]\u003c/sup\u003e.By utilizing Image Pro Plus to calculate the quantity of osteocytes and the proportion of collagen fibers to the surface area of the whole tissue (Collagen Volume Fraction (CVF)), bone mineralization formation was evaluated semi-quantitatively\u003csup\u003e[16]\u003c/sup\u003e.The analysis included microscopic images at a magnification of 100x, Masson staining with high color resolution, the same HSI mode for all images, representation of the blue-stained collagen fiber regions, calculation of the CVF, and statistical analysis of all results. The same microscopic 100x pictures were chosen for HE-stained slices in order to manually count the osteocytes. (Figure 2)\u003c/p\u003e\n\u003cp\u003eAt two weeks after surgery, both control and experimental groups showed blue-red stained areas in Masson staining, with more proliferated blue collagen fibrous tissue. The mean collagen fibrous area ratio was 0.2820\u0026plusmn;093 in the control group and 0.2370\u0026plusmn;039 in the experimental group, with the irregular interwoven arrangement of fibers. Osteoblasts were also visible in the fibrous tissue, scattered throughout the bone traps. HE In both groups, staining revealed dense connective tissue and osteoblasts on the surface of the bone trabeculae; osteocytes, new capillaries, and Haver\u0026apos;s system bone were uniformly distributed within the trabeculae, and the trabeculae were grouped irregularly.\u003c/p\u003e\n\u003cp\u003eAt one month following surgery, Masson staining revealed more new bone in the experimental group compared to the control group, with collagen fibers arranged in a parallel pattern, and more fibrous tissue in the control group compared to the experimental group, albeit with an uneven pattern. In the experimental group, the mean collagen fiber area ratio was 0.1390\u0026plusmn;019, compared to 0.2000\u0026plusmn;074 in the control group. Osteocytes in bone traps were visible within the trabeculae, and the osteocytes in the experimental group had smaller cytostomes with a flat oval shape compared to the control group. Bone trabeculae were more regularly arranged in HE staining than at 2 weeks, connective tissue was reduced than before, and osteocytes were visible within the trabeculae.\u003c/p\u003e\n\u003cp\u003eIn Masson staining at 3 months after surgery, the experimental group had collagen fibers arranged parallel to one another, with obvious bone scab formation and woven bone forming lamellar bone, whereas the control group still had more fibrous tissue, primarily woven bone. The mean collagen fiber area ratio was 0.4290\u0026plusmn;006 in the control group and 0.1140\u0026plusmn;090 in the experimental group. There was no connective tissue visible and the bone trabeculae were properly structured. They also contained numerous osteocytes and osteoblasts. In both groups, the level of bone scab reconstruction was comparable(Figure3,Figure4).\u003c/p\u003e\n\u003cp\u003eAt 2 weeks and 1 month postoperatively, there was no statistically significant difference between the two groups when comparing the CVF data, however at 3 months postoperatively, there was a statistically significant difference between the control and experimental groups (P\u0026lt;0.05)(Figure5).With passing time, there was no statistically significant difference in CVF between the two groups (Figure6).\u003c/p\u003e\n\u003cp\u003eOsteocyte counts in the control and experimental groups were 137.5\u0026plusmn;23.3, 121.5\u0026plusmn;31.8, 88.0\u0026plusmn;2.8, 108.0\u0026plusmn;1.4, 44.5\u0026plusmn;17.6, and 37.5\u0026plusmn;16.2, respectively, after 2 weeks, 1 month, and 3 months following surgery(Figure7) .Statistical analysis of osteocyte counts showed a statistical difference between the two groups at 1 month postoperatively (P\u0026lt; 0.05). When compared longitudinally, osteocytes in the control group were statistically different (P\u0026lt; 0.05) when compared at 2 weeks and 3 months (Figure8).