Journal of Hard Tissue Biology Vol. 26(2017) No. 2

Advanced extraction and purification techniques were found to be essential tools for obtaining sufficient DNA from bones. However, in case of short tandem repeats (STRs) typing failure especially with old skeletal remains, mitochondrial DNA (mtDNA) analysis is the ultimate solution for individual and species identification. Thirty six bone samples were collected from human remains. DNA was extracted using the organic method after special sample preparations and quantified using a Real-time polymerase chain reaction (RT-PCR). Polymerase chain reaction (PCR) was done using Identifiler Plus PCR Amplification Kit and amplified products were typed using a Genetic Analyzer for autosomal STR typing. Samples with DNA concentration above 0.04 ng were efficient in obtaining their complete STR profiles. MtDNA control region was also amplified and sequenced. MtDNA was more efficient than autosomal STR profiling in discriminating among human bone samples especially those which have low and/or degraded DNA content.

The minimally invasive surgery (MIS) yields favorable clinical outcomes for periodontal regeneration therapy. However, there have been few reports on the histological analysis underlying these outcomes. In this study, we created periodontal defects for investigate two different surgical procedures by histological assessment at the early wound healing process. The maxillary second molars of Sprague-Dawley (SD) male rats at twelve-week-old were used. In the control (open flap) left maxillary, mucoperiosteal flap resection was performed on the first to third molars of both the buccal and palate sides. The right maxillary served as the experimental (minimally invasive) side and the resection was performed only on the second molar of the palatal side. Periodontal defects were then created to the palatal root of second molar at the both sides with round bur. On each of days 1, 3, and 5 after surgery, five rats were euthanized. Sections were prepared and subjected to hematoxylin–eosin staining, and immunohistochemical staining on type III collagen followed by microscopic observations and statistical analysis. In the experimental side, the inflammatory cell infiltration disappeared earlier compared to the control side. The defect area was positive for type III collagen staining in the experimental side, which were significantly greater on days 1, 3, and 5 after surgery. The results suggest that wound healing accelerated by wound stability caused by fibrous tissue generation with type III collagen formation, using minimally invasion by single flap line periodontal surgery, caused by fibrous tissue generation.

We previously developed a hamster oral papilloma virus (HOPV) tumor model. We also showed that immunization with a DNA vaccine consisting of naked plasmid DNA (pDNA) encoding the L1 region of the HOPV genome (pHOPV-L1) had a cancer suppressing effect in this model. Here, we investigated if the use of electroporation as a vaccine delivery system could enhance the effect of this vaccine. A pHOPV-L1 vaccine was generated and was inoculated (100 µg/animal) intramuscularly into hamsters (n=10 per group), who then underwent eight rounds of electroporation of 50, 100, or 200 V/cm (VE50, VE100, and VE200 group, respectively). Control groups (n=10 per group) were untreated (N), or were treated with electroporation alone (E50, E100, and E200 groups) or vaccination alone (V group). The animals then underwent carcinogenic treatment over the next 69 days, during which 9,10-dimethyl-1,2-benzanthracene (DMBA) was applied onto lingual mucosa and a lingual wound was created. Histopathological analysis of lingual tissue indicated that all of the animals in the N and E groups developed lingual carcinoma that was accompanied by koilocytosis in the squamous carcinoma lesions. In contrast, 4, 5, 7 and 9 animals in the V, VE50, VE100 and VE200 groups, respectively, showed no carcinoma lesions. All of the animals with carcinomas ultimately showed a decreasing trend in weight, as well as HOPV infection that was detected using real-time PCR analysis. On the other hand, all animals without carcinomas increased in weight over the entire course of the experiment and did not display HOPV infection. These data confirm that a vaccine consisting of naked pDNA from the L1 region of HOPV can suppress HOPV-associated cancer, and show that its cancer suppressing effects can be enhanced by low-voltage electroporation following inoculation. We conclude that low-voltage electroporation is a useful and safe delivery system for DNA vaccines and should be further exploited.

The purpose of this study was to investigate the appearance of lymph nodes draining areas of periodontitis in the mandible using axial short T1 inversion recovery magnetic resonance imaging, to see if there is a characteristic pattern that may aid in diagnosis and treatment monitoring. The number and short-axis diameter of submental lymph nodes, submandibular nodes, superior internal jugular nodes, and spinal accessory nodes were measured on magnetic resonance images in 216 subjects (97 patients diagnosed with periodontitis, age 21–81 years and 119 patients undergoing magnetic resonance imaging of the brain without any diseases that would affect the mandible or lymph nodes, age 29-79 years). Between-group differences in the number and diameter of the nodes were analyzed. The size and number of submental nodes, submandibular nodes, and superior internal jugular nodes were significantly different between the periodontitis group and the non-periodontitis group (p < 0.01). The size and number of spinal accessory nodes were not significantly different between the two groups (p > 0.05). Our study found that a definite pattern of lymphadenopathy is associated with periodontitis. These findings indicate that lymphadenopathy should be considered as an inflammation condition commonly associated with periodontitis.

