自体骨髓基质细胞用于牙周骨缺损移植的动物实验研究

目的：应用分离的骨髓干细胞（ＭＳＣ）进行牙周骨缺损的自体移植动物实验,观察ＭＳＣ用于牙周再生治疗的可行性。方法：体外分离狗髂骨的ＭＳＣ,体外培养,移植前进行ＭＳＣ的体外钙化实验,以胶原膜为移植载体,将ＭＳＣ移植到狗下颌后牙的根分叉骨缺损中,覆盖国产聚四氟乙烯膜,３０ｄ后进行组织学观察。结果：分离的ＭＳＣ在体外均出现了不同程度的钙化;动物实验结果发现,ＭＳＣ移植组达到了骨缺损的完全修复,无根面吸收;     

Regenerative potential and healing dynamics of the periodontium: a critical‐size supra‐alveolar periodontal defect study

Objectives: The nature and characteristics of the newly formed periodontium obtained following regenerative procedures remain a matter of controversy. The objective of this study was to evaluate the regenerative potential of the periodontal attachment and healing dynamics as observed from the spatial distribution of newly formed cementum, periodontal ligament (PDL) and alveolar bone following optimal circumstances for wound healing/regeneration in a discriminating animal model. Material and Methods: Critical‐size, 6‐mm, supra‐alveolar, periodontal defects were surgically created in six young adult Beagle dogs. Space‐providing ePTFE devices with 300‐μm laser‐drilled pores were implanted to support wound stability and space provision in one jaw quadrant/animal. Treatments were alternated between left and right jaw quadrants in subsequent animals. The gingival flaps were advanced to submerge the defect sites for primary intention healing. Histometric analysis followed an 8‐week healing interval. Results: Healing was uneventful in all animals. The histometric analysis showed that cementum regeneration (2.99 ± 0.22 mm) was significantly greater than PDL (2.54 ± 0.18 mm, p=0.03) and bone regeneration (2.46 ± 0.26 mm, p=0.03). The wound area showed significant positive non‐linear effect on cementum (log β=1.25, p<0.001), PDL (log β=1.24, p<0.001) and new bone formation (log β=1.36, p<0.001). A high degree of concordance and significant linear relationship was observed between cementum, PDL and bone regeneration indicating that their formation virtually occurred in parallel. Conclusions: Cementum, PDL and alveolar bone virtually regenerate in parallel under optimal circumstances for periodontal wound healing/regeneration. Moreover, space provision positively influences the extent of periodontal regeneration.     

Engineered autologous bone marrow mesenchymal stem cells: alternative to cleft alveolar bone graft surgery

ABSTRACT: Human recombinant bone morphogenetic protein 2 (rhBMP-2) accelerates bone regeneration but is associated with limited cementum and periodontal ligament regeneration, local root resorption, and ankylosis. This study assessed a new approach to the regeneration of the alveolar bone and periodontal attachment apparatus using a combination of ex vivo autologous bone marrow mesenchymal stem cells (MSCs) engineered by replication defective adenovirus to express the BMP-2 gene and pluronic F127 (PF127) in a large mammalian animal model. Bilateral maxillary periodontal defects were created over the premolar area in 9 mature male miniature swine. The 18 defects were randomly assigned to receive either BMP-2-expressing MSCs in the advBMP-2 group or MSCs alone in the MSC group. The regenerated periodontal attachment apparatus was evaluated histologically, and the total regenerated bone volume was calculated from three-dimensional computed tomography analysis. Three months after implantation, significant bone volume was regenerated in the advBMP-2 group. Periodontal apparatus regeneration was significantly better in the advBMP-2 group. New cementum and Sharpey fibers were observed on the denuded root surfaces in the advBMP-2 group, whereas incomplete healing with localized root surface resorption was noted in the control group. The use of ex vivo BMP-2-engineered autologous MSCs enhanced bone and periodontal apparatus regeneration in maxillary alveolar and periodontal defects in swine. This novel integrated approach might be suitable for clinical periodontal apparatus repair. This may be an alternative for cleft alveolar bone graft surgery.     

