Proliferation,migration and apoptosis of periodontal ligament cells after tooth replantation

Oral Diseases (2010) 16 , 263–268 Objective: The aim of this study was to investigate the proliferation, migration and death of periodontal ligament (PDL) cells after tooth replantation. Materials and methods: Maxillary first molars were extracted from 4‐week‐old male (n = 28) Sprague–Dawley rats and immediately replanted, after which, proliferation, migration and death of PDL cells were investigated. Results: At 3 days after tooth replantation, many proliferative cell nuclear antigen (PCNA)‐positive PDL cells were observed on the alveolar bone side, but fewer on the root side. However, while a gradual decrease was observed in number of PCNA‐positive PDL cells on the alveolar bone side until 7 days, an increase was seen on the root side. At 3 weeks, cells labeled with PKH26 (fluorescent dye into plasma membrane) were located in the middle of the PDL space. However, these PKH26‐labeled cells did not spread to the surface of the cementum or the alveolar bone. TUNEL‐positive cells were observed on both the bone and root sides at 3 days. Number of apoptotic cells increased until 7 days on the bone sides, but decreased on root sides. Conclusion: These results suggest that both cell proliferation and apoptosis occur in different patterns and at different times to maintain regular spacing of the PDL after tooth replantation.     

The influence of storage conditions on the clonogenic capacity of periodontal ligament cells: implications for tooth replantation

Viable periodontal ligament (PL) cells are required for PL healing of avulsed teeth following replantation. If immediate replantation cannot be accomplished, the ability of PL progenitor cells to reproduce (clonogenic capacity) and recolonize the wound may be extended by prevention of desiccation and storage in physiological media. This investigation examined the effects of storage in saliva, milk, Hank's balanced salt solution (HBSS) and Eagle's medium (αMEM) on the clonogenic capacity of human PL progenitor cells at 30 and 60 min extra-alveolar time. Twenty erupted human premolar teeth extracted as atraumatically as possible for orthodontic purposes were used in the present study. Fifteen premolars were placed immediately in freshly collected autologous saliva at room temperature, (+ 23°C) for 15 min. These 15 premolars were next divided into three groups of five and stored in either saliva, milk or HBSS at + 4°C in plastic cups surrounded by ice. The remaining five teeth served as positive controls and were immediately placed in αMEM at + 4°C. PL tissue was scraped from one-half of the root surface with a scalpel at 30 and 60 min total extra-alveolar duration. Cells were released from the tissue sample with a 30 min enzymatic digestion procedure and the cells from the tissue samples analyzed for clonogenic capacity. There was a reduction in clonogenic capacity with time for all protocols. Periodontal ligament cells stored in αMEM showed the least reduction between 30 and 60 min and the greatest reduction was observed for PL cells stored in saliva. The difference in clonogenic capacity following transfer from saliva to milk or HBSS was not significant at 30 min. At 60 min, cells transferred from saliva to HBSS had a statistically higher percentage of clonogenic cells than those transferred to milk (5.9% vs. 3.5%; P < 0.05). We conclude that immediate storage of avulsed teeth in autologous saliva, followed by transfer to chilled milk, preserves the presence of sufficient progenitor cells in the PL to warrant replantation and the possibility of PL healing at 60 min extra-alveolar duration.     

完全脱位牙再植的牙髓、牙周膜愈合:从生物学基础到牙外伤指南

牙完全脱位又称牙撕脱伤,是一种严重的牙外伤,指在外力作用下,牙齿从牙槽窝中脱出,导致牙髓血运中断,牙周膜细胞暴露于体外。首选治疗方法是根据国际牙外伤协会(IADT,2012年)和美国牙髓病协会(AAE,2013年)修订的完全脱位牙再植治疗指南对患牙进行再植,以增大牙周膜愈合及牙髓血运重建的可能。但是完全脱位牙的预后受到多种临床因素影响。在某些情况下,即使按照指南进行规范治疗,完全脱位牙的术后并发症仍无法完全避免。因此,本文详细介绍牙外伤指南制定的相关生物学基础,结合临床病例,帮助临床医生理解和应用上述指南来治疗完全脱位牙及评估患牙的预后,以期为临床提供参考。     

再植牙复合牙周膜细胞的实验研究

目的:研究体外培养的牙周膜细胞与牙根共同再植入人造牙槽窝内,其组织形成的能力与促进牙周再生的潜能。方法:拔除2只杂种狗的第三、第四前磨牙,得到牙周膜细胞并进行体外培养。拔牙后1个月,将体外培养的牙周膜细胞与自体牙根共同再植入右侧下颌无牙区的人造牙槽窝内,左侧下颌作为对照。再植2个月后,处死动物,标本制成石蜡切片,HE染色,光镜下进行组织学观察。结果:实验组标本中,在与近根尖部分牙根相对的一些区域可以看到垂直于骨表面的纤维束包埋于牙槽窝壁中。结论:即使被植入人造牙槽窝中,体外培养的牙周膜细胞仍保留了在体内形成类牙周膜组织的能力。     