The objective of this study was to investigate spontaneous changes in the mandibular intermolar widths concurrent with protraction facemask treatment combined with slow maxillary expansion in skeletal Class III children, and to evaluate whether slow expansion has favorable effects on maxillary protraction. Twenty-three patients were divided into expansion and non-expansion groups. The expansion group comprised 11 children (mean age, 6.9 ± 1.0 years) who underwent protraction facemask treatment combined with slow maxillary expansion. The non-expansion group consisted of 12 children (mean age, 7.8 ± 1.1 years) who underwent protraction only. Dental casts and lateral cephalograms obtained before and after protraction were used to analyze occlusal and skeletal changes during approximately 1 year of treatment. The expansion group showed significantly larger increments than the non-expansion group in all mandibular intermolar measures (P < .01). No significant differences in skeletal changes were seen for any cephalometric measures between groups. In conclusion, spontaneous increases in mandibular intermolar widths were found during maxillary protraction combined with slow expansion treatment. No favorable skeletal effects of slow expansion on protraction were confirmed.

In the human molar, multirooted tooth formation occurs through the formation of dentin islands, termed subpulpal lobus, in the dental papilla. However, the mechanisms underlying subpulpal lobus formation currently remain unclear. Thus, using the rat molar, in which multirooted teeth formed through subpulpal lobus formation, similar to that in humans, we observed the process of subpulpal lobus formation over multirooted tooth formation histologically and immunohistologically, and discussed accessory root canal formation in the furcation area. The upper second molar (M2) was examined from rats 8 to 18 days after birth, stained with hematoxylin, and observed under a stereoscopic microscope. The maxilla including M2 was dissected out en bloc and decalcified, and paraffin-embedded sections were prepared following the standard method. Sections were subjected to hematoxylin-eosin double staining and immunohistochemical staining with anti-pan-keratin (PK), anti-heat shock protein 27 (HSP27), and anti-sonic hedgehog (Shh) antibodies. After the completion of tooth crown formation, a part of Hertwig’s epithelial root sheath (HERS) in the cervical region extended and formed an epithelial projection (EP) that was reactive with the anti-PK antibody. The EP induced the differentiation of dental papilla cells facing it into anti-Hsp27 antibody-reactive odontoblasts and formed the subpulpal lobus. EP and subpulpal lobus-forming odontoblasts reacted with the anti-Shh antibody, suggesting that cell differentiation in subpulpal lobus formation occurs through the epithelial-mesenchymal interaction. While forming subpulpal lobus, the EP extended without making contact with the opposing EP or HERS in the cervical region. The furcation area was formed by fusion between the dentin projection (DP) formed in the cervical region and subpulpal lobus and between subpulpal lobus. HERS in the cervical region and EP formed the furcation area without making contact with each other, suggesting that the accessory root canal is formed where DP and the subpulpal lobus do not fuse.

Porcine placenta extract (P-placenta) is widely applied in medicine and cosmetics. However, few studies have examined the effect of the extract on the cellular behavior of the osteoblastic cell line Saos-2. Here, we demonstrated that P-placenta enhances the proliferation, collagen type I production, and alkaline phosphatase (ALP) secretion of Saos-2 in vitro. Proliferation of Saos-2 was assessed by MTT and DNA synthesis assays. Type I collagen production and ALP secretion were evaluated using enzyme-linked immunosorbent assay and ALP assays. The cells were treated with/without 20, 200 and 2000 μg/ml of P-placenta for 24 h. We found that 200 μg/ml P-placenta significantly induced the proliferation of Saos-2 and enhanced type I collagen production and ALP secretion. The results indicate that P-placenta controls the cellular behavior of osteoblasts, resulting in the secretion of early bone-related biomarkers.

