Detection of unilateral and bilateral cleft alveolus on panoramic radiographs using a deep-learning system.

OBJECTIVES: The purpose of this study was to evaluate the difference in performance of deep-learning (DL) models with respect to the image classes and amount of training data to create an effective DL model for detecting both unilateral cleft alveoli (UCAs) and bilateral cleft alveoli (BCAs) on panoramic radiographs. METHODS: Model U was created using UCA and normal images, and Model B was created using BCA and normal images. Models C1 and C2 were created using the combined data of UCA, BCA, and normal images. The same number of CAs was used for training Models U, B, and C1, whereas Model C2 was created with a larger amount of data. The performance of all four models was evaluated with the same test data and compared with those of two human observers. RESULTS: The recall values were 0.60, 0.73, 0.80, and 0.88 for Models A, B, C1, and C2, respectively. The results of Model C2 were highest in precision and F-measure (0.98 and 0.92) and almost the same as those of human observers. Significant differences were found in the ratios of detected to undetected CAs of Models U and C1 (p = 0.01), Models U and C2 (p < 0.001), and Models B and C2 (p = 0.036). CONCLUSIONS: The DL models trained using both UCA and BCA data (Models C1 and C2) achieved high detection performance. Moreover, the performance of a DL model may depend on the amount of training data.

Deep-learning systems for diagnosing cleft palate on panoramic radiographs in patients with cleft alveolus.

OBJECTIVES: The aim of the present study was to create effective deep learning-based models for diagnosing the presence or absence of cleft palate (CP) in patients with unilateral or bilateral cleft alveolus (CA) on panoramic radiographs. METHODS: The panoramic images of 491 patients who had unilateral or bilateral cleft alveolus were used to create two models. Model A, which detects the upper incisor area on panoramic radiographs and classifies the areas into the presence or absence of CP, was created using both object detection and classification functions of DetectNet. Using the same data for developing Model A, Model B, which directly classifies the presence or absence of CP on panoramic radiographs, was created using classification function of VGG-16. The performances of both models were evaluated with the same test data and compared with those of two radiologists. RESULTS: The recall, precision, and F-measure were all 1.00 in Model A. The area under the receiver operating characteristic curve (AUC) values were 0.95, 0.93, 0.70, and 0.63 for Model A, Model B, and the radiologists, respectively. The AUCs of the models were significantly higher than those of the radiologists. CONCLUSIONS: The deep learning-based models developed in the present study have potential for use in supporting observer interpretations of the presence of cleft palate on panoramic radiographs.

Transplantation of cultured dental pulp stem cells into the skeletal muscles ameliorated diabetic polyneuropathy: therapeutic plausibility of freshly isolated and cryopreserved dental pulp stem cells.

INTRODUCTION: Dental pulp stem cells (DPSCs) are mesenchymal stem cells located in dental pulp and are thought to be a potential source for cell therapy since DPSCs can be easily obtained from teeth extracted for orthodontic reasons. Obtained DPSCs can be cryopreserved until necessary and thawed and expanded when needed. The aim of this study is to evaluate the therapeutic potential of DPSC transplantation for diabetic polyneuropathy. METHODS: DPSCs isolated from the dental pulp of extracted incisors of Sprague-Dawley rats were partly frozen in a -80 °C freezer for 6 months. Cultured DPSCs were transplanted into the unilateral hindlimb skeletal muscles 8 weeks after streptozotocine injection and the effects of DPSC transplantation were evaluated 4 weeks after the transplantation. RESULTS: Transplantation of DPSCs significantly improved the impaired sciatic nerve blood flow, sciatic motor/sensory nerve conduction velocity, capillary number to muscle fiber ratio and intra-epidermal nerve fiber density in the transplanted side of diabetic rats. Cryopreservation of DPSCs did not impair their proliferative or differential ability. The transplantation of cryopreserved DPSCs ameliorated sciatic nerve blood flow and sciatic nerve conduction velocity as well as freshly isolated DPSCs. CONCLUSIONS: We demonstrated the effectiveness of DPSC transplantation for diabetic polyneuropathy even when using cryopreserved DPSCs, suggesting that the transplantation of DPSCs could be a promising tool for the treatment of diabetic neuropathy.