The effect of saline storage before replantation upon dry damage of the periodontal ligament

Abstract The effect of 30 min of saline storage before replantation of teeth, which had been dried out for 30 min, was studied in 10 Green Vervet monkeys (Cercopithecus aethiops). Maxillary central incisors were extracted and dried out for 30 min, after which 1 incisor was replanted and the other transferred to a saline solution for 30 min before replantation. No splinting or endodontic treatment was carried out. The teeth were examined after 8 wk. Histometric analysis showed identical extent of root resorption in the 2 groups, with ankylosis being the dominant resorption type, and with very limited pulpal repair in either group. It is concluded that saline storage under the experimental conditions chosen had no effect on development of root resorption or pulpal repair, presumably because 30 min dry storage had inflicted close to maximal damage on the periodontal ligament at the root surface. On the other hand, it is of clinical importance that a certain delay in the replantation procedure does not influence periodontal and pulpal healing, as long as the tooth is kept in saline storage. This suggests that replantation under these conditions can be reserved for dental professionals, rather than favoring immediate replantation by anyone ‘on-the-spot’.     

Apical tooth germ cell-conditioned medium enhances the differentiation of periodontal ligament stem cells into cementum/periodontal ligament-like tissues

Background and Objective: Limitations of current periodontal regeneration modalities in both predictability and extent of healing response, especially on new cementum and attachment formation, underscore the importance of restoring or providing a microenvironment that is capable of promoting the differentiatiation of periodontal ligament stem cells (PDLSCs) towards cementoblast‐like cells and the formation of cementum/periodontal ligament‐like tissues. The aim of this study was to investigate the biological effect of conditioned medium from developing apical tooth germ cells (APTG‐CM) on the differentiation and cementogenesis of PDLSCs both in vitro and in vivo. Material and Methods: Using the limiting dilution technique, single‐colony‐derived human PDLSCs were isolated and expanded to obtain homogeneous populations of PDLSCs. Morphological appearance, cell cycle analysis, bromodeoxyuridine incorporation, alkaline phosphatase (ALP) activity, mineralization behavior, gene expression of cementoblast phenotype and in vivo differentiation capacities of PDLSCs co‐cultured with APTG‐CM were evaluated. Results: The induced PDLSCs exhibited several characteristics of cementoblast lineages, as indicated by the morphological changes, increased proliferation, high ALP activity, and the expression of cementum‐related genes and calcified nodule formation in vitro. When transplanted into immunocompromised mice, the induced PDLSCs showed tissue‐regenerative capacity to produce cementum/periodontal ligament‐like structures, characterized by a layer of cementum‐like mineralized tissues and associated periodontal ligament‐like collagen fibers connecting with the newly formed cementum‐like deposits, whereas control, untreated PDLSCs transplants mainly formed connective tissues. Conclusion: Our findings suggest that APTG‐CM is able to provide a cementogenic microenvironment and induce differentiation of PDLSCs along the cementoblastic lineage. This has important implications for periodontal engineering.     

釉质基质蛋白诱导延迟再植牙牙周愈合的研究

目的：研究应用釉基质蛋白对延迟再植比格犬恒切牙牙周再生的影响。方法：选取3只比格犬的15颗干燥60min的切牙随机分为两组,实验组10颗牙根涂釉基质蛋白;对照组5颗牙根不涂釉基质蛋白,进行再植。8周后处死犬,每隔500μm切取5μm厚的横断切片作组织学观察。结果：与对照组相比,实验组牙再植后能显著地增加牙周膜性愈合的数量（P〈0．001）,并减少替代性吸收（P〈0．001）。结论：釉基质蛋白能对延迟再植牙的牙周愈合有促进作用。     

Mechanical removal of necrotic periodontal ligament by either Robinson bristle brush with pumice or scalpel blade. Histomorphometric analysis and scanning electron microscopy