Introduction of a Mixture of β‐Tricalcium Phosphate Into a Complex of Bone Marrow Mesenchymal Stem Cells and Type I Collagen Can Augment the Volume of Alveolar Bone Without Impairing Cementum Regeneration

Background: The purpose of this study is to evaluate whether β‐tricalcium phosphate (β‐TCP) could be a promising modality to help augment alveolar bone in periodontal tissue regeneration by bone marrow mesenchymal stem cells (BMMSCs). Methods: Expanded BMMSCs and atelocollagen (Col) were mixed together (MSC/Col). A combination of β‐TCP with MSC/Col was also prepared (MSC/Col/TCP). MSC/Col/TCP or MSC/Col was transplanted into experimental periodontal Class III furcation defects that had been exposed to inflammation in beagle dogs. Periodontal tissue regeneration was evaluated by histologic and morphometric analyses at 4 and 8 weeks after transplantation. Results: MSC/Col and MSC/Col/TCP enhanced periodontal tissue regeneration compared to Col and TCP/Col according to hematoxylin and eosin staining. The percentage of new cementum length in the MSC/Col/TCP group was not significantly different from that in the MSC/Col group at 4 and 8 weeks. On the other hand, the percentage of new bone area in the MSC/Col/TCP group was much higher than that in the MSC/TCP group at 4 weeks. However, at 8 weeks, no significant difference in new bone area was found between the two groups. In the MSC/Col/TCP group, β‐TCP was surrounded by newly formed bone. Multinucleated cells, which were positive for osteopontin and tartrate‐resistant acid phosphatase, were present in the interconnected macropores of β‐TCP. Conclusion: These findings suggest that β‐TCP is applicable as a scaffold for BMMSCs transplantation and helps augment alveolar bone without impairing regeneration of cementum.     

Application of periodontal ligament cell sheet for periodontal regeneration: a pilot study in beagle dogs

OBJECTIVE: The ultimate goal of periodontal treatment is to regenerate the damaged periodontal support. Although periodontal ligament (PDL) cells are essential for periodontal regeneration, few studies have reported the transplantation of periodontal ligament cells to periodontal defects. We developed a new method to apply periodontal ligament cells as a sheet to the defect. The aim of this study was to investigate the periodontal healing after application of the periodontal ligament cell sheet in beagle dogs. METHODS: Autologous periodontal ligament cells were obtained from extracted premolars of each beagle dog. Periodontal ligament cell sheets were fabricated using a temperature-responsive cell culture dish. Dehiscence defects were surgically created on the buccal surface of the mesial roots of bilateral mandibular first molars of each dog. In the experimental group (five defects), periodontal ligament cell sheet with reinforced hyaluronic acid carrier was applied to the defect. Only the hyaluronic acid carrier was applied to the contralateral side as a control (five defects). Eight weeks after surgery, the animals were sacrificed and decalcified specimens were prepared. Healing of the periodontal defects was evaluated histologically and histometrically. RESULTS: No clinical signs of inflammation or recession of gingiva were observed in both experimental and control groups. In the experimental group, periodontal tissue healing with bone, periodontal ligament and cementum formation was observed in three out of five defects. In the control group, such periodontal tissue formation was not observed except in one defect. Histometric analysis revealed that the formation of new cementum in the experimental group was significantly higher than that in the control group. CONCLUSION: The periodontal ligament cell sheet has a potential to regenerate periodontal tissue and may become a novel regenerative therapy.     

