Wound healing and revascularization

Wound healing and revascularization

Wound healing and revascularization A histologic observation of experimental tooth root fracture Hangqing Jin, DDS, PhD, a Huw F. Thomas, BDS, MS, PhD...

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Wound healing and revascularization A histologic observation of experimental tooth root fracture Hangqing Jin, DDS, PhD, a Huw F. Thomas, BDS, MS, PhD, b and Jinkun Chen, DMD, MS, PhD, c San Antonio, Tex. DENTAL SCHOOL, THE UNIVERSITY OF TEXAS HEALTH SCIENCE CENTER AT SAN ANTONIO

We used dogs as an animal model to generate tooth root fracture and to observe the wound-healing process of the fracture. I-tistologic examination of the specimens revealed that the early reaction of the wound healing was infiltration of inflammatory cells particularly at the coronal part of the fracture, whereas less inflammation but more abundant collagen fibers were seen at the apical part of the fracture (15 and 30 days). Inflammation lasted for more than 90 days and then subsided. At day 180, bone tissue healing was observed. Revascularization of the pulp tissues reached a high level at the same stage that bone healing took place. Our data suggest that in tooth root fracture, the regeneration of blood vessels is important in the wound-healing process and the revascularization is synchronized with the fracture wound healing. In this animal model the complete hard tissue healing could take as long as 6 months. (ORALSURGORAL MED ORAL PATHOLORAL RADIOLENDOD 1996;81:26-30)

Tooth root fracture is a common dental disorder. In the permanent dentition horizontal root fracture has been reported to comprise from 0.2% to 7% of all traumatic injuries to teeth surveyed in different populations.i, 2 Root fracture can be caused by trauma after blows to teeth or jaw or other injuries from automobile accidents, contact sports, malocclusion, or other pathologic factors. From their clinical radiographic observation of 50 cases of tooth root fracture, Andreasen and Hjorting-Hansen 3 reported three different healing patterns. The fractured tooth roots are healed by interposition of (1) hard tissue, (2) connective tissue, or (3) granulation tissue. The type of healing of a fractured tooth root is dependent on the amount of damage to the coronal pulp. In the case that pulpal damage is minor and the pulp tissue remains vital, hard tissue healing in the fracture site usually takes place. Whereas more greatly damaged pulp results in connective tissue healing. Granulation tissue healing occurs in the cases where severe damage and necrosis are seen in the pulpal tissue. 4 The clinical parameters relating to root fracture healing are luxation of the coronal fragment and separation of fragments before and after repositioning. Root healing may also be related to the age of the patient at the time of injury, stage of root formation, and diameter of the apical foramen. Moreover, marginal periodontal disapostdoctoral Fellow. bprofessor and Chairman. CAssistant Professor. Department of Pediatric Dentistry. Received for publication Apr. 14, 1995; returned for revision June 14, 1995; accepted for publication Aug. 28, 1995. Copyright 9 1996 by Mosby-Year Book, Inc. 1079-2104/96/$5.00 + 0 7/12/68934

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ease and orthodontic band fixation also affect the consequence of root fracture. 5-1~ In these previous investigations, however, tooth root fractures have been studied by limited means such as clinical or posttreatment observations often with radiophotography or simply case reports with demographic analysis. Only a few reports in the literature deal with studies of experimental tooth root fracturell, 12 in which certain questions can be addressed in depth. In the present work we used an animal model to study healing patterns of fractured tooth roots. Clinically, in some cases only simple tooth root fracture occurs, whereas in others the fracture is combined with alveolar bone fracture and even torn gingiva and periosteum) 3 In the present study our approach was more similar to the latter condition. After generating fractures in dog tooth roots we investigated the wound-healing process histopathologically. We found that cells in adjacent tissues are involved in the hard tissue formation in the fracture sites, regeneration of blood vessels in pulp is correlated with the fracture-healing process, and the complete healing of a fractured tooth root could take as long as 6 months in this model.