Marx was the first to report osteonecrosis of the jaw due to administration of BPs in 2003. Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is the result of an adverse drug reaction. Moreover, many studies worldwide have reported an association between a range of serious dental diseases and the use of bisphosphonates (BPs). Few studies, however, have evaluated BRONJ based on an abnormal signal detected by MRI in the bone marrow of the mandibular condyle. The aim of this study was to assess changes in the magnetic resonance imaging (MRI) signal from the mandibular condyle that could be due to BRONJ. In particular, we focused on the presence of an abnormal MRI signal emanating from mandibular condyle bone marrow. Twenty-eight patients (11 men, 17 women; 56 temporomandibular joints) with BRONJ were evaluated for jaw pain. The patients, whose mean age was 72.9 ± 9.4 years (range 48 - 88 years), underwent MRI examination of the jawbone at our hospital from August 2006 to December 2015 and were included in the study. Overall, 80.0% of the patients diagnosed with BRONJ exhibited an abnormal bone marrow signal in the mandibular condyle on the same side of the face that suffered jaw pain. This abnormal signal was present significantly more frequently on the side of the face with the jaw symptoms than on the side without symptoms. Patients with BRONJ displayed an abnormal MRI signal in the mandibular condyle on the side of the face with jaw symptoms, suggesting that MRI findings could be useful clinically for detecting BRONJ in the mandibular condyle.

In this study, we performed animal experiment using calcium hydroxide paste, a root canal filling material (Vitapex®, Neo Dental Chemical Products Co., Tokyo), on dentin-pulp complex and observed several tissue reactions. While rats were under general anesthesia, pulp exposure was done by drilling a cavity on the occlusal part of the maxillary molar using 1/2 round bur. Thereafter, calcium hydroxide paste was injected into the cavity, temporarily sealed with composite resin and photographed using m_CT. After 4 weeks, the experimental part was surgically excised as a whole and examined histologically. A thick tertiary dentin was formed in the area where iodoform calcium hydroxide paste was directly applied. The newly formed hard tissue is composed of extremely irregular dentinal tubules. Although only few samples confirmed the formation of the so-called ‘dentin bridge’, the hard tissue could not be absolutely classified as a ‘reparative dentin’ but it can be recognized as a hard tissue connecting the dentin walls as it filled or covered the exposed pulp. The tertiary dentin formed underneath was thick. No necrotic layer was observed. Photographs of m_CT of the experimental side showed that the hard tissue formed in the root canal was radiopaque. However in the part of root canal where no hard tissue was formed, a radiolucent image was observed in the center. In this regard, a thick tertiary dentin was formed rather than a dentin bridge filling the gap. Since this is uncommon for calcium hydroxide, it is regarded as a distinct characteristic of Vitapex when applied to an exposed pulp. This phenomenon was thought to be due to the silicone oil component. The results suggest that the silicone oil reduced the alkalinity of the material thereby making it more amiable as a pulp capping agent. Furthermore, it is believed that the odontoblasts were promptly activated which led to the formation of large amounts of dentin.

Transforming growth factor (TGF)-β are strongly associated with osteoblast differentiation. Mechanical stress including compressive force (CF) also involve in osteoblast differentiation via the alteration of the expression of bone-specific transcription factors, namely Runx2 and Osterix. However, the role of TGF-βs in mediating the effects of CF on osteoblasts remains unclear. In the present study, we examined the effects of CF on the expression of TGF-β1, TGF-β2, TGF-β type 1 and 2 receptor (Tβr1 and Tβr2), and Runx2 and Osterix in osteoblasts. We also investigated the effects of CF on the phosphorylation of Smad2, Smad3 and p38; which were located on the downstream of Tβrs. Effect of Tβr inhibitor (LY2109761) on expression of Runx2 and Osterix, and phosphorylation of Smad2, Smad3 and p38 were additionally examined. Cultured MC3T3-E1 osteoblast-like cells were subjected, or not, to continuous CF (1.0 g/cm² or 2.0 g/cm²) for 1–9 h. TGF-β1, TGF-β2, Tβr1, Tβr2, Runx2 and Osterix expression were measured by real-time polymerase chain reaction and Western blot analysis. Phosphorylation levels of Smad2, Smad3 and p38 were determined by Western blot analysis. The mRNA and protein expression of TGF-β1 and TGF-β2 were significantly increased by 1.0 g/cm², but not 2.0 g/cm², CF for 3–6 h, relative to control cells; Tβr1 and Tβr2 expression were unaffected by either CF condition. The 1.0 g/cm² CF also increased the phosphorylation of Smad2, Smad3 and p38 and the expression of Runx2 and Osterix, and these increases were attenuated by pretreatment with LY2109761. The present findings indicate that 1.0 g/cm² CF can induce bone-specific transcription factors via autocrine action of CF-induced TGF-β signaling in osteoblasts.