自体牙周膜细胞移植对狗牙周组织再生的影响

目的　对应用自体牙周细胞移植结合e PTFE膜引导的牙周组织再生的动物实验进行评价。方法　将 6只成年狗的 36颗牙分为实验组和对照组 ,每组 18颗牙。在人工制造的牙周缺损中 ,进行体外培养的自体牙周细胞移植结合GTR法为实验组 ,单纯应用GTR法为对照组。 6周后切片行牙周组织学观察。结果　实验组新生牙槽骨、牙周膜组织及牙骨质的修复再生效果明显好于对照组 (P <0 0 5 ) ;实验组牙槽骨再生高度平均为 (4 0 0± 0 13)mm ,对照组为 (3 0 9± 0 2 8)mm。结论应用自体牙周膜细胞移植结合e PTFE膜引导牙周组织再生可促进狗牙周组织的再生     

Periodontal regeneration following implantation of cementum and periodontal ligament-derived cells

Nuñez J, Sanz‐Blasco S, Vignoletti F, Muñoz F, Arzate H, Villalobos C, Nuñez L, Caffesse RG, Sanz M. Periodontal regeneration following implantation of cementum and periodontal ligament‐derived cells. J Periodont Res 2012; 47: 33–44. © 2011 John Wiley & Sons A/S Background and Objective: The periodontal regeneration of bone defects is often unsatisfactory and could be largely improved by cell therapy. Therefore, the purpose of this study was to evaluate the regenerative potential of implanting canine cementum‐derived cells (CDCs) and canine periodontal ligament‐derived cells (PDLDCs) in experimentally created periodontal intrabony defects in beagle dogs. Material and Methods: Cells were obtained from premolars extracted from four beagle dogs. Three‐wall intrabony periodontal defects, 3 mm wide and 4 mm deep, were surgically created in their second and fourth premolars and plaque was allowed to accumulate. Once the defects were surgically debrided, periodontal regeneration was attempted by random implantation of collagen sponges embedded with 750,000 CDCs, 750,000 PDLDCs or culture medium. After 3 mo of healing, specimens were obtained and periodontal regenerative outcomes were assessed histologically and histometrically. Results: The histological analysis showed that a minimal amount of new cementum was formed in the control group (1.56 ± 0.39 mm), whereas in both test groups, significantly higher amounts of new cementum were formed (3.98 ± 0.59 mm in the CDC group and 4.07 ± 0.97 mm in the PDLDC group). The test groups also demonstrated a larger dimension of new connective tissue, resulting in a significantly more coronal level of histological attachment. Conclusion: This proof‐of‐principle study suggests that cellular therapy, in combination with a collagen sponge, promoted periodontal regeneration in experimental intrabony periodontal defects.     

Enhancement of periodontal tissue regeneration by transplantation of bone marrow mesenchymal stem cells

BACKGROUND: The use of suitable cells transplanted into periodontal osseous defects appears to be a powerful strategy to promote periodontal tissue regeneration. Mesenchymal stem cells (MSCs) isolated from bone marrow have the potential for multilineage differentiation. The purpose of this study was to examine whether auto-transplantation of MSCs into periodontal osseous defects would be useful for periodontal tissue regeneration. METHODS: Bone marrow MSCs were isolated from beagle dogs and expanded in vitro. The expanded MSCs were mixed with atelocollagen (2% type I collagen) at final concentrations of 2 x 10(6), 5 x 10(6), 1 x 10(7), or 2 x 10(7) cells/ml, and auto-transplanted into experimental Class III defects. Atelocollagen alone was implanted into the defects as a control. Periodontal tissue healing was evaluated by histological and morphometric analyses 1 month after transplantation. RESULTS: The defects were regenerated with cementum, periodontal ligament, and alveolar bone in the MSC-atelocollagen groups. Less periodontal tissue regeneration was observed in the control group compared to the MSC-atelocollagen groups. Morphometric analysis revealed that the percentage of new cementum length in the 5 x 10(6) and 2 x 10(7) cells/ml groups and the percentage of new bone area in the 2 x 10(7) cells/ml group were significantly higher than in the control group (P < 0.01). CONCLUSION: These findings suggest that auto-transplantation of bone marrow mesenchymal stem cells is a novel option for periodontal tissue regeneration.     

