Are cementoblasts a subpopulation of osteoblasts or a unique phenotype?

Experimental studies have shown a great potential for periodontal regeneration. The limitations of periodontal regeneration largely depend on the regenerative potential at the root surface. Cellular intrinsic fiber cementum (CIFC), so-called bone-like tissue, may form instead of the desired acellular extrinsic fiber cementum (AEFC), and the interfacial tissue bonding may be weak. The periodontal ligament harbors progenitor cells that can differentiate into periodontal ligament fibroblasts, osteoblasts, and cementoblasts, but their precise location is unknown. It is also not known whether osteoblasts and cementoblasts arise from a common precursor cell line, or whether distinct precursor cell lines exist. Thus, there is limited knowledge about how cell diversity evolves in the space between the developing root and the alveolar bone. This review supports the hypothesis that AEFC is a unique tissue, while CIFC and bone share some similarities. Morphologically, functionally, and biochemically, however, CIFC is distinctly different from any bone type. There are several lines of evidence to propose that cementoblasts that produce both AEFC and CIFC are unique phenotypes that are unrelated to osteoblasts. Cementum attachment protein appears to be cementum-specific, and the expression of two proteoglycans, fibromodulin and lumican, appears to be stronger in CIFC than in bone. A theory is presented that may help explain how cell diversity evolves in the periodontal ligament. It proposes that Hertwig's epithelial root sheath and cells derived from it play an essential role in the development and maintenance of the periodontium. The role of enamel matrix proteins in cementoblast and osteoblast differentiation and their potential use for tissue engineering are discussed.     

Periodontal regeneration of transplanted rat molars after cryopreservation

The effects of cryopreservation on periodontal regeneration of transplanted rat molars were investigated histologically and histochemically in rats. Bilateral first and second maxillary molars of 4-week-old Wistar rats were gently extracted and transplanted into the abdominal subcutaneous connective tissue immediately or after cryopreservation in liquid nitrogen overnight. Donor teeth were slowly frozen by a rate-controlling freezer (program freezer) using 5% dimethylsulfoxide (DMSO) and 6% hydroxyethyl starch (HES) as cryoprotectants. One-four weeks after transplantation, they were carefully excised with the surrounding tissues. Regeneration of acellular cementum, periodontal ligament, and alveolar bone were observed 2 weeks after immediate transplantation. The pulp was repaired by the ingrowth of granulation tissue from the root apex followed by the formation of calcified tissue. The regenerated periodontal ligament was positive for alkaline phosphatase (ALP). Small or mononuclear tartrate resistant acid phosphatase (TRAP) positive cells were scattered on the newly formed alveolar bone and on the hard tissue in the pulp, but there was no external or internal progressive root resorption at 4 weeks. Cryopreserved teeth had acellular cementum with a rough surface at 1 week, but with the increase of cementoblasts and the appearance of periodontal ligament and alveolar bone, the surface became smooth at 3 weeks. Epithelial rests of Malassez (ERM) also revived. After regeneration of the periodontal tissues at 4 weeks, there was no evidence of root resorption. Although the process proceeded slowly, the cryopreserved teeth showed the periodontal regeneration substantially similar to that of the immediately transplanted teeth without progressive root resorption, indicating that they could be applicable for clinical use.     

Runt相关基因2/核心结合因子a1在小鼠牙周组织发育中的免疫组化定位研究

目的探讨Runt相关基因2/核心结合因子a1(Runx2/Cbfa1)在小鼠牙周组织发育过程中的时空表达及意义。方法建立BALB/c小鼠牙周发育动物模型,采用免疫组化方法检测Runx2/Cbfa1在小鼠出生后各期牙周组织发育中的表达及特点。结果Runx2/Cbfa1在牙齿发育过程中的表达具有时空特异性,在牙根开始发育之前,仅在牙槽骨及成骨细胞中表达;当牙根开始发育即从第11天以后各期,在根部牙周膜细胞、成牙骨质细胞及成骨细胞中均呈阳性表达,但牙槽骨呈阴性表达。结论Runx2/Cbfa1在成牙骨质细胞及成骨细胞的分化及牙植骨的形成具有重要作用,可能在牙周组织的发育中发挥作用。     