MATERIAL AND METHODS In this study we used 18 dogs of both sexes that weighed 22 to 30 pounds each. General anesthesia was given by intraperitoneal injection of pentobarbital (15 mg per pound of body weight) and unilateral surgical operation was performed on the left side of the mandibles. The operative area was shaved and 2% tincture of iodine and 75% ethanol was applied. A submandibular incision was made to expose the masseter and medial pterygoid muscles. A sharp incision

ORAL SURGERY ORAL MEDICINEORAL PATHOLOGY Volume 81, Number 1 was then made on the inferior border of mandibular body and the masseter and medial pterygoid muscles reflected from the surface of mandibular body. Two holes were drilled and a tread saw was pulled over through the holes. The teeth at the left lower jaw and their surrounding bone tissues were transversally cut through with the saw. Copious irrigation was used at the time of operation. The artificial fracture gap was about 1 mm and sited at the level of root middle (Fig. 1). Closure was accomplished by suturing with 3-0 silk thread. These 18 dogs were then randomly divided into six groups; three dogs in each group were killed on day 7, 15, 30, 60, 90, and 180 after surgery. Block samples of fractured teeth as well as adjacent bone tissues were dissected and fixed in 10% neutral buffered formalin solution for 2 days. After fixation the tissues were demineralized and embedded in paraffin. Consecutive sections 6 ]am thick were cut longitudinally with a microtome, and representative sections stained with hemotoxylin-eosin were examined with a light microscope. Microphotographs focused on the tooth fracture areas were taken with a microscopic camera (Zeiss GMBH, Jena Germany).

RESULTS At day 7 after surgery microscopic examination of root fracture specimens revealed a large number of neutrophilic leukocytes at the root fracture site and in the adjacent bone tissue (Fig. 2). Fibroblasts in the periodontal ligament started migrating toward the fracture space. The coronal segment of pulp also demonstrated substantial inflammatory reaction with enlarged and congestive capillaries, tissue swelling, and a heavy infiltration of polymorphonucleated neutrophils. Odontoblasts, however, showed essentially normal morphologic conditions, and the odontoblastic processes in the dentinal tubules were also intact. On day 15, more abundant collagen fibers were seen at the apical part of the root fracture. At the coronal part of the root fracture, on the contrary, there were still a large number of inflammatory cells but few and sparse collagen fibers. Pulp tissue showed the same features as on day 7. On day 30, the trabecular bone was prevailing at the apical part of the root fracture. Inflammatory cells and sparse collagen fibers were still seen at the coronal half of the fracture sites (Fig. 3). Bone tissue was first seen at the sites adjacent to the bone fracture and then toward the tooth root fracture sites. These delicate bone trabecula that appear at the apical side of the fracture were lined by osteoblastic cells possibly derived from the adjacent fractured bone or periodontal

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Fig. 1. Schematic diagram shows surgical incision to generate tooth root fractures in dog mandibles. Teeth at left lower jaw and surrounding bone tissues are transversally cut through. Artificial fracture gap is about 1 mm and sited at the level of the root middle. (M, mandible; R, tooth root; S, surgical incision line; C, mandibular canine.) ligament tissue (Fig. 4). No change was observed in the pulp tissue. On day 60, there was no evidence of the inflammatory reaction subsiding at the root fracture site. A few normal fibroblastic cells, however, appeared in the coronal pulpal chamber in the specimens on day 90. The newly formed woven bone at the fractured site underwent remodeling and oriented in a trabecular fashion. On day 180, the root fracture gap was filled with the bone tissue (data not shown). It was of interest to note that the new bone at the apical part of the fracture site was more mature than its counterpart at the coronal part where bone Structure was relatively loose and poorly organized. Importantly, the coronal pulpal tissue demonstrated the inflammation subsiding with recovery of pulp cell population and normal blood vessel distribution. Only at this point was the woundhealing completed. The adjacent fractured bone site was also filled with mature lamellar bone with haversian system. DISCUSSION In the present investigation we used dogs as the animal model to study the wound-healing process of tooth root fracture. Eighteen dogs were used in this study to generate artificial tooth root fracture. The tissues were harvested at different times after surgery and processed for histologic analysis. We believed that this experimental model would provide insights into understanding the cellular reaction and tissue regeneration in tooth root fracture; these can then be related to clinic symptoms, treatment, and prognosis. In all experimental specimens, the first tissue reaction for root fracture was inflammation that occurred both in the injured pulp and at the fracture line. Microscopically, the inflammatory reaction is charac-

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Fig. 2. Microphotograph of root fracture specimen at day 7 after surgery. Large number of neutrophilic leukocytes at root fracture site and in adjacent bone tissue is seen. (A, apical part of the root.) (Hematoxylineosin stain; original magnification x70.)