Participation of periodontal ligament cells with regeneration of alveolar bone

BACKGROUND: It is important to clarify the participation of periodontal ligament (PDL) cells in the regeneration of alveolar bone to establish a reliable approach for obtaining periodontal regeneration. The aim of this study was to determine whether PDL cells play an important role in alveolar bone repair during the course of periodontal regeneration. METHODS: In an in vitro study, the expression of the osteoblast phenotype, such as alkaline phosphatase activity and parathyroid hormone-dependent 3',5'-cyclic adenosine monophosphate accumulation, was investigated in dog PDL cells (DPLC) and dog bone cells isolated from mandibles (DBC). In a related study, the roots of mandibular third premolars extracted from aged dogs were divided into a PDL(+) group, in which the PDL was preserved, and a PDL(-) group, in which the PDL was removed. These roots were respectively transplanted into surgically created bone cavities with buccal and interproximal bone defects in an edentulous area, prepared in advance by extraction of mandibular fourth premolars. These bone defects with the transplanted roots were completely covered with submerged physical barrier membranes. New bone formation and new connective tissue attachment, which require new cementum and insertion of functionally oriented new collagen fibers of periodontal ligament, were histomorphometrically assessed, and were compared between the PDL(+) and PDL(-) groups 6 weeks after transplantation. RESULTS: Both cultured DPLC and DBC exhibited the osteoblast phenotype. New connective tissue attachment was observed only in the PDL(+) group. However, alveolar bone was almost completely regenerated to the original bone height in both the PDL(+) and PDL(-) groups, and the amount of newly formed bone was not significantly different between the 2 groups. CONCLUSIONS: DPLC retain the capability to differentiate into an osteoblast lineage and may act in the regeneration of periodontal ligament with new cementum formation, whereas these cells may have a limited influence on alveolar bone formation during the course of periodontal regeneration.     

Stem cells in craniofacial and dental tissue engineering

Mesenchymal stem cells (MSC) have been identified in a variety of adult tissues as a population of pluripotential self-renewing cells. Based on their adherence and colony forming properties, a small number of MSC can be isolated from most mesenchymal tissues as well as bone marrow. In the presence of one or more growth factors, these cells commit to lineages that lead to the formation of bone, cartilage, muscle, tendon and adipose tissue; recent studies indicate that stem cells for cementum, dentine and the periodontal ligament also exist. All of these cells can be expanded in vitro, and, embedded in a scaffold, inserted into defects to promote healing and tissue replacement. Increased understanding of the molecular mechanism directing lineage specification and morphogenesis is providing a rational approach for the regeneration of craniofacial tissues and oral structures.     

Biomaterials for promoting periodontal regeneration in human intrabony defects: a systematic review

Intrabony periodontal defects are a frequent complication of periodontitis and, if left untreated, may negatively affect long‐term tooth prognosis. The optimal outcome of treatment in intrabony defects is considered to be the absence of bleeding on probing, the presence of shallow pockets associated with periodontal regeneration (i.e. formation of new root cementum with functionally orientated inserting periodontal ligament fibers connected to new alveolar bone) and no soft‐tissue recession. A plethora of different surgical techniques, often including implantation of various types of bone graft and/or bone substitutes, root surface demineralization, guided tissue regeneration, growth and differentiation factors, enamel matrix proteins or various combinations thereof, have been employed to achieve periodontal regeneration. Despite positive observations in animal models and successful outcomes reported for many of the available regenerative techniques and materials in patients, including histologic reports, robust information on the degree to which reported clinical improvements reflect true periodontal regeneration does not exist. Thus, the aim of this review was to summarize, in a systematic manner, the available histologic evidence on the effect of reconstructive periodontal surgery using various types of biomaterials to enhance periodontal wound healing/regeneration in human intrabony defects. In addition, the inherent problems associated with performing human histologic studies and in interpreting the results, as well as certain ethical considerations, are discussed. The results of the present systematic review indicate that periodontal regeneration in human intrabony defects can be achieved to a variable extent using a range of methods and materials. Periodontal regeneration has been observed following the use of a variety of bone grafts and substitutes, guided tissue regeneration, biological factors and combinations thereof. Combination approaches appear to provide the best outcomes, whilst implantation of alloplastic material alone demonstrated limited, to no, periodontal regeneration.     