WNT/β-连环蛋白信号转导通路在牙周组织再生中的作用

中重度牙周炎的牙周膜、牙槽骨和牙骨质等牙周组织丧失严重,其最有效的治疗是牙周组织再生技术。WNT/β-连环蛋白信号转导通路可以促进牙周膜细胞、成骨细胞和成牙骨质细胞增殖和分化,以维持牙周膜不断更新,促进牙槽骨和牙骨质的形成和再生。本文就WNT/β-连环蛋白信号转导通路以及WNT/β-连环蛋白信号转导通路在牙槽骨和牙骨质形成及再生中的作用等研究进展作一综述。     

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.     

骨膜蛋白与牙周组织再生

骨膜蛋白为成骨细胞及其前体细胞的细胞黏附分子,在骨膜中强表达,间接地参与骨形成和骨修复过程,还表达于牙周膜、肌腱、心脏瓣膜和皮肤等承受压力的纤维结缔组织。人类的骨膜蛋白基因定位于6p21,鼠类的骨膜蛋白基因位于17号染色体。骨膜蛋白有一个N-末端结构,一个富含半胱氨酸结构,四个同源性重复区,一个C-末端。N-末端有一个典型的信号序列,提示其可能是一种分泌性蛋白。在牙胚的帽状期,骨膜蛋白表达于内釉上皮和前期成牙本质细胞层之间以及颈环周围的间充质。在牙胚钟状期的牙囊细胞中也有骨膜蛋白表达。在牙体移动过程中,骨膜蛋白是机械应力下骨和牙周组织改建过程中的局部促进因素之一。牙周组织修复再生的关键是促使来源于牙周膜的细胞优先附着于根面并增殖分化,进而形成新的牙周膜、牙骨质和牙槽骨,即形成新的牙周附着。骨膜蛋白是具有骨膜及牙周膜组织表达特异性的骨黏附蛋白,对成骨细胞的分化及组织的早期矿化有重要作用。     

经典Wnt信号通路与牙周膜干细胞成骨分化

牙周炎是口腔最常见的疾病之一,累及牙周支持组织,随着疾病的进展将引起附着丧失、牙周袋形成、牙槽骨吸收,最终导致牙齿松动脱落。因被牙周炎破坏吸收的牙槽骨自愈能力十分有限,所以牙周炎的治疗目标是在彻底清除菌斑生物膜的基础上,争取获得较多的牙周组织再生。牙周膜干细胞作为最适宜进行牙周组织再生的细胞,被广泛研究。Wnt信号通路分为经典Wnt通路和非经典Wnt信号通路,为十分复杂而高度保守的通路传导途径。该通路与牙周膜干细胞成骨分化的关系十分密切,牙周膜干细胞的成骨分化又对牙周组织再生有重要意义。该文对经典Wnt信号通路与牙周膜干细胞成骨分化的研究概况作一综述。     

Dental regenerative therapy targeting sphingosine-1-phosphate (S1P) signaling pathway in endodontics

The establishment of regenerative therapy in endodontics targeting the dentin-pulp complex, cementum, periodontal ligament tissue, and alveolar bone will provide valuable information to preserve teeth. It is well known that the application of stem cells such as induced pluripotent stem cells, embryonic stem cells, and somatic stem cells is effective in regenerative medicine. There are many somatic stem cells in teeth and periodontal tissues including dental pulp stem cells (DPSCs), stem cells from the apical papilla, and periodontal ligament stem cells. Particularly, several studies have reported the regeneration of clinical pulp tissue and alveolar bone by DPSCs transplantation. However, further scientific issues for practical implementation remain to be addressed. Sphingosine-1-phosphate (S1P) acts as a bioactive signaling molecule that has multiple biological functions including cellular differentiation, and has been shown to be responsible for bone resorption and formation. Here we discuss a strategy for bone regeneration and a possibility for regenerative endodontics targeting S1P signaling pathway as one of approaches for induction of regeneration by improving the regenerative capacity of endogenous cells.Scientific field of dental scienceEndodontology     