One of the important factors accounting for successful delayed replantation of avulsed teeth is seemingly the type of root surface treatment. Removal of necrotic cemental periodontal ligament remnants may prevent the occurrence of external root resorption, which is the major cause of loss of teeth replanted in such conditions. The purpose of this study was to compare the efficacy of two mechanical techniques for removal of root-adhered periodontal ligament. Preservation or removal of the cementum layer concomitantly with these procedures was also assessed. Forty-five roots of healthy premolars extracted for orthodontic purposes were selected. After extraction, the teeth were kept dry at room temperature for 1 h and then immersed in saline for rehydration for an additional 10 min. Thereafter, the roots were assigned to three groups, as follows: group 1 (control)--the cemental periodontal ligament was preserved; group 2--removal of the periodontal ligament by scraping root surface with a scalpel blade (SBS); group 3--periodontal ligament remnants were removed using a Robinson bristle brush at low-speed with pumice/water slurry (RBP). The specimens were analysed histomorphometrically and examined by scanning electron microscopy. The quantitative and qualitative analyses of the results showed that the RBP technique was significantly more effective than the SBS technique for removal of the periodontal ligament remnants adhered to root surface. Both techniques preserved the cementum layer.     

鼠根端乳头条件培养基诱导下的牙周膜细胞膜片与牙本质管和煅烧骨间的牙周膜再生

目的 利用鼠根端乳头条件培养基(APTG-CM)与牙周膜细胞(PDLCs)建立间接共培养体系,探讨牙周组织再生的研究.方法 胶原酶联合组织块法获得人PDLCs,用波形蛋白和角蛋白鉴定PDLCs的来源.APTG-CM诱导PDLCs 28d,通过免疫细胞化学法检测PDLCs中骨钙素(OCN)、Ⅰ型胶原(COL Ⅰ)、骨涎蛋...     

Effect on periodontal healing of saline irrigation of the tooth socket before replantation

Abstract The following investigation was designed to study the healing of replanted teeth when the formation of a blood coagulum in the alveolar socket is avoided as much as possible before replantation. Eighteen maxillary incisors from 4 beagle dogs were used. The teeth were extracted and stored dry for 30 min in order to induce a standardized damage to the periodontal ligament cells, and were then replanted and splinted. Nine of the sockets were continuously irrigated with saline to achieve hemostasis before replantation. In 9 sockets a coagulum was allowed to form. After 2 wk the root canals were cleansed and filled with Ca(OH)₂ and the splints were removed. Three months later the dogs were sacrified and the premaxillas were removed and processed according to routine histologic methods. Transverse sections were cut and one section at every 500 μm interval was analyzed using light microscopy. Ankylosis was found in 3 teeth replanted in saline-irrigated sockets, while 8 teeth replanted in untreated sockets were ankylosed. Resorption without ankylosis was found in all teeth, irrespective of type of treatment of the alveolar sockets, but a tendency toward less extensive resorption areas was noted in the teeth replanted in saline-irrigated sockets. Thus, the present study indicates that continuous irrigation of the alveolar socket with saline before replantation promotes the normal healing of experimentally exarticulated teeth in dogs.     

Apoptosis of periodontal ligament cells induced by mechanical stress during tooth movement

The mechanical force generated during tooth movement creates compressed and cell-free areas in the periodontal membrane. The way in which periodontal ligament cells disappear at the compressed area during tooth movement remains unclear. In the present study we examined whether periodontal ligament cells undergo apoptosis by mechanical stress during tooth movement using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end-labelling method (TUNEL). TUNEL-positive stainings of periodontal ligament cells began to appear at the compressed areas 12 h after tooth movement, and the number of those cells reached maximum at 24 h after tooth movement. Thereafter TUNEL-positive cells disappeared at 48 h, and direct and undermining bone resorption began at the same area 72 h after tooth movement. These results showed that compressed periodontal ligament cells were eliminated by apoptosis in the early phase of tooth movement.     

Reinnervation in the canine periodontal ligament of replanted teeth using an antibody to protein gene product 9.5: an immunohistochemical study

Abstract— The re-innervation process in the periodontal ligament of replanted canine teeth was examined by immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general marker for neurons, and by electron microscopy. Within 1 week of replantation, the periodontal fibers had regenerated, filling the narrow spaces between the alveolar bone and the root surface around the cervical and apical regions. Near the root apex, however, no PGP 9.5-im-munopositive nerve fibers were found in the regenerated periodontal ligament except for those in the alveolar half of the ligament. At 2 weeks after replantation, many nerve fibers positive for PGP 9.5 had ascended the periodontal ligament from the thick nerve bundles located near the root apex. Fine nerve endings showing complicated ramification were also present in the apical region. By 3 or 4 weeks after replantation, the vascular network was regenerated and principal periodontal fibers were re-established throughout the entire length of the periodontal ligament. The extensively ramified PGP 9.5-immunopositive structures had increased in thickness and density and showed characteristic treelike profiles by 3 weeks. Electron microscopy confirmed that most of these structures were Ruffini-like endings, and demonstrated that such nerve terminals were almost regenerated by 4 weeks post-replantation. These results indicated that, in the periodontal ligament of replanted canine teeth, the regeneration of the nerve fibers including mechanosensory receptors first showed signs of regeneration by 2 weeks following tooth replantation and proceeded rapidly thereafter. Regeneration of the periodontal ligament including fiber architecture as well as vascular and neural elements was almost complete by 4 weeks after replantation.     