Periodontal ligament cell sheet promotes periodontal regeneration in athymic rats

Aim: The primary goal of periodontal treatment is regeneration of the periodontium. Current theories suggest that the periodontal ligament (PDL) cells have the capacity to participate in restoring connective and mineralized tissues, when appropriately triggered. We evaluated whether human PDL cell sheets could reconstruct periodontal tissue. Material and Methods: To obtain the cell sheet, human PDL cells were cultured on temperature‐responsive culture dishes with or without osteogenic differentiation medium. The cell sheets were transplanted on periodontal fenestration defects of immunodeficient rats. Forty rats were divided in two groups: in one group, cell sheets cultured with control medium were transplanted and in the other, cell sheets cultured with osteogenic differentiation medium were transplanted. The defects were analysed histologically and histomorphologically after healing. Results: Most of the experimental group exhibited a new cementum‐like layer and new attachment of collagen fibres to the layer. Histomorphological analyses indicated significant periodontal regeneration. The control group revealed dense extracellular matrix and fibre formation, but an obvious cementum layer was not observed. Conclusions: Transplanted PDL cell sheets cultured with osteogenic differentiation medium induced periodontal regeneration containing an obvious cementum layer and Sharpey's fibres. Thus, the method could be feasible as a new therapeutic approach for periodontal regeneration.     

Periodontal ligament cells and gingival fibroblasts respond differently to attachment factors in vitro

One of the initial events required for regeneration of periodontal tissues lost due to disease is the establishment of connective tissue attachment to root surfaces. Thus, considerable research efforts have focused on developing reliable procedures to gain new connective tissue attachment. Our studies focus on evaluating agents for their ability to promote cell attachment and spreading using an in vitro assay. For these studies human gingival fibroblasts (GF) and human periodontal ligament (PDL) cells, after exposure to fibronectin; 44 kilodalton bone phosphoprotein (44K BPP-osteopontin) or guanidine EDTA extracts of bone, cementum, or dentin, were compared as to degree of cell attachment and spreading. Fibronectin equally enhanced attachment and spreading PDL cells and GF. In contrast, 44K BPP, as well as guanidine EDTA extracts of bone and cementum, preferentially promoted attachment of GF when compared with attachment of PDL cells. For both PDL cells and GF the attached cells exhibited spreading. The guanidine EDTA extract of dentin did not promote attachment of either cell type. These results suggest that PDL cells and GF have different attachment properties which need to be considered for investigations directed at developing regenerative periodontal treatments.     

Regeneration of periodontal tissues by basic fibroblast growth factor

Several growth factors (or cytokines) have recently received attention because of their ability to actively regulate various cellular functions of periodontal ligament (PDL) cells and the effects of topical application of such factor(s) on periodontal tissue regeneration has been evaluated. In this study, we examined the role of basic fibroblast growth factor (bFGF) in the wound healing and regeneration of periodontal tissues. Alveolar bone defects (such as 2-wall, 3-wall and furcation class II bone defects) were created surgically in beagle dogs and primates. Recombinant bFGF was topically applied to the artificial bony defects. Six or 8 wk after application, the periodontal regeneration was morphologically and histomorphometrically analyzed. In all sites where bFGF was applied, significant periodontal ligament formation with new cementum deposits and new bone formation was observed in amounts greater than in the control sites. We found it noteworthy that no instances of epithelial down growth, ankylosis or root resorption were observed in the bFGF sites. In vitro studies demonstrated that bFGF enhances the proliferative responses of human PDL cells, which express FGF receptor-1 and -2, but inhibits the induction of alkaline phosphatase activity and mineralized nodule formation by PDL cells. Interestingly, we observed that the mRNA level of laminin in PDL cells, which plays an important role in angiogenesis, was specifically upregulated by bFGF stimulation, but that of type I collagen was downregulated. The present study demonstrates that bFGF can be applied as one of the therapeutic modalities which actively induce periodontal tissue regeneration. The results of in vitro studies suggest that by suppressing the cytodifferentiation of PDL cells into mineralized tissue forming cells, bFGF may play important roles in wound healing by promoting angiogenesis and inducing the growth of immature PDL cells, and may in turn accelerate periodontal regeneration.     