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Bone mineral density results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess bone scab growth and determine whether there was a difference in bone mineral content between the two groups, BMD was measured using dual-energy X-ray absorptiometry and coupled with imaging.The mean BMD of the experimental group two weeks after surgery was 0.898\u0026plusmn;0.231 and that of the control group was 0.532\u0026plusmn;0.182, no statistically significant difference was seen between the two groups (P=0.221). The mean BMD of the experimental group at 1 month postoperatively was 0.765\u0026plusmn;0.076 and that of the control group was 0.142\u0026plusmn;0.024, which was statistically different between the two groups (P\u0026lt;0.05). The mean BMD at 3 months postoperatively was 0.444\u0026plusmn;0.086 in the experimental group and 0.250\u0026plusmn;0.074 in the control group, with no statistically significant difference between the two groups (P=0.139)(Figure9).BMD values gradually increased with the extension of the fracture healing period in both groups, and no statistical difference was observed(Figure10). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Biomechanical measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe fracture energy, stiffness, and maximum load of fracture specimens were assessed using a four-point bending experiment. The experimental group\u0026apos;s mean fracture energy measures at two weeks, one month, and three months postoperatively were 61.6\u0026plusmn;4.0,89.0\u0026plusmn;3.4, and125.3\u0026plusmn;6.8,while the control group\u0026apos;s mean values were 57.0\u0026plusmn;3.0,77.8\u0026plusmn;2.5,and110.4\u0026plusmn;6.1,respectively.The two groups were statistically different at one month postoperatively (P\u0026lt;0.05), at which point the fracture energy of the experimental group was higher than that of the control group. Stiffness measurements were not statistically different between the two groups at two weeks and one month postoperatively, and the stiffness of the experimental group was greater than that of the control group at three months postoperatively (P\u0026lt;0.05). Maximum load measurements were not statistically different at two weeks postoperatively and were higher in the experimental group than in the control group at 1 month and 3 months postoperatively (P\u0026lt;0.05) (Figure11). The fracture energy, stiffness and maximum load were significantly higher and the data were statistically different in both groups as the fracture healing time increased. (Figure12)\u003c/p\u003e"},{"header":"4.\tDiscussions","content":"\u003cp\u003eWhen comparing the two groups laterally, it could be seen that there were no significant differences between the two groups during the pre-healing period. At 1 month postoperatively, the osteocytes, bone density, fracture energy, and maximum load were greater in the experimental group than in the control group, and at 3 mon boths mandibular body lamellar/oblique fractures and linear fractures achieved good healing.maximum volume proportion of collagen fibers The experimental group exhibited higher bone maturity and better mechanical characteristics, as evidenced by the CVF of the experimental group being\u0026nbsp;lower than that of the control group and the maximum load of the experimental group being higher than that of the control group. The fracture line gradually blurred, the osteoblasts gradually decreased, the bone matrix gradually increased, and the bone trabeculae were regularly and orderly arranged in parallel when compared longitudinally within groups. Additionally, both groups\u0026apos; bone density and mechanical properties gradually increased over time.\u003c/p\u003e\n\u003cp\u003eAt the shattered ends of the bone, more collagen fibers can be seen in the early stages of fracture healing. As healing time advances, however, there is a steady decrease in collagen fibers and an increase in bone tissue. Bone scab size, bone density, and biomechanical qualities are all directly related to fibrous tissue area and bone tissue area, which are important stage indicators of fracture healing\u003csup\u003e[17]\u003c/sup\u003e.Cell differentiation and fracture healing may be impacted by changes in the mechanical environment of the fracture end due to the fracture inclination\u003csup\u003e[18]\u003c/sup\u003e. Ramasamy\u003csup\u003e[19]\u003c/sup\u003e discovered that the mechanical function of bone in mice is significantly influenced by the orientation of collagen fibers in the anatomical loading axis, with longitudinal fibers forming a tissue with better tensile properties and maximum strain. The lamellar/oblique group in our trials had a fracture inclination angle of 25\u0026deg;, and the outcomes demonstrated superior mechanical qualities to the linear group, which is also consistent with the Miramini et al. study.