Periodontal Ligament-like Tissue Research Articles

Histological observations of pulpal replacement tissue in immature dog teeth after revascularization of infected pulps.

Many studies have examined the nature of tissue formed in the canals of immature necrotic teeth, following revascularization in animals and humans. While speculations have been made that regeneration of the pulp tissue might take place in the canal, the tissue has been found to be cementum-like, bone-like, and periodontal ligament-like. The purpose of this study was to histologically examine the tissue in the root canals in immature dog teeth that had been artificially infected and then revascularized. Two 4- to 5-month-old mongrel dogs with immature teeth were used in the study. In one dog, four maxillary and four mandibular anterior teeth, and in another dog, four maxillary and five mandibular anterior teeth were used in the experiment. Pulp infection was artificially induced in the immature teeth. Revascularization was performed on all teeth by disinfecting the root canals with sodium hypochlorite irrigation and triple antibiotic intracanal dressing, completed with induction of intracanal bleeding, and sealed with an MTA plug. The access cavity was restored with silver amalgam. The animals were sacrificed 3months after revascularization procedures. The revascularized teeth and surrounding periodontal tissues were removed and prepared for histological examination. Besides cementum-like, bone-like, and periodontal ligament-like tissues formed in the canals, residual remaining pulp tissue was observed in two revascularized teeth. In four teeth, ingrowth of alveolar bone into the canals was seen; presence of bone in the root canals has the potential for ankylosis. Within the limitation of this study, it can be concluded that residual pulp tissue can remain in the canals after revascularization procedures of immature teeth with artificially induced pulp infection. This can lead to the misinterpretation that true pulpal regeneration has occurred. Ingrowth of apical bone into the root canals undergoing revascularization can interfere with normal tooth eruption if ankylosis occurs.

1 荷重下における三次元In Vitroヒト歯根膜様組織の細胞生存と遺伝子発現(第544回大阪歯科学会例会)

1 荷重下における三次元In Vitroヒト歯根膜様組織の細胞生存と遺伝子発現(第544回大阪歯科学会例会)

Mesenchymal Stem/Progenitor Cell Isolation from Tooth Extraction Sockets

Bone marrow–derived mesenchymal stem/progenitor cells (BMSCs) are commonly used in regeneration therapy. The current primary source of BMSCs is the iliac crest; however, the procedure is associated with various burdens on the patient, including the risk of pain and infection. Hence, the possibility to collect BMSCs from other, more accessible, sources would be an attractive approach. It is well known that stem cells migrate from surrounding tissues and play important roles in wound healing. We thus hypothesized that stem/progenitor cells could be isolated from granulation tissue in the dental socket, and we subsequently collected granulation tissue from dog dental socket 3 d after tooth extraction. After enzyme digestion of the collected tissue, the cells forming colonies constituted the dental socket–derived stem/progenitor cells (dDSCs). Next, dDSCs were compared with dog BMSCs (dBMSCs) for phenotype characterization. A flow cytometric analysis showed that dDSCs were positive for CD44, CD90, and CD271 but negative for CD34 and CD45, similar to dBMSCs. dDSCs also exhibited osteogenic, adipogenic, and chondrogenic differentiation ability, similar to dBMSCs, with a higher capacity for colony formation, proliferation, and motility than dBMSCs. In addition, an in vivo ectopic bone formation assay showed that dDSCs and dBMSCs both induced hard tissue formation, although only dDSCs formed a fibrous tissue-like structure connected to the newly formed bone. Finally, we tested the ability of dDSCs to regenerate periodontal tissue in a one-wall defect model. The defects in the dDSC-transplanted group (β-TCP/PGA/dDSCs) were regenerated with cementum-like and periodontal ligament-like tissues and alveolar bone, whereas only bony tissue was observed in the control group (β-TCP/PGA). In conclusion, we identified and characterized a population of stem/progenitor cells in granulation tissue obtained from the dental socket that exhibited several characteristics similar to those of BMSCs. Dental sockets could therefore be a novel source for isolating stem/progenitor cells from bone.