N-acetyl cysteine as an osteogenesis-enhancing molecule for bone regeneration.

Bone regeneration often requires cues from osteogenesis-inducing factors for successful outcome. N-acetyl cysteine (NAC), an anti-oxidant small molecule, possibly modulates osteoblastic differentiation. This study investigated the potential of NAC as an osteogenesis-enhancing molecule in vitro and in vivo. Various concentrations of NAC (0, 2.5, 5.0, and 10 mM) were added to rat bone marrow stromal cell or osteoblastic cell culture in media with or without dexamethasone. The results showed marked enhancement of alkaline phosphatase activity and mineralized matrix formation together with consistent upregulation of bone-related gene markers such as collagen I, osteopontin, and osteocalcin in the osteoblastic culture with addition of 2.5 or 5.0 mM NAC regardless of the presence of dexamethasone. Micro-CT-based analysis and histological observation revealed that addition of NAC to a collagenous sponge implanted in a critical size cortical bone defect (3.0 mm × 5.0 mm) in rat femur yielded acceleration and completion of defect closure, with thick, compact, and contiguous bone after 6 weeks of healing. In contrast, with sponge alone, only sparse and incomplete bone regeneration was observed during the matching healing period. These results indicate that NAC can function as an osteogenesis-enhancing molecule to accelerate bone regeneration by activating differentiation of osteogenic lineages.

Mechanical stretch increases the proliferation while inhibiting the osteogenic differentiation in dental pulp stem cells.

Dental pulp stem cells (DPSCs), which can differentiate into several types of cells, are subjected to mechanical stress by jaw movement and occlusal forces. In this study, we evaluated how the uniaxial mechanical stretch influences proliferation and differentiation of DPSCs. DPSCs were isolated and cultured from male Sprague-Dawley rats. Cultured DPSCs were identified by surface markers and the differentiation capabilities as adipocytes or osteoblasts. To examine the response to mechanical stress, uniaxial stretch was exposed to cultured DPSCs. We evaluated the impact of stretch on the intracellular signaling, proliferation, osteogenic differentiation, and gene expressions of DPSCs. Stretch increased the phosphorylation of Akt, ERK1/2, and p38 MAP kinase as well as the proliferation of DPSCs. The stretch-induced proliferation of DPSCs was abolished by the inhibition of the ERK pathway. On the other hand, stretch significantly decreased the osteogenic differentiation of DPSCs, but did not affect the adipogenic differentiation. We also confirmed mRNA expressions of osteocalcin and osteopontin were significantly suppressed by stretch. In conclusion, uniaxial stretch increased the proliferation of DPSCs, while suppressing osteogenic differentiation. These results suggest a crucial role of mechanical stretch in the preservation of DPSCs in dentin. Furthermore, mechanical stretch may be a useful tool for increasing the quantity of DPSCs in vitro for regenerative medicine.

An experimental study of bone grafting using rat milled tooth.

PURPOSE: The aim of this study was to develop a novel bone graft material that used extracted teeth. MATERIALS AND METHODS: Thirty-six 10-week-old male Wistar rats were used. The incisors were extracted, immediately frozen and milled, mixed with hydroxypropyl cellulose (HPC), and injected into the socket. The remaining rats received HPC alone, or the socket was left to heal untreated. Socket healing and bone formation in all three groups were evaluated by three-dimensional image analysis from microcomputed tomography examination and histologic observation. RESULTS: Quantitative morphologic measurements demonstrated that bone formation was significantly stimulated in the group that received milled tooth and HPC at 2 and 4 weeks after extraction compared to that of the control (untreated) group, which showed normal healing without any intervention. Histologic observation revealed that the compound of milled tooth and HPC promoted early healing of the socket and initiation of bone formation in the surrounding area. Interestingly, HPC injection alone decreased bone formation and bone mineral content at 2 weeks and then increased bone formation at 4 weeks. CONCLUSION: A bone graft material composed of milled tooth promotes early healing and bone formation, while HPC, which is chemically stable in vivo, affects bone formation in the extraction socket.