\u003c/p\u003e\n\u003cp\u003eWhen the mandibular fracture is correctly and steadily repositioned anatomically, studies have shown improved bone healing and fewer complications\u003csup\u003e\u0026nbsp;[20\u0026ndash;21]\u003c/sup\u003e. Restoring occlusion and ensuring bone healing have long been the main objectives of jaw fracture treatment\u0026nbsp;\u003csup\u003e[22]\u003c/sup\u003e. Both groups of animals in this experiment received care in accordance with the OR/IF standardized fracture treatment approach\u003csup\u003e\u0026nbsp;[23]\u003c/sup\u003e. The gap at the fracture break after fixation may be larger than in the linear fracture group because the laminar/oblique group had trouble resetting the lingual bone portion under direct view.According to Tetsuo\u003csup\u003e[24]\u003c/sup\u003e experiments on sheep with metatarsal fractures, significant micromotion (0.7 mm) results in an increase in the amount of new bone growth when the fracture gap is small (2 mm), but on the other hand, when the fracture gap is large (6 mm), the micromotion magnitude can result in a delay in the healing process. The most suitable micromovement at the fracture end is currently thought to be a 0.2 to 1.0 mm axial movement\u003csup\u003e[25]\u003c/sup\u003e. This implies that the right kind of intermittent motion between fractures will encourage the development of bone scabs and hasten the healing of fractures.\u003c/p\u003e\n\u003cp\u003eA four-point bending test, which involves applying two downward forces on the skeletal scaffold equidistant from the center of the bone, was used in this experiment. This test is crucial for determining the final fracture repair because it allows a more uniform propagation of the bending moment between the two points where the force is applied\u003csup\u003e[17]\u003c/sup\u003e. The strains caused by weight-bearing activity or muscle contraction at the fracture site cause interfragmentary movement (IFM), which alters the size of the initial fracture gap and causes interfragmentary strain \u0026epsilon;\u003csup\u003e[26]\u003c/sup\u003e.Moderate-sized IFM can promote bone healing.Axial motion, as opposed to the\u0026nbsp;translational and torsional shear motion, which slows bone healing, is found by some researchers to produce better healing in mechanical simulations and finite element analysis\u003csup\u003e[27]\u003c/sup\u003e.The maximum mineralized bone crust area, four-point bending experimental stiffness, and mineralized tissue density, on the other hand, were higher in the torsional shear group than in the axial group in Bishop\u0026apos;s experiments in a sheep tibial fracture model, suggesting that interfragmentary shear motion does not necessarily delay bone healing but instead stimulates bone crust formation and promotes fracture healing\u003csup\u003e[28]\u003c/sup\u003e.Our research also supports the notion that shear does not always prevent bone healing because the stiffness and maximum load of the lamina/bevel group were higher in the late phase of fracture healing than those of the linear group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBMD can react to the development of a bone scab and changes with the speed and depth of healing. The mandible shares several characteristics with long bones, and BMD of long bones is frequently used to evaluate changes in the balance between osteoblast bone creation and osteoclast bone resorption\u003csup\u003e[29]\u003c/sup\u003e. A number of writers have employed BMD measurements obtained from DXA in animal studies recently to quantify fracture healing and statistically examine healing variations\u003csup\u003e[30-32]\u003c/sup\u003e.Our study demonstrates that at the middle stage of fracture union, the experimental group\u0026apos;s BMD was higher than the control group\u0026apos;s, and at the same time, the experimental group\u0026apos;s callus hyperplasia was visible on X-rays. This means that the control group\u0026apos;s bone maturity was lower than the experimental group\u0026apos;s at this stage of fracture union.