Cell survival and gene expression under compressive stress in a three-dimensional in vitro human periodontal ligament-like tissue model.

This study investigated cell survival and gene expression under various compressive stress conditions mimicking orthodontic force by using a newly developed in vitro model of human periodontal ligament-like tissue (HPdLLT). The HPdLLT was developed by three-dimensional culturing of human periodontal ligament fibroblasts in a porous poly-L-lactide matrix with threefold increased culture media permeability due to hydrophilic modification. In vitro HPdLLTs in experimental groups were subjected to 5, 15, 25 and 35g/cm(2) compressive stress for 1, 3, 7 or 14days; controls were cultured over the same periods without compressive stress. Cell morphology and cell apoptosis in the experimental and control groups were investigated using scanning electron microscopy and caspase-3/7 detection. Real-time polymerase chain reaction was performed for seven osteogenic and osteoclastic genes. Similar extracellular matrix and spindle-shaped cells were observed inside or on the surface of in vitro HPdLLTs, with no relation to compressive stress duration or intensity. Similar caspase-3/7 activity indicating comparable apoptosis levels was observed in all samples. Receptor activator of nuclear factor kappa-B ligand and bone morphogenetic protein 2 genes showed characteristic "double-peak" expression at 15 and 35g/cm(2) on day 14, and alkaline phosphatase and periodontal ligament-associated protein 1 expression peaked at 5g/cm(2) on day 14; other genes also showed time-dependent and load-dependent expression patterns. The in vitro HPdLLT model system effectively mimicked the reaction and gene expression of the human periodontal ligament in response to orthodontic force. This work provides new information on the effects of compressive stress on human periodontal ligament tissue.

In vitro human periodontal ligament-like tissue formation with porous poly-l-lactide matrix

This study aimed to establish an in vitro human periodontal ligament-like tissue (HPdLLT) by three-dimensional culturing of human periodontal ligament fibroblasts (HPdLFs) in a porous poly-l-lactide (PLLA) matrix modified hydrophilically with ammonia solution. After ammonia modification, the surface roughness and culture-medium-soaking-up ability of the PLLA matrix increased, whereas the contact angle of water drops decreased. The thickness, porosity, and pore size of the PLLA matrix were 400±50μm, 83.3%, and 75–150μm, respectively. HPdLFs (1×105 cells) were seeded on the modified PLLA matrix and centrifuged to facilitate seeding into its interior and cultured for 14days. Scanning electron microscope (SEM) observation, proliferation assay, picrosirius-red staining, and real-time polymerase chain reaction (RT-PCR) for type-1 collagen (COL1), periodontal ligament associated protein-1 (PLAP-1), fibroblast growth factor-2 (FGF-2), and alkaline phosphatase (ALP) mRNA were conducted on days 1, 3, 7, and 14. HPdLFs were observed entirely from the surface to the rear side of the matrix. Cell proliferation analysis, SEM observation, and picrosirius-red staining showed both progressive growth of 3D-cultured HPdLFs and extracellular matrix maturation by the secretion of COL1 and type 3 collagen (COL3) from days 1 to 14. Expressions of COL1, PLAP-1, and FGF-2 mRNA suggested the formation of cellular components and supplementation of extracellular components. Expressions of ALP, COL1, and PLAP-1 mRNA suggested the osteogenic potential of the HPdLLT. The results indicated in vitro HPdLLT formation, and it could be used in future periodontal ligament tissue engineering to achieve optimal periodontal regeneration.

Effect of basic fibroblast growth factor on root resorption after delayed autotransplantation of tooth in dogs

The aim of this study was to investigate the effect of basic fibroblast growth factor (FGF-2) on root resorption after delayed autotransplantation in dogs. Mandibular second and third premolars of beagle dogs were extracted to create sites for autotransplantation. After 2 months, in the experimental sites the first and fourth mandibular premolars were extracted and air dried before autotransplantation with the application of recombinant FGF-2; the control sites received teeth without FGF-2. At 2, 4, or 8 weeks after surgery, the animals were killed and specimens collected and processed for histologic examination. Autotransplantation with FGF-2 yielded formation of new periodontal ligament-like tissues with inserting collagen fibers, associated cementum, and bone. The occurrence of replacement resorption in the FGF-2 treated group was significantly lower than in the control group (P < .01). It was demonstrated that topical application of FGF-2 reduced the occurrence of ankylosis and root resorption after delayed autotransplantation in this experimental model.