Clinical periodontal status is among the major determinants of the volumetric features of GCF. Previous studies have shown that probing depth, presence/severity of gingival and periodontal inflammation affect the biodynamics of GCF. The aim of the present study was to evaluate the impact of the clinical periodontal status on volumetric features of gingival crevicular fluid. Thirty-six patients were equally divided into healthy group, gingivitis group and periodontitis group. Clinical periodontal status was assessed by recording: papillary bleeding index, probing pocket depth and clinical attachment level. Gingival crevicular fluid (GCF) samples were obtained from 4 sites in each patient. The mean volume of GCF was determined in each group for comparison. Further in periodontitis group, GCF samples were also collected from healthy sites, gingivitis sites and periodontitis sites. Mean GCF volume recorded by using Periotron® 8000 in healthy gingival group, gingivitis group and periodontitis group were: 0.04, 0.09, 0.78 µl respectively. There was increase in GCF volume in a diseased related pattern, healthy gingival group < gingivitis group < periodontitis group. In periodontitis group, the sites with healthy gingiva showed significantly higher GCF volume than healthy gingival group (0.11µl Vs 0.04µl), similarly, the sites with gingivitis in periodontitis group also showed significantly higher GCF volume than gingivitis group (0.32µl Vs 0.11µl). The present study suggests that the volumetric analysis of GCF could be sensitive and reliable indicator of periodontal health. The ability of GCF volume as a chair side measure to differentiate healthy sites from sites with mild disease within the same mouth is considered noteworthy.

The purpose of this study was to evaluate the dental arch relationship of non-syndromic Malay unilateral cleft lip and palate children and assess the various congenital and postnatal treatment factors that affect dental arch relationship. Study models of 107 UCLP children were included in this study that was treated in Hospital Universiti Sains Malaysia over a period of 10 years (2000-2012). The mean age was 7.69± 2.46 (mean± SD). The dental arch relationship was assessed by mHB scoring system which comprises five categories; named-excellent; good; fair; poor and very poor. All the subjects were divided into two groups; favorable (category ratings excellent, good and fair) and unfavorable (category ratings poor and very poor) groups. The mean mHB score was - 10.7. Total 60 subjects (68% of all subjects) were categorized into unfavourable group (category ratings poor and very poor) using mHB scoring system. Intra- and inter-examiner agreements were very good. Cheiloplasty seemed to be correlated with favourable dental arch relationship using crude regression analysis but no significant associations were found. This multivariate study shows no significant association between various congenital and postnatal treatment factors and dental arch relationship.

The purpose of this study was to investigate the property of hydroxyapatite (HA)/gelatin hydrogel (GH) composite granules as a scaffold and controlled-release carrier of basic fibroblast growth factor (bFGF) for bone regeneration. HA granules and GH microsphere at various weight ratios to the HA granules were mixed. The composite provided the controlled release of bFGF in vitro. The critical-sized bone defects of 6 mm diameter were created in the skull of New Zealand white rabbits and the composite granules with or without incorporated bFGF, were implanted into the defects. Bone regeneration and were evaluated by computed tomography and H-E staining. The composite granules incorporating bFGF promoted significantly higher bone regeneration at the defect site as compared to the bFGF-free composites. Further, the amount of new bone increased in proportion to the amount of GH microspheres incorporated into the composite. We conclude that the bFGF-impregnated HA/GH composite granules is useful for bone regeneration.

Osteoporosis is a major problem in the elderly population worldwide. Low calcium intake and vitamin D blood level are risk factors for osteoporosis, and improving their intake is effective in patients with micronutrients deficiency. However, the effect of these interventions is ambiguous. Additive formula diet (AFD) contains fructo-oligosaccharide (FOS), isoflavone (ISO) and 1.0% citric acid Ca as a supplement for patients with osteoporosis. We aimed to investigate the effect of AFD on bone structure of the femur in ovariectomized rats. Sixteen 20-week old ovariectomized rats were randomly distributed into 2 groups; one group was fed normal diet (N, n = 8) and the second group was fed AFD (A, n = 8). Both groups were fed for 24 weeks, and body weight was measured at 8 and 24 weeks. After measuring the weight at 24 weeks, rats were euthanized using carbon dioxide. Lateral femur bone was extracted, and bone mineral density (BMD) and bone mineral content (BMC) were measured via micro computed tomography. Non-decalcified ground sections of the femur were examined via polarized-light microscopy. At 24 weeks, BMD and BMC were significantly higher for the A group than in the N group. The A group showed significantly better structural values with respect to Tb.Th, Tb.N, Tb.sp, Tb.spac and SMI than the N group. The A group showed significantly denser trabecular observations than the N group. Examination of the non-decalcified ground section from the A group showed strong polarized light properties of orange compared with sections from the N group. AFD may improve bone turnover in osteoporosis with the expectant decrease in the incidence of falls and bone fractures, which may enhance the quality of life of the elderly.