\u003c/p\u003e\n\u003cp\u003eOsteocytes are the primary cells of bone metabolism, and bone reconstruction involves more than just the interaction of osteoblasts and osteoclasts\u003csup\u003e[33]\u003c/sup\u003e. Through a network of luminal tubules, osteocyte protrusions come into contact with the extracellular matrix and are responsive to a variety of mechanical and chemical stimuli\u003csup\u003e[34]\u003c/sup\u003e.In order to influence bone reconstruction, osteoclasts can transform mechanical shear stresses into biochemical signals that can be communicated with by other cells. Osteocytes trigger the production of growth factors, extracellular matrix synthesis, and the stimulation of mesenchymal cells to develop into osteoblasts in response to mechanical forces\u003csup\u003e[35]\u003c/sup\u003e.In our findings, we demonstrated that during mid-fracture healing, the lamellar/oblique group had more osteocytes, fracture energy, maximum loading force, and BMD than the linear group. This suggests that mechanical shear stress may have encouraged osteoblast osteogenesis in the lamellar/oblique fracture group, yielding a result resembling mandibular distraction osteogenesis. Schwarz\u003csup\u003e[36]\u003c/sup\u003edemonstrated that compared to resorbed fixed fractures, the fracture load of mandibular distraction osteogenesis was much higher, and the development of high-density bone mineralization was increased in the distraction osteogenesis regeneration group.\u003c/p\u003e\n\u003cp\u003eThis research has some restrictions: 1) The fracture line in the animal experimental model was artificially osteotomized, which makes it impossible to accurately simulate the real fracture environment. This will have an impact on the experiment\u0026apos;s accuracy, and future mechanical loading experiments should be improved to simulate a fracture subjected to mechanical impact in a more realistic manner. 2) Because of the sample\u0026apos;s small size and potential for error.\u003c/p\u003e\n\u003cp\u003eIn conclusion, we created models of lamellar/oblique and linear fractures of the mandible in Beagle dogs, assessed and compared the cellular, bone density, and biomechanical functions during the healing process of these two fracture types, both of which were converted into the new bone through osteoblast-osteoclast conversion, and observed faster bone scab formation and higher mechanical properties, such as BMD and stiffness of the bone, in lamellar/oblique fractures. This study offers evidence in favor of improving the kinds and management of mandibular fractures.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe experimental study protocol was approved by the Ethics Committee of the People\u0026apos;s Hospital of the Xinjiang Uygur Autonomous Region (KY2018060613).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe experimental procedures complied with \u0026nbsp;relevant guidelines and regulations pertaining to animal care.\u003c/p\u003e\n\u003cp\u003eAll methods are reported in accordance with\u0026nbsp;ARRIVE guidelines\u0026nbsp;for the reporting of animal experiments.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of Xinjiang [Grant Number: 2018D01C099].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthors\u0026rsquo;Contributions\u003c/p\u003e\n\u003cp\u003eQian Xu designed the study, Qian Xu and Tingting Xin analyzed the data, prepared the figures, and wrote the original draft. Jun Li critically reviewed the manuscript. All Authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors owe a debt of gratitude to Xinjiang Medical University Animal Experimental Research Center due to their support and intensive effort to our project.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEscott EJ, Branstetter BF. Incidence and characterization of unifocal mandible fractures on CT. AJNR Am J Neuroradiol. 2008. 29(5): 890-4.\u003c/li\u003e\n\u003cli\u003eAhmed A, Wu E, Sarai R, Williams R, Breeze J. Potentially modifiable patient factors in mandible fracture complications: a systematic review and meta-analysis. Br J Oral Maxillofac Surg. 2022. 60(3): 266-270.\u003c/li\u003e\n\u003cli\u003eNaeem A, Gemal H, Reed D. Imaging in traumatic mandibular fractures. Quant Imaging Med Surg. 2017. 7(4): 469-479.\u003c/li\u003e\n\u003cli\u003eOdom EB, Snyder-Warwick AK. Mandible Fracture Complications and Infection: The Influence of Demographics and Modifiable Factors. Plast Reconstr Surg. 2016. 138(2): 282e-289e.\u003c/li\u003e\n\u003cli\u003ePickrell BB, Hollier LH Jr. Evidence-Based Medicine: Mandible Fractures. Plast Reconstr Surg. 2017. 140(1): 192e-200e.\u003c/li\u003e\n\u003cli\u003eMunante-Cardenas JL, Facchina Nunes PH, Passeri LA. Etiology, treatment, and complications of mandibular fractures. J Craniofac Surg. 2015. 26(3): 611-5.\u003c/li\u003e\n\u003cli\u003eKolar P, Schmidt-Bleek K, Schell H, et al. The early fracture hematoma and its potential role in fracture healing. Tissue Eng Part B Rev. 2010. 16(4): 427-34.\u003c/li\u003e\n\u003cli\u003eLeucht P, Kim JB, Amasha R, James AW, Girod S, Helms JA. Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration. Development. 