Cementum- and periodontal ligament-like tissue formation by dental follicle cell sheets co-cultured with Hertwig's epithelial root sheath cells

Dental follicle cells (DFCs) are believed contain the precursor cells of the periodontium and can form cell sheets by secreting extracellular matrix (ECM) proteins. Cell sheet engineering has been recently developed and applied successfully in the field of tissue regeneration. However, research on the in vitro characteristics of DFC sheets is lacking and an assessment of whether DFC sheets can produce periodontal tissues in vivo has not been reported. To test the characteristics and applicability of DFC sheets in this field, we established a co-culture system of rat DFCs and Hertwig's epithelial root sheath (HERS) cells in vitro, and included the following controls: a co-culture of DFCs and alveolar mucosa epithelial cells, DFCs with no cells in the upper chamber, and DFCs cultured without an upper chamber. After 3 weeks of co-culturing the cells, the DFC sheets were transplanted into adult male rats' omenta. One week after co-culturing DFCs with HERS cells, mRNA levels of collagen type I (COL-1), alkaline phosphatase (ALP), runt related transcription factor 2 (Runx 2) and bone sialoprotein (BSP) were increased significantly. In addition, after 3 weeks of co-culturing the cells, the numbers of ALP-, osteocalcin (OCN)-, BSP- and osteoprotegerin (OPG)-positive DFCs increased. The DFCs also produced more calcified nodules and exhibited an increased number of subcellular organelles, which are important for protein synthesis and secretion. Moreover, gap junctions were found between the experimental DFCs within the sheet. Five weeks of in vivo growth of DFC sheets pre-exposed to HERS cells led to the formation of cementum-like tissues, which were positive for OCN, BSP and OPG, as well as the formation of periodontal ligament-like tissues, which were positive for COL-1. In contrast, control cells only produced fibrous tissues. These results indicate that the DFC sheets induced by HERS cells are able to produce periodontal tissues through epithelial-mesenchymal interactions. Therefore, DFC sheets may be useful in the field of periodontium regeneration.

Regenerative effect of basic fibroblast growth factor on periodontal healing in two‐wall intrabony defects in dogs

The aim of the present study was to evaluate the effect of a basic fibroblast growth factor (bFGF) candidate treatment on periodontal healing in two-wall intrabony defects in dogs. Two-wall intrabony defects (5 x 5 x 5 mm) were created surgically on the distal and mesial sides of bilateral mandibular second and fourth premolars in four Beagle dogs. bFGF, enamel matrix derivative (EMD) and platelet-derived growth factor with beta-tricalcium phosphate (PDGF/beta-TCP) treatments, and sham-surgery (OFD) were rotated among the four defects in each animal, EMD and PDGF/beta-TCP serving as benchmark controls. The animals were euthanized for radiographic and histologic evaluation at 8 weeks. Bone formation was significantly greater in the bFGF group (4.11 +/- 0.77 mm) than in the EMD (3.32 +/- 0.71 mm; p<0.05) and OFD (3.09 +/- 0.52 mm; p<0.01) groups. The EMD (4.59 +/- 1.19 mm) and PDGF/beta-TCP (4.66 +/- 0.7 mm) groups exhibited significantly greater cementum regeneration with periodontal ligament-like tissue than the OFD group (2.96 +/- 0.69 mm; p<0.01). No significant differences were observed between the bFGF and the PDGF/beta-TCP groups in any of the histometric parameters. The candidate bFGF treatment supported periodontal regeneration comparable with that of established benchmarks: EMD and PDGF/beta-TCP.