Effects of UV photofunctionalization on the nanotopography enhanced initial bioactivity of titanium.

This study addresses the control of the biological capabilities of titanium through specific nanosurface features and its potential modulation by UV photofunctionalization. Rat bone marrow derived osteoblasts were cultured on titanium disks with micropits alone, micropits with 100 nm nodules, micropits with 300 nm nodules, or micropits with 500 nm nodules, with or without UV treatment. After a 24 h incubation protein adsorption, as well as the attachment, retention, and spread of osteoblasts were examined in correlation with the topographical parameters of the titanium substrates. Each of the biological events was governed by a different set of multiple surface topographical factors with a distinctive pattern of regulation. For instance, without UV treatment the protein adsorption and cell attachment capability of titanium substrates increased linearly with increasing average roughness (Ra) and surface area of titanium disks, but increased polynomially with increasing nanonodule diameter. The cell retention capability increased polynomially with increasing nanonodular diameter and Ra, but increased linearly with increasing surface area. Consequently, the micropits with 300 nm nodules created the most favorable environment for this initial osteoblast behavior and response. UV treatment of the nanonodular titanium surfaces resulted in considerable enhancement of all biological events. However, the pattern of UV-mediated enhancement was disproportionate; exponential and overriding effects were observed depending upon the biological event and topographical parameter. As an example of overriding enhancement, the cell retention capability, which fluctuated with changes in various topographical parameters, became invariably high after UV treatment. The present data provide a basis for understanding how to optimize nanostructures to create titanium surfaces with increased biological capabilities and uncover a novel advantage of UV photofunctionalization of titanium substrates that synergistically increases its nanotopography enhanced biological capabilities whereby most of the initial biological events of osteoblasts were overwhelmingly enhanced beyond a simple proportional increase.

Improvement in the osteoblastic cellular response to a commercial collagen membrane and demineralized freeze-dried bone by an amino acid derivative: an in vitro study.

PURPOSE: The objectives of this in vitro study were (1) to determine whether a commercially available collagen membrane (CM) or human demineralized freeze-dried bone (DFDB) particles adversely affected viability or function in cultured osteoblasts through oxidative stress, and, if so, (2) to determine whether N-acetyl cysteine (NAC) successfully prevented loss of viability and dysfunction in osteoblasts. MATERIALS AND METHODS: Rat calvaria-derived osteoblasts were seeded onto polystyrene and commercially available CM (Cytoplast ®) or DFDB (DynaGraft ™) with or without pretreatment with NAC solution. The osteoblastic response was evaluated using a flow cytometric cell viability assay, measurement of attached viable cell number, quantification of reactive oxygen species (ROS) and alkaline phosphatase (ALP) staining. RESULTS: The percentage of viable cells on CM was <50% at 24 h after seeding. However, this increased to 70% by pretreatment with NAC. The numbers of attached osteoblasts on DFDB remained at 60% the level of that on polystyrene at 24 h after seeding, but increased to up to 90% the level of that on polystyrene with NAC pretreatment. Although collagen materials increased intracellular ROS generation 1.5-5 times that with polystyrene, this was significantly reduced by NAC pretreatment. The percentage of the ALP-positive area was consistently 7% or less on CM and DFDB at days 7 and 14, which was restored by NAC pretreatment up to 60% or more. CONCLUSIONS: Commercially available CM and DFDB impaired osteoblastic viability and function and markedly increased intracellular ROS, indicating an oxidative stress-mediated negative impact on osteoblasts. Pretreatment with NAC substantially alleviated these cytotoxic effects.

Nonvolatile buffer coating of titanium to prevent its biological aging and for drug delivery.

The osseointegration capability of titanium decreases over time. This phenomenon, defined as biological aging of titanium, is associated with the disappearance of hydrophilicity and the progressive accumulation of hydrocarbons on titanium surfaces. The objective of this study was to examine whether coating of titanium surfaces with 4-(2-