Fig. 3. Specimen on day 30 after surgery. Bone tissue starts forming at apical portion of fracture gap. Inflammatory reaction is still present at the coronal part of the fracture. (A, apical part of the root; C, coronal part of the root.) (Hematoxylin-eosin stain; original magnification x35.) terized by infiltration of neutrophilic leukocytes, interstitial swelling, and congestion of blood vessels. The extent of inflammatory infiltration seemed to be in reverse proportion to the amount of new bone formed in the wound site. This is probably a result of the fact that inflammatory elements released from the inflammatory cells inhibit the tissue regeneration.

This finding is analogous with our previous studies of autogenous bone graft in dog mandibles. In these studies we surgically moved a bone block from the left site to the right site of the dog mandibles. On day 15 after transplantation, there were a large number of inflammatory cells near the grafted bone. However, collagen fibers were initially seen only in the area

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near the recipient site. From day 30 to 180 as the inflammatory reaction was subsiding, the grafted bone was gradually resorpted and bone-forming cells migrating from adjacent area grew into the resorptive space and eventually the newly formed bone replaced the whole graft. In these studies we found that as long as the inflammation existed the bone regeneration and wound healing could not be completely accomplished. 14-t6 Similarly in the present study we showed that from day 15 to day 90 collagen fibers were generally formed in the apical segment of a fractured tooth and intensive inflammatory infiltration was seen in the coronal part of the fracture. As soon as the inflammatory debris cleared at the fracture site (day 180), bone tissue formed to fill the fracture space. In this study inflammatory reaction proved to be important in the healing of tooth root fracture; the identification of the inflammatory elements, such as cytokines, involved in this process is therefore of interest. In this study we also observed the process of the revascularization in the remaining pulp tissue and the correlation between the establishment of new blood vessels and wound healing in the root fracture. The pulp artery coming from the apical foramen passes through the tiny root canal and forms a dense clump adjacent to the odontoblasts. The small vein after capillaries drains the blood to the pulp vein that also goes out from the apical foramen. Thus the damage of the fragile pulp vessels at the fracture site will actually cut the blood supply to the coronal pulp but will probably not obstruct or affect the blood supply to the apical segment. Clinically, the chance of hard tissue healing in tooth root fracture was significantly increased by decreasing the luxation of the coronal fragment. 5 In our experimental model the root fracture was produced in such a way that the luxation of the tooth crown was minimal and the fractured fragments were almost in alignment with each other. The hard tissue healing achieved in this model might be attributed to this favorable positioning. In our animal model the apical portion of the pulp apparently remained vital; this might contribute to a more rapid repair at the apical portion of the fracture than the coronal portion. We also observed a reestablishment of blood supply in the coronal pulp chamber after the severe inflammatory reaction withdrew. It was interesting to note that the revascularization of the pulp tissues reached even higher level at the 180 days when bony healing at the fracture site took place. Thus the revascularization is synchronized with the fracture wound healing and can serve as an indicator for the fracture-repairing process. In our model, because the root pulp tissue was transversely cleaved,

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Fig. 4. Adjacent bone tissues are healed, and trabecular bone in the fracture site is derived from the periodontal bone tissue. (A, apical part of the root; C, coronal part of the root.) (Hematoxylin-eosin stain; original magnification x35.) the blood vessel elements that enter the coronal pulp seemed to be derived from the periodontal ligament. Other studies showed that the root fracture was healed with dentin, cementum, or cementoid tissues in extracted human teeth. 3 The present study revealed that in almost all the experimental samples the fracture sites were filled with bone tissue. The newly formed bone showed characteristics of osteoid tissue and subsequently exhibited woven structure. At day 180 mature bone with haversian structure predominated the fracture space. Although several cementum particles and islands were seen in endosteal space of bone tissue they were not conspicuous. In our animal model, the periodontal and surrounding bone tissues were incised simultaneously together with the tooth root and thus the osteoprogenitor cells at the bone fracture site could be stimulated and subsequently entered the fracture site forming new bone tissue.