Experimental and clinical studies on regenerative periodontal therapy

The recognition of a periodontal therapy as a regenerative procedure requires the demonstration of new cementum, periodontal ligament, and bone coronal to the base of the defect. A diversity of regenerative strategies has been evaluated, including root surface conditioning, bone grafts and bone substitute materials, guided tissue regeneration, enamel matrix proteins, growth/differentiation factors, combined therapies and, more recently, tissue‐engineering approaches. The aim of this chapter of Periodontology 2000 is to review the research carried out in Latin America in the field of periodontal regeneration, focusing mainly on studies using preclinical models (animal models) and randomized controlled clinical trials. This review may help clinicians and researchers to evaluate the current status of the therapies available and to discuss the challenges that must be faced in order to achieve predictable periodontal regeneration in clinical practice.     

The use of demineralized freeze-dried bone-glycoprotein matrix grafts in treating baboon periodontal infrabony defects

In an effort to improve regenerative capacity and establish higher levels of predictability, the use of a new graft material was investigated in baboon infrabony periodontal defects. A graft matrix composed of human demineralized freeze-dried bone combined with human connective tissue glycoprotein was implanted in 20 defects in 9 baboons. Control, debrided-only sites in the same animal served as a comparison. Grafted sites achieved a 1.8-mm greater reduction in probing depth and attachment gain compared to controls. In addition, 25.3% greater defect fill was found in grafted sites. Histologic analyses provided evidence of bone induction by the graft matrix and regeneration of new cementum, periodontal ligament, and bone in the grafted sites.     

The study of calcification of autogenous bone marrow stem cell transplantation on alveolar bone defect in dogs]

OBJECTIVE: To evaluate the effect of calcification of autogenous bone marrow stem cell transplantation in periodontal tissue regeneration. METHODS: Bone marrow stem cells derived from the same dog were cultured with alpha-MEM. 1 x 10(7) cells of first passage were allowed to attach to the collagen membrane for 24 hours. The membrane-cells were transplanted into periodontal defect in the same dog. Then the defects were covered with e-pTFE membranes. The defects covered only with e-pTFE without membrane-cells were served as control. Eighteen teeth of 6 dogs for every group were studied. The dogs were sacrificed after 6 weeks. RESULTS: The results showed that new bone formation in test group was significantly higher than that of control group. The calcification of new bone in test group was better than control group. CONCLUSIONS: The results suggested that autogenous bone marrow stem cell transplantation with guided tissue regeneration technique could enhance periodontal tissue regeneration and could form new bone tissue fast and could shorten times of periodontal tissue regeneration in dogs.     

Cellular therapy in periodontal regeneration

Periodontitis is a chronic inflammatory condition leading to destruction of the tooth supporting tissues, which if left untreated may cause tooth loss. The treatment of periodontitis mainly aims to arrest the inflammatory process by infection control measures, although in some specific lesions a limited periodontal regeneration can also be attained. Current regenerative approaches are aimed to guide the cells with regenerative capacity to repopulate the lesion and promote new cementum and new connective tissue attachment. The first phase in periodontal tissue regeneration involves the differentiation of mesenchymal cells into cementoblasts to promote new cementum, thus facilitating the attachment of new periodontal ligament fibers to the root and the alveolar bone. Current regenerative approaches limit themselves to the confines of the lesion by promoting the self‐regenerative potential of periodontal tissues. With the advent of bioengineered therapies, several studies have investigated the potential use of cell therapies, mainly the use of undifferentiated mesenchymal cells combined with different scaffolds. The understanding of the origin and differentiation patterns of these cells is, therefore, important to elucidate their potential therapeutic use and their comparative efficacy with current technologies. This paper aims to review the in vitro and experimental studies using cell therapies based on application of cementoblasts and mesenchymal stem cells isolated from oral tissues when combined with different scaffolds.