全脱位牙延期再植根面牙周膜不同去除方法的比较

目的:研究全脱位牙延期再植前去除其根面坏死牙周膜的有效方法.方法:取35颗因正畸需要拔除的健康右下颌第一前磨牙,室温下干燥保存超过1h,随机平均分为5组.①阴性对照组;②机械去除组:用手术刀片刮除根面坏死牙周膜;③化学去除组1以1％NaClO溶液浸泡实验牙5 min;④化学去除组2以2％NaClO溶液浸泡实验牙5 min;⑤化学去除组3以1％NaClO溶液浸泡实验牙10 min.然后用扫描电镜和组织学切片观察评价各组间牙周膜残留和牙骨质剥脱情况,采用SAS 8.02软件包对数据进行统计学分析.结果:牙周膜残留方面,阴性对照组显著高于机械去除组和3个化学去除组(P＜0.05),机械去除组显著高于3个化学去除组(P＜0.05),但3个化学去除组之间无显著差异.牙骨质缺损方面,机械去除组显著高于阴性对照组和3个化学去除组(P＜0.05);阴性对照组和3个化学去除组之间无显著差异.结论:在本实验条件下,机械去除法既无法去净根面坏死牙周膜,又会导致根面牙骨质层破坏.化学去除法能在有效去除根面牙周膜的同时,很好地保持牙骨质层的完整性,较机械去除法更佳,是一种较理想的方法.     

Periodontal healing by periodontal ligament cell sheets in a teeth replantation model

ObjectiveSuccessful transplantation of avulsed teeth is to restore the attachment and regenerate the periodontal support. Different strategies have been applied in treatment from modification of teeth storage, antibiotic usage to peridontium tissue replacement. We developed a novel periodontal ligament cell-sheet delivery system to apply on delayed replanted teeth in promoting periodontal healing in a canine model.DesignAutologous periodontal ligament (PDL) fibroblasts were isolated from extracted premolars of beagle dog. The cell-sheets were fabricated using normal culture dish after stimulation of extracellular matrix formation. Teeth were surgically extracted and attached soft tissues were removed. After root canal treatment, the root of teeth were wrapped by the PDL cell-sheets and replanted back to prior socket accordingly whilst teeth without cell sheets as a control. Eight weeks after surgery, the animals were sacrificed and decalcified specimens were prepared. Regeneration of periodontal tissue was evaluated through histology assay.ResultsMulti-layered PDL cell-sheet could be attached on tooth root and most cells on sheet-tooth constructs were viable before replantation. Minimum clinical signs of inflammation were observed in experiment. PDL cell-sheets group show significant higher occurrence of favourable healing (88.4%) than control group with low healing (5.3%). Periodontal ligament and cememtum tissue regeneration was observed in the experimental group, and the regenerated tissues showed high collagen type III, type I and fibronectin expression.ConclusionThe periodontal ligament cell-sheets fabricated through normal cell culture dish has a potential for regeneration of periodontal ligament and may become a novel therapy for avulsed teeth replantation.     

Calcification of degenerating tissues in the periodontal ligament during tooth movement

OBJECTIVE: Calcification of degenerating tissues in the periodontal ligament (PDL) during tooth movement was investigated longitudinally. MATERIALS AND METHODS: Upper first molars of male Wistar rats were moved lingually for 1, 7 and 21 d, following which unfixed undecalcified sections of the lingual PDL (in the pressure zone) were examined histologically, histochemically (autoradiography and electron probe microanalysis). RESULTS: On d 1 of tooth movement, degenerating tissues, together with some calcified particles, were visible in the pressure zone of the lingual PDL. On d 7, substantial calcified aggregations were seen in the degenerating tissues, predominantly situated between the bone and root. This was confirmed by the 45Ca autoradiography. On d 21 of tooth movement, large calcified aggregations were still clearly evident between the bone and root. CONCLUSIONS: This calcification of the degenerating tissues is a self-defense response of the living body to prevent direct contact between alveolar bone and the tooth root during compression of the PDL, so preventing friction between them and the development of ankylosis.     