Injectable Calcium Phosphate Bone Cement Provides Favorable Space and a Scaffold for Periodontal Regeneration in Dogs

An earlier study showed that an injectable calcium phosphate cement (CPC) served as a stable scaffold for bone formation and promoted histocompatible healing of periodontal tissue in dogs. In this study, we evaluated the influence of CPC on regeneration of periodontal defects with experimental periodontitis in dogs. Experimental periodontitis was induced by placing stainless-steel mesh on the mesial side of maxillary canines in six adult, healthy beagle dogs. Subsequently, intrabony defects were resized so as to be standard, and CPC was injected in the experimental bone defects. Non-grafted defects on the contralateral side served as controls. Twelve weeks after surgery, the animals were sacrificed and histologic specimens were prepared. Periodontal tissue healing was evaluated histologically and histometrically. Healing of periodontal tissues, in terms of bone and cementum formation, was consistently observed in the CPC-applied sites. CPC was partly replaced by new bone. New cementum and periodontal ligament-like tissue were observed between CPC and the root surface. New bone (P <0.05), new cementum (P <0.01), and new connective tissue attachment and adhesion (P <0.05) were significantly enhanced in the experimental sites. Calcium phosphate cement provides stable wound healing and enhanced periodontal regeneration in periodontal defects in dogs with experimental periodontitis.

Human Periodontal Ligament Cell Sheets Can Regenerate Periodontal Ligament Tissue in an Athymic Rat Model

Conventional periodontal regeneration methods remain insufficient to attain complete and reliable clinical regeneration of periodontal tissues. We have developed a new method of cell transplantation using cell sheet engineering and have applied it to this problem. The purpose of this study was to investigate the characteristics of human periodontal ligament (HPDL) cell sheets retrieved from culture on unique temperature-responsive culture dishes, and to examine whether these cell sheets can regenerate periodontal tissues. The HPDL cell sheets were examined histologically and biochemically, and also were transplanted into a mesial dehiscence model in athymic rats. HPDL cells were harvested from culture dishes as a contiguous cell sheet with abundant extracellular matrix and retained intact integrins that are susceptible to trypsin-EDTA treatment. In the animal study, periodontal ligament-like tissues that include an acellular cementum-like layer and fibrils anchoring into this layer were identified in all the athymic rats transplanted with HPDL cell sheets. This fibril anchoring highly resembles native periodontal ligament fibers; such regeneration was not observed in nontransplanted controls. These results suggest that this technique, based on the concept of cell sheet engineering, can be useful for periodontal tissue regeneration.

骨形成タンパク質添加ＤＮＡ／アテロコラーゲンによるビーグル犬歯周組織の再生

A unique biomaterial, DNA/atelocollagen membrane, was newly developed. The effect of DNA/atelocollagen combined with bovine derived-bone morphogenetic proteins (BMP) on osteogenesis was evaluated histologically in subcutaneous tissue and supra-alveolar and periodontal defects. DNA/atelocollagen was prepared as a composite of DNA from salmon milt and pepsin-digested type I collagen (atelocollagen) from bovine dermis. The composite fiber showed structural characteristics of DNA-coated atelocollagen on phase-contrast and fluorescence microscopy. The time-course of bone induction was investigated, using a rat ectopic bone formation assay. DNA/collagen (10×5×1mm) was subcutaneously implanted with 100μg of BMP induced osteoblast differentiation 1 week after operation. Histological examination of the BMP/DNA/collagen showed direct bone formation, and carrier DNA/collagen fibers were found in woven bone. Wounds through the supra-alveolar bone and periodontal ligament (10×5×5mm) were surgically created on the mesial root of the mandibular second and third premolars in 6 beagle dogs. The defects were immediately filled with either DNA/collagen (10×5×5mm) combined with 500μg of BMP or DNA/collagen alone, or were left unfilled (controls). Animals treated with BMP/DNA/collagen showed regeneration of alveolar bone, periodontal ligament, and cementum on the exposed dentin at 4 and 6 weeks, while the other groups showed virtually no regeneration. In addition, the newly formed bone and cementum were separated by a periodontal ligament-like tissue, and no ankylosis was seen. These results indicate that DNA/collagen is an effective absorbable material that can be used as a carrier matrix for BMP. DNA/collagen sponge combined with BMP may promote tissue regeneration without disturbing periodontal homeostasis in supra-alveolar and periodontal defects.