2008. 135(17): 2845-54.\u003c/li\u003e\n\u003cli\u003eWong SA, Hu DP, Slocum J, et al. Chondrocyte-to-osteoblast transformation in mandibular fracture repair. J Orthop Res. 2021. 39(8): 1622-1632.\u003c/li\u003e\n\u003cli\u003eCheng C, Shoback D. Mechanisms Underlying Normal Fracture Healing and Risk Factors for Delayed Healing. Curr Osteoporos Rep. 2019. 17(1): 36-47.\u003c/li\u003e\n\u003cli\u003ePapachristou DJ, Georgopoulos S, Giannoudis PV, Panagiotopoulos E. Insights into the Cellular and Molecular Mechanisms That Govern the Fracture-Healing Process: A Narrative Review. J Clin Med. 2021. 10(16).\u003c/li\u003e\n\u003cli\u003eStigler RG, Becker K, Kloss FR, Gassner R, Lepperdinger G. Long-lived murine osteocytes are embodied by craniofacial skeleton in young and old animals whereas they decrease in number in postcranial skeletons at older ages. Gerodontology. 2018. 35(4): 391-397.\u003c/li\u003e\n\u003cli\u003eCornelius CP, Audig\u0026eacute; L, Kunz C, et al. The Comprehensive AOCMF Classification System: Mandible Fractures-Level 3 Tutorial. Craniomaxillofac Trauma Reconstr. 2014. 7(Suppl 1): S031-43.\u003c/li\u003e\n\u003cli\u003eZhang Yi.Discussion on misdiagnosis and difficulties in the treatment of jaw fracture.Chinese Journal of Stomatology.2004. (01): 24-26.\u003c/li\u003e\n\u003cli\u003eLi Y, Su J, Sun W, Cai L, Deng Z. AMP-activated protein kinase stimulates osteoblast differentiation and mineralization through autophagy induction. Int J Mol Med. 2018. 41(5): 2535-2544.\u003c/li\u003e\n\u003cli\u003eHorai Y, Kakimoto T, Takemoto K, Tanaka M. Quantitative analysis of histopathological findings using image processing software. J Toxicol Pathol. 2017. 30(4): 351-358.\u003c/li\u003e\n\u003cli\u003eKnox AM, McGuire AC, Natoli RM, Kacena MA, Collier CD. Methodology, selection, and integration of fracture healing assessments in mice. J Orthop Res. 2021. 39(11): 2295-2309.\u003c/li\u003e\n\u003cli\u003eMiramini S, Zhang L, Richardson M, Mendis P, Ebeling PR. Influence of fracture geometry on bone healing under locking plate fixations: A comparison between oblique and transverse tibial fractures. Med Eng Phys. 2016. 38(10): 1100-8.\u003c/li\u003e\n\u003cli\u003eRamasamy JG, Akkus O. Local variations in the micromechanical properties of mouse femur: the involvement of collagen fiber orientation and mineralization. J Biomech. 2007. 40(4): 910-8.\u003c/li\u003e\n\u003cli\u003eHisting T, Heerschop K, Klein M, et al. Characterization of the healing process in non-stabilized and stabilized femur fractures in mice. Arch Orthop Trauma Surg. 2016. 136(2): 203-11.\u003c/li\u003e\n\u003cli\u003eBatbayar EO, Malwand S, Dijkstra PU, Bos R, van Minnen B. Accuracy and outcome of mandibular fracture reduction without and with an aid of a repositioning forceps. Oral Maxillofac Surg. 2019. 23(2): 201-208.\u003c/li\u003e\n\u003cli\u003ePham Dang N, Barth\u0026eacute;l\u0026eacute;my I, Bekara F. From rigid bone plate fixation to stable dynamic osteosynthesis in mandibular and craniomaxillo-facial surgery: Historical evolution of concepts and technical developments. J Stomatol Oral Maxillofac Surg. 2019. 120(3): 229-233.\u003c/li\u003e\n\u003cli\u003eEl-Anwar MW, Sayed El-Ahl MA, Amer HS. Open Reduction and Internal Fixation of Mandibular Fracture without Rigid Maxillomandibular Fixation. Int Arch Otorhinolaryngol. 2015. 19(4): 314-8.\u003c/li\u003e\n\u003cli\u003eYamaji T, Ando K, Wolf S, Augat P, Claes L. The effect of micromovement on callus formation. J Orthop Sci. 2001. 6(6): 571-5.\u003c/li\u003e\n\u003cli\u003eShi J, Xiao Y, Wu M, Guan J. [Research on the nature of micromovement and the biomechanical staging of fracture healing]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2021. 35(9): 1205-1211.\u003c/li\u003e\n\u003cli\u003eAugat P, Hollensteiner M, von R\u0026uuml;den C. The role of mechanical stimulation in the enhancement of bone healing. Injury. 2021. 52 Suppl 2: S78-S83.\u003c/li\u003e\n\u003cli\u003eSteiner M, Claes L, Ignatius A, Simon U, Wehner T. Disadvantages of interfragmentary shear on fracture healing--mechanical insights through numerical simulation. J Orthop Res. 2014. 32(7): 865-72.\u003c/li\u003e\n\u003cli\u003eBishop NE, van Rhijn M, Tami I, Corveleijn R, Schneider E, Ito K. Shear does not necessarily inhibit bone healing. Clin Orthop Relat Res. 2006. 443: 307-14.\u003c/li\u003e\n\u003cli\u003eLucisano MP, Nelson-Filho P, Morse L, et al. Radiodensitometric and DXA analyses for the measurement of bone mineral density after systemic alendronate therapy. Braz Oral Res. 2013. 