牙周膜牵张成骨正畸牙快速移动的研究

牙周膜牵张成骨正畸牙快速移动与腭中缝快速打开的过程类似.拔牙后,可立即去除移动牙远中的牙槽间隔以减小骨阻力并黏结口内牵张器以快速移动正畸牙.通过此方法,正畸牙可在2～3周内移动到位.牙周膜牵张成骨术节省了支抗并大大缩短了正畸治疗时间.     

The dynamic healing profile of human periodontal ligament stem cells: histological and immunohistochemical analysis using an ectopic transplantation model

Kim Y‐T, Park J‐C, Choi S‐H, Cho K‐S, Im G‐I, Kim B‐S, Kim C‐S. The dynamic healing profile of human periodontal ligament stem cells: histological and immunohistochemical analysis using an ectopic transplantation model. J Periodont Res 2012; 47: 514–524. © 2012 John Wiley & Sons A/S Background and Objective: Human periodontal ligament stem cells (hPDLSCs) have been reported to play the pivotal role in periodontal regeneration. However, the dynamic cellular healing process initiated by hPDLSCs still remains to be elucidated. In the present study, the sequence of regeneration by hPDLSCs was assessed using histological and immunohistochemical observation in an ectopic transplantation model, which is a well‐standardized assessment tool that excludes the innate healing factors from the animals. Material and Methods: Human periodontal ligament stem cells that were isolated and characterized from teeth (n = 12) extracted for the purpose of orthodontic treatment were transplanted with carriers into ectopic subcutaneous pouches in immunocompromised mice (n = 20). Animals were killed after several different healing periods: 3 d (n = 4), 1 (n = 4), 2 (n = 4), 4 (n = 4) and 8 wk (n = 4). Histological analysis for regenerated tissues formed by hPDLSCs was conducted using hematoxylin and eosin, Masson’s trichrome and picrosirius red staining. In addition, immunohistochemical staining was performed to observe the sequential expression of osteogenic/cementogenic and periodontal ligament tissue‐specific markers associated with periodontal regeneration. Results: The whole healing process by transplanted hPDLSCs could be broadly divided into four distinctive phases. In the first phase, proliferated hPDLSCs migrated evenly all over the carrier, and collagenous tissues appeared in the form of amorphous collagen matrices. In the second phase, collagen fibers were well arranged among the carriers, and cementoid‐like tissues were observed. In the third phase, the formation of mature collagen fibers, resembling Sharpey’s fibers, was associated with active mineralization of cementum‐like tissues, and in the fourth phase, the maturation of cementum‐like tissues was observed on carrier surfaces. Various osteogenic/cementogenic markers related to the regeneration processes were expressed in a well‐orchestrated time order. Interestingly, well‐organized cementum‐like and periodontal ligament fiber‐like tissues and cells with early and late osteogenic/cementogenic markers were frequently observed in the secluded area of carrier surfaces. We termed this area the cell‐rich zone. Conclusion: The results from this study clearly demonstrated the sequential histological changes during periodontal tissue regeneration by hPDLSCs. Understanding of this process would potentially enable us to develop better cell‐based treatment techniques.     

Periodontal response to orthodontic tooth movement in diabetes‐induced rats with or without periodontal disease

1 Background

Systemic conditions can influence orthodontic tooth movement. This study evaluates histologic periodontal responses to orthodontic tooth movement in diabetes‐induced rats with or without periodontal disease.

2 Methods

Forty Wistar rats were divided according their systemic condition (SC) into diabetic (D) and non‐diabetic (ND) groups. Each group was subdivided into control (C), orthodontic tooth movement (OM), ligature‐induced periodontitis (P) and ligature‐induced periodontitis with orthodontic movement (P+OM) groups. Diabetes mellitus (DM) was induced with alloxan monohydrate, and after 30 days, the P group received a cotton ligature around their first lower molar crown. An orthodontic device was placed in OM and P+OM groups for 7 days, and the animals were then euthanized.

3 Results

Differences in OM between D and ND groups were not significant (6.87± 3.55 mm and 6.81 ± 3.28 mm, respectively), but intragroup analysis revealed statistically significant differences between the P+OM groups for both SCs. Bone loss was greater in the D group (0.16 ± 0.07 mm²) than in the ND group (0.10 ± 0.03 mm²). In intragroup analysis of the D condition, the P+OM group differed statistically from the other groups, while in the ND condition, the P+OM group was different from the C and OM groups. There was a statistically significant difference in bone density between D and ND conditions (18.03 ± 8.09% and 22.53 ± 7.72%) in the C, P, and P+OM groups.

4 Conclusion

DM has deleterious effects on bone density and bone loss in the furcation region. These effects are maximized when associated with ligature‐induced periodontitis with orthodontic movement.