Histocompatible healing of periodontal defects after application of an injectable calcium phosphate bone cement. A preliminary study in dogs.

A novel injectable, fast setting calcium phosphate cement (CPC) is currently used in orthopedic therapy for bone fractures. This study evaluated the possibility of applying this cement to healing periodontal defects. Fenestrations and 3-walled periodontal defects were surgically created on bilateral first molars and canines in 5 beagle dogs. CPC was applied to the defects on one side of the mandible. Untreated defects on the contralateral side served as controls. CPC was applied to all defects in the maxilla. Twelve weeks after surgery, the animals were sacrificed and decalcified and undecalcified specimens were prepared. Periodontal tissue healing was evaluated histologically and histometrically under a light microscope. Healing of periodontal tissues in terms of bone and cementum formation was consistently observed in the CPC-applied sites. CPC was partly replaced by new bone. The residual CPC appeared detached from the denuded root surface. New cementum and periodontal ligament-like tissue were observed between the detached CPC and root surface. No unfavorable reaction was noted in the CPC-applied sites. No statistically significant difference was noted in the experimental or control sites under histometric analysis. Although there were no statistically significant differences between the 2 treatment groups, histological observation indicated that CPC seemed to act as a scaffold for bone formation and provided histocompatible healing of periodontal tissues in this study. This cement might be applicable to periodontal therapy; however, further investigations are required.

ハイブリッド型人工歯根に関する基礎的研究 ヒト歯根膜由来細胞の増殖および分化機能に及ぼす人工歯根用結晶化ガラスの影響について

The hybrid-type dental implants which are composed of substrates and periodontal ligament-derived cells are expected to form the periodontal ligament-like tissue around the dental implant. The purpose of this study was to investigate the possibility of the application of glass-ceramics (GC) as the substrate using the human periodontal ligament-derived cells (PL cells). The growth and differentiation of the PL cells on the GC were investigated, as compared to the hydroxyapatite plates and the conventional plastic dishes. The Ca concentration of the media was also measured. The results were as follows: 1) The PL cells were fibroblastic of spindle shape. The population doubling time was approximately 26 hours. 2) The PL cells exhibited an alkaline phosphatase (ALPase) activity, collagen productivity and responsiveness to 1 alpha,25-dihydroxyvitamin D3. 3) The PL cells proliferated favorably both on the mirror-polished and #2000-polished GC plates as well as on the hydroxyapatite plates. 4) The ALPase activity of the PL cells on the GC increased in a time-dependent manner. 5) The GC released Ca into the media. The PL cells possessed some characteristics of periodontal ligaments, and the growth and differentiation of the PL cells were not impaired significantly on the GC. The GC also provided the dental implant with enough biocompatibility and strength. These results promised the usefulness of the GC as the possible substrate for the hybrid-type dental implants.

Intentional vital root transection: A 52-week histopathologic study in Macaca mulatta

Past studies dealing with the vascular supply to surgically mobilized dentoalveolar segments have mentioned occasional encounters with vital root transection and have noted varying degrees of pulpal response to this insult. The purpose of this investigation was to observe the pulpal and periapical responses of monkey dental tissues to intentional vital root transection over a 1-year postoperative period. Four adult Macaca mulatta monkeys were used in this study. All roots were surgically transected within the apical third with a bur. Maxillary and mandibular quadrants for 1, 2, 3, 4, 6, 12, 24, and 52 weeks following surgery were obtained after the animals were killed by perfusion of the left ventricle. The tissue blocks were prepared by routine histologic methods. The results demonstrated a disruption of the normal pulpal architecture, with initial pulpal degeneration and subsequent early replacement by the periodontal ligament tissue. A cellular cemental lining of the root canal occurred in all specimens. The periodontal ligament-like tissue continued to deposit cementum, resulting in almost total obliteration of the root canal and pulp chamber and leaving an intact but much reduced blood supply to the pulp chamber containing periodontal tissues.