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January 1996 Recent studies both in vitro and in vivo 17' 18 also demonstrated that fibroblasts in rat periodontal ligament have the potential to differentiate into osteoblasts forming bone tissue. The difference between the types of bridging tissue that heal the root fractures may also reflect the different time of observation and tissue sample collection. We cannot rule out the possibility that the conflict m a y also represent patterns of root-fracture healing in different species. Wound healing of a fractured tooth root is a relatively long process. I t may also be related to the age of the patient at the time of injury, stage of root formation, and diameter of the apical foramen. Some authors reported that hard tissue healing could be diagnosed approximately 6 weeks after trauma. 5 It was reported that hard tissue healing in a 15-year-old b o y ' s right central incisor with nondisplaced coronal fragment took only 4 weeks. 5 However, the same authors reported another case of a 20-year-old man with root fracture of incisor that showed radiolucency 2 years after injury and became radiopaque 5 years after injury in the follow-up study. 5 Our data suggest that the complete bony healing of a fractured tooth root could take as long as 180 days in this model. We thus suggest that in clinic treatment of tooth root fracture accompanied by alveolar bone damage the fixation and reducing of occlusion force can be maintained for 6 months.

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fractured permanent incisors: prediction of healing modalities. Endod Dent Traumatol 1989;5:11-22. Andreasen FM, Vestergaard Pedersen B. Prognosis of luxated permanent teeth: the development of pulp nectrosis. Endod Dent Traumatolo 1985; 1:207-20. Andreasen FM, Yu Z, Thomsen BL, Andersen PK. The occurrence of pulp canal obliteration after luxation injuries in the permanent dentition. Endod Dent Traumatol 1987;3:10315. Andreasen FM, Yu Z, Thomsen BL. The relationship between pulpal dimensions and the development of pulp necrosis after luxation injuries in the permanent dentition. Endod Dent Traumatol 1986;2:90-8. Jacobsen I. Root fractures in permanent anterior teeth with incomplete root formation; Scand J Dent Res 1976;84:210-7. Zachrisson BU, Jacobsen I. Long-term prognosis of 66 permanent anterior teeth with root fracture. Scand J Dent Res 1975;83:345-54. Michanowicz AE. Histologic evaluation of experimentally produced intra-alveolar root fractures. In: Gutmann JL, Harrison JW, eds. Proceedings of the International Conference on Oral Trauma. Chicago: American Association of Endodontists Endowment & Memorial Foundation, 1986: 101-28. Revelander G. Tissue reactions in experimental tooth fracture. J Dent Res 1942;21:481-7. Gao ZR, Si JN. Traumatic root fracture in permanent teeth (review). I Med Abroad [Section of Stomatology] 1987; 14:210-3. Jin H, Qiu W, Lin G. Comparative study on healing process between vascularized and nonvascularized autogenous bone grafts. Shanghai J Stomatol 1992;1:47-50. Jin H, Qiu W, Lin G. A bone induction comparison of vascularized and nonvascularized bone grafts. J Practical Stomatol 1993;9:70-4. Jin H, Qiu W, Lin G. A bone strength comparison of vascularized and nonvascularized bone grafts. J Oral Maxillofac Surg 1993;3:1-4. Lin WL, McCulloch CA, Cho MI. Differentiation of periodontal ligament fibroblasts into osteoblasts during socket healing after tooth extraction in the rat. Anat Rec 1994; 240:49 2-506. Cho MI, Matsuda N, Lin WL, Moshier A, Ramakrishnan PR. In vitro formation of mineralized nodules by periodontal ligament cells from the rat. Calcif Tissue Int 1992;50:459-67.

Reprint requests: Hangqing Jin, DDS, PhD Department of Pediatric Dentistry Dental School, The University of Texas Health Science Center San Antonio, Texas 78284-7888