27(3): 252-7.\u003c/li\u003e\n\u003cli\u003eLiu QH, Liao LM, Wu H, Lin YP, Yu S. PTH promotes rabbit tibial fracture healing via the Notch signaling pathway. Eur Rev Med Pharmacol Sci. 2020. 24(4): 1616-1623.\u003c/li\u003e\n\u003cli\u003eDeng J, Wu J, Zhu Y. Inhibition of MicroRNA-9 Improves Fracture Healing by Modulating the Bone Morphogenetic Protein-7 Pathway. Pharmacology. 2019. 104(5-6): 352-358.\u003c/li\u003e\n\u003cli\u003eGirard N, Cauvin E, Gauthier O, Gault S. Biphasic Calcium Phosphate Microparticles Mixed With Autologous Blood: Application for the Reconstruction of a Large Mandibular Bone Defect in a Dog. J Vet Dent. 2020. 37(4): 201-209.\u003c/li\u003e\n\u003cli\u003eRupp M, Merboth F, Daghma DE, Biehl C, El Khassawna T, Hei\u0026szlig; C. Osteocytes. Z Orthop Unfall. 2019. 157(2): 154-163.\u003c/li\u003e\n\u003cli\u003eCamal Ruggieri IN, C\u0026iacute;cero AM, Issa J, Feldman S. Bone fracture healing: perspectives according to molecular basis. J Bone Miner Metab. 2021. 39(3): 311-331.\u003c/li\u003e\n\u003cli\u003eSchwarz DA, Arman KG, Kakwan MS, Jamali AM, Elmeligy AA, Buchman SR. Regenerate healing outcomes in unilateral mandibular distraction osteogenesis using quantitative histomorphometry. Plast Reconstr Surg. 2010. 126(3): 795-805.\u003c/li\u003e\n\u003cli\u003eSchwarz DA, Arman KG, Kakwan MS, Jamali AM, Buchman SR. Analysis of the biomechanical properties of the mandible after unilateral distraction osteogenesis. Plast Reconstr Surg. 2010. 126(2): 533-542.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Mandibular fractures, Lamellar/oblique fractures, Histology, Bone mineral density, Biomechanics","lastPublishedDoi":"10.21203/rs.3.rs-2556512/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2556512/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eObjective\u003c/em\u003e: The goal of this study was to determine whether there were any differences in the healing processes for linear fractures and lamellar/oblique mandibular fractures.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMaterials and methods\u003c/em\u003e: Using a random number generator, two groups of six beagle dogs each were created from a group of twelve.The experimental group was the lamellar/oblique fracture of the mandible, while the control group was the traditional linear fracture. The first premolar and the first molar were where the fracture occurred. Two 4-hole bone plates were used to repair the fracture line.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eResults\u003c/em\u003e: X-ray:the experimental group's fracture space was fuzzier and eventually disappeared between the two groups. Histology: the Collagen Volume Fraction in the experimental group was lower than that in the control group three months after surgery. The experimental group had more bone cells than the control group did one month following the surgery.Both groups' trabecular arrangements became more regular as the healing process progressed.Bone mineral density: the experimental group's bone mineral density was higher than the control group's one month following surgery. Four-point bending test:the experimental group's fracture energy was higher than the control group's one month following the surgery. The experimental group's stiffness was substantially greater than the control group's three months after the procedure. The maximal loading of the experimental group was higher than that of the control group at one and three months following the operation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConclusion\u003c/em\u003e: In comparison to traditional linear fractures, lamellar/oblique fractures of the mandible have higher bone mineral density, are more rigid, and heal more quickly.\u003c/p\u003e","manuscriptTitle":"Histological and bone mineral density analyses of the mandibular lamellar/oblique fracture healing in beagles.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-02-14 16:00:21","doi":"10.21203/rs.3.rs-2556512/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"44d1b9e0-f9a8-4bcd-b008-cfbd2ee3892d","owner":[],"postedDate":"February 14th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-24T07:34:31+00:00","versionOfRecord":[],"versionCreatedAt":"2023-02-14 16:00:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2556512","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2556512","identity":"rs-2556512","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}