牙周膜干细胞的研究进展

牙周膜干细胞可分化为成骨细胞或成牙骨质细胞、脂肪细胞和胶原形成细胞,是牙周组织工程中新的种子细胞。笔者就牙周膜干细胞的生物学特性、牙周膜干细胞的多向分化潜能、牙周膜干细胞的移植及其潜在的临床应用价值作一综述。     

Synthesis of cementum-like tissue in vitro by cells cultured from bone: a light and electron microscope study

Cells obtained from calvariae of fetal rats, human gingival connective tissue and periodontal ligament of rat molars were each co-cultured in vitro with non-demineralized and partly demineralized root slices in nutrient medium containing 50 μGg/ml ascorbic acid and 10 mM B-glyeerophosphate, in order to determine whether the root slices could affect the phenotype expressed by the cells. The cultures were examined by light and transmission electron microscopy. Nodules of bone-like tissue, resembling those previously described by others, were observed in the multilayers of cells obtained from rat calvariae. but not in the multilayers of gingival and periodontal ligament cells, after 30 days of culture. Calvaria cells associated with the root slices produced tissues exhibiting ultrastructure that resembled that of bone or cellular cementum. acellular cemenlum and afibrillar cementum in vivo when examined after the same length of time. The tissues were never found in cultures of gingival fibroblasts or periodontal ligament cells. These observations suggest, first, that cells derived from bone can express in vitro the phenotype for the cementums; second, that cells obtained from human gingival and rat periodontal ligament do not do so when cultured under similar conditions; and third, the possibility that the osteoblasts, cementoblasts and their progenitors that are found in the periodontal ligament could, at least in part, have their origin and migrate there from the endosteal spaces of the alveolar process.     

Healing of periodontal tissues following transplantation of cells in a rat orthodontic tooth movement model

OBJECTIVE: To determine the fate and differentiation of transplanted periodontal ligament (PL) precursor cells and mouse embryonic stem (ES) cells and their relative capacity to regenerate wounded periodontium. MATERIALS AND METHODS: Orthodontic tooth movement was introduced 24 hours before transplantation of PL or ES cells, and rats were euthanized either 24 hours or 72 hours after cell transplantation. The control rats received either no tooth movement and no cell transplantation or tooth movement and no cell transplantation. Differentiation of transplanted cells was assessed from mandibular periodontal histological tissue sections by immunohistochemical methods using monoclonal antibodies against PL cell differentiation markers. Data were analyzed using Student's t-test at a significance level of P = .05. RESULTS: Transplantation of PL and ES cells resulted in a higher number of osteopontin, bone sialoprotein, and alpha-smooth muscle actin labeled transplanted cells, predominantly around the blood vessels of the periodontium in study rats compared with control rats (cell transplantation but no orthodontic tooth movement, P = .05). Combined treatments of tooth movement and cell transplantation resulted in enhanced regeneration of the periodontium as a result of tooth movement. Transplantation of PL cells induced a higher number of differentiating cells in the PL and alveolar bone than did transplantation of ES cells. CONCLUSIONS: Orthodontic tooth movement promotes the differentiation of transplanted cells, and the differentiation occurs predominantly in the paravascular areas of the periodontium. In terms of regeneration of wounded periodontium, transplantation of PL cells produced a higher level of regeneration than ES cells, possibly because of PL cell plasticity and the capacity to undergo effective differentiation in the periodontal cellular microenvironment.     

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.     