The aim of this study was to evaluate the osteogenic potential of demineralized dentin matrix (DDM) in supporting the osteogenic activity of MG-63 cells and compare it to a mixture of inorganic bovine bone and collagen (Bio-Oss Collagen^®). Cell morphology, differentiation, adhesion, and growth were investigated via scanning electron microscopy and immunofluorescence analyses of F-actin, osteocalcin, and osteonectin. Cell adhesion and growth on the DDM as a bone graft material were more abundant, with cells adopting a flat shape and uniform distribution, than on the Bio-Oss Collagen^® at all the observation time points (i.e., 6, 12, 24, 48, 72 h and 14 days). Cell attachment and cytoskeleton organization assessed using confocal laser scanning microscopy revealed that the DDM provided better attachment with cytoplasmic propagation than the control. Immunofluorescence assays showed that the fluorescent intensities of osteocalcin and osteonectin as biomarkers of cellular differentiation were higher on the DDM than on Bio-Oss Collagen^® at 6, 12 and 24 h. We showed that the DDM had an enhanced osteogenic potential as a bone substitute for bone augmentation procedures in the dental and medical fields.

This retrospective study evaluated the utility of an orbital wall reconstruction plate system for orbital fractures with relatively large orbital-wall defects. We analyzed the clinical and radiological data of seven patients with orbital fractures. All of the patients suffered midfacial trauma with fractures extending to the orbit. Ophthalmologists diagnosed functional eye complications, including diplopia, enophthalmos, restricted eyeball mobility, and reduced globe motility. The fractures with orbital-wall defects were located in the orbital floor in four (57.1%) and the floor/medial wall in three (42.9%). A 0.4-mm-thick orbital wall reconstruction plate (anatomically preformed titanium mesh plate system) was used for orbital reconstruction together with midfacial fracture reduction with stable internal fixation at the inferior orbital rim. The fractures were assessed preoperatively using computed tomography (CT). The accuracy of the three-dimensional (3D) reconstructed orbital volume with the correct positioning of the anatomically preformed titanium mesh plate system was assessed postoperatively with CT. All of the patients had satisfactory clinical results with no complications associated with this system and the open reduction and internal fixation. There was full recovery of ophthalmological function and active eyeball mobility, without enophthalmos or diplopia. The postoperative CT data showed that the injured orbits had been corrected anatomically based on a precise postoperative 3D volume reconstruction using this system. There was no significant difference compared with the uninjured orbital 3D volume. In conclusion, the reconstruction of orbital wall fractures related to midfacial factures with the anatomically preformed titanium mesh plate system for the orbital reconstruction is a promising surgical technique.

Combined orbital floor and medial wall defect fractures can be challenging to repair. The aim of this retrospective clinical study was to evaluate the feasibility of single folded uncalcined and unsintered hydroxyapatite (u-HA) particles and poly-L-lactide (PLLA; u-HA/PLLA) composite sheets with a tack fixation technique in computer-assisted combined orbital floor and medial wall complicated fracture reconstruction. This study included five patients (mean age, 27.2 years) with relatively large complex orbital floor and medial wall defects (three type III defects and two type IV defects). A single folded u-HA/PLLA sheet was fabricated and prepared with an average optimal orbital floor to mesial walls angle (OHMW-angle) of 130.84° ± 5.42; this was followed by adaption and stable fixation with tacks to the inferior orbital rim using computer-assisted and intraoperative navigation-assisted orbital reconstructive surgery. The clinical and radiological data were analyzed, and ophthalmologists diagnosed functional eye complications with a mean follow-up period of 9.1 months (range, 6 to 18 months). Although the postoperative computed tomography (CT) study revealed accurate three-dimensional (3D) orbital wall reconstruction, a slight OHMW-angle increase, to 133.68°± 4.28, between the orbital floor and medial reconstructed walls was observed 1 month postoperatively compared with immediately postoperatively (p < .05), but no other angle changes were observed in the postoperative follow-up study (p > .05). There was full recovery of ophthalmological function and active eyeball mobility without enophthalmos or diplopia. Overall, computer-assisted single folded u-HA/PLLA sheets with a tack fixation technique provide stable and satisfactory ophthalmologic functional results for combined orbital floor and medial wall fracture reconstruction with no intraoperative or postoperative complications. Furthermore, this u-HA/PLLA composite sheet could be an optimal next-generation bioactive osteoconductive implant material for the reconstruction of relatively large orbital wall defects, which could be applied using computer-assisted surgery.