Collagen implants do not preserve periodontal ligament homeostasis in periodontal wounds

An improved understanding of the differentiation of periodontal ligament cells could facilitate the development of new treatment approaches for overcoming the loss of specialized cell types caused by periodontitis. To study healing of wounded periodontal tissues and the differentiation of mineralizing connective tissue cells in periodontal ligament, we have examined the influence of wound size and collagen implantation on the regeneration of periodontium and on immunohistochemical staining for osteopontin and bone sialoprotein. Four groups of Wistar rats were wounded by drilling through the alveolar bone and by extirpation of the periodontal ligament. Wounds were 0.6 or 1.8 mm in diameter and defects were either implanted with collagen gels or were treated without implants. Rats were killed at 1 wk or 2 months after wounding and tissue sections were stained with monoclonal antibodies against rat osteopontin and bone sialoprotein. Collagen implants strongly increased staining for osteopontin and bone sialoprotein in defects at 1 wk. By 2 months alveolar bone healed completely regardless of the wound size but in large defects, periodontal ligament width was significantly reduced with or without implants. In large wounds at 2 months, collagen implants inhibited bone regeneration and there was stronger staining for osteopontin and bone sialoprotein in the bone replacing the implant, indicating that collagen prolonged bone remodelling. We conclude that implantation of exogenous collagen affects alveolar bone healing but does not preserve the width of the regenerated periodontal ligament. Therefore collagen does not appear to contribute to homeostasis in the periodontium following wounding.     

Non‐surgical model for alveolar bone regeneration by bone morphogenetic protein‐2/7 gene therapy

1 Background

Alveolar bone is a critical tissue for tooth retention; however, once alveolar bone is lost, it may not spontaneously regenerate. Currently, bone grafts or artificial bone is commonly used for alveolar bone regeneration therapy. However, these therapies require surgical procedures, which present risks, particularly in elderly patients. Therefore, development of alveolar bone regeneration techniques that do not require surgical procedures is critical. It is well known that stem cells present in the periosteal and periodontal ligament may be induced to differentiate into osteogenic cells. This study hypothesizes that transfer of the bone morphogenetic protein‐2/7 (BMP‐2/7) gene into periodontal tissues via in vivo electroporation induces exogenous BMP production and causes stem cells in periodontal tissues to differentiate into osteogenic cells, enabling generation of new alveolar bone.

2 Method

The BMP‐2/7 gene expression vector was introduced via electroporation into the target site in periodontal tissues of the first molar of rat maxillae.

3 Results

Exogenous BMP‐2 and ‐7 were detected in the target areas, and growth of new alveolar bone tissue was observed 5 days after gene transfer. On day 7, new alveolar bone tissues were found to connect to the original bone tissues. Moreover, mineral apposition rates of the alveolar bone after BMP‐2/7 gene transfer were significantly higher than those in the control group after LacZ gene transfer.

4 Conclusion

The present findings indicate that a combination of the BMP‐2/7 non‐viral vector and in vivo electroporation represents a promising non‐surgical option for alveolar bone regeneration therapy.     

利用细胞膜片技术进行牙周重建的研究进展

牙周炎是人类牙齿缺失的最主要病因,危害人类口腔及全身健康.牙周再生的目标是再生牙周组织和重建功能性牙槽骨-牙周膜-牙骨质牙周复合体.随着组织工程学和牙周再生技术的发展,细胞膜片技术的出现为牙周重建提供了新思路.现就细胞膜片技术在细胞膜片构建和不同来源种子细胞两方面的研究进展作一综述.     

1α,25-Dihydroxyvitamin D3 inhibits osteoblastic differentiation of mouse periodontal fibroblasts

ObjectivePeriodontal ligament (PDL) is a non-mineralized tissue connecting cementum and alveolar bone. Recent studies have suggested that periodontal fibroblasts can differentiate into mineralized tissue-forming cells such as osteoblasts and cementoblasts. We elucidated the immunolocalization of vitamin D receptor (VDR) and the effects of vitamin D₃ (VD₃) on mouse periodontal fibroblasts to clarify the role of VDR and VD₃ in the differentiation of periodontal fibroblasts.DesignImmunohistochemical analysis using anti-VDR antibody was performed in paraffin sections of mouse mandibles at E13, E14, E17, P10, and P35. The roles of VD₃ in osteoblastic differentiation and mineralization were estimated by alkaline phosphatase (ALP) and alizarin red (AR) staining. In addition, the mRNA expression of osteoblast markers and mineralization inhibitors was examined by real-time PCR.ResultsAt the bud, cap and early bell stages, immunoreactivity for VDR was scarcely seen in the cells of dental follicles. Labelling was detected in the nuclei of cemetoblasts, periodontal fibroblasts and osteoblasts during root formation. VD₃ inhibited ALP activity and AR-positive mineralized nodule formation of periodontal fibroblasts in osteogenic culture medium. Real-time PCR revealed that VD₃ down-regulated the levels of osteoblast markers. In contrast, VD₃ significantly up-regulated the level of periodontal ligament associated protein (PLAP)-1, a negative regulator of osteoblastic differentiation.ConclusionThese results suggest that VD₃ negatively regulates the osteoblastic differentiation of mouse periodontal fibroblasts and prevents the periodontal ligament from mineralization via increase of PLAP-1.     

The therapeutic role of bone marrow stem cell local injection in rat experimental periodontitis

Mesenchymal stem cell therapy brings hope for regenerating damaged periodontal tissues. The present study aimed to investigate the therapeutic role of local bone marrow stem cell (BMSC) injection in ligation-induced periodontitis and the underlying mechanisms. Alveolar bone lesion was induced by placing ligatures subgingivally around the bilateral maxillary second molars for 28 days. The alveolar bone lesion was confirmed by micro-CT analysis and bone histomorphometry. Allogeneic BMSC transplantation was carried out at 28 day after ligation. The survival state of the transplanted BMSC was observed by bioluminescent imaging. The implantation of the BMSC into the gingival tissues and periodontal ligament was confirmed by green fluorescent protein (GFP) immunohistochemical staining. The expression level of pro-inflammatory, tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and receptor activator of nuclear factor-κ B ligand (RANKL) and osteoprotegerin (OPG) in periodontal tissues were evaluated by immunohistochemical staining and real-time PCR. Significant reverse of alveolar bone lesion was observed after BMSC transplantation. The expression of TNF-α and IL-1β was down-regulated by BMSC transplantation. The number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in the periodontal ligament was reduced, and the increased RANKL expression and decreased OPG expression were also reversed after BMSC transplantation. It is concluded that allogeneic BMSC local injection could inhibit the inflammation of the periodontitis tissue and promote periodontal tissue regeneration.     

Periodontal tissue regeneration by transplantation of adipose tissue-derived stem cells

Periodontitis is the most common cause of tooth loss, and therefore great effort is directed towards developing therapies to counteract its progression. Recent advances have been made to regenerate periodontal tissue damaged by periodontitis. The topical application of recombinant cytokines such as platelet-derived growth factor and fibroblast growth factor-2 can activate endogenous stem cells existing within the periodontal ligament, resulting in enhancement of periodontal regeneration. These cytokine therapies have studied in both preclinical and clinical studies, and concentrated efforts are now being made to translate these techniques to cell-based therapies for enhancing periodontal regeneration. Although recent studies reported that transplantation of stem cells from various tissues, including bone marrow and periodontal ligaments, had positive effects on periodontal regeneration, these techniques are limited by problems with stem cell isolation and availability. Thus, we focused on adipose tissue-derived stem cells (ADSCs) because adipose tissue is abundant and is easily and safely accessible. Recently, we demonstrated that transplantation of ADSCs enhances periodontal tissue regeneration in beagle dogs. We have already completed preparations to begin clinical studies to evaluate the safety and efficacy of ADSCs in periodontal tissue regeneration. This review summarizes our knowledge of adipose stem cell therapies and explores its potential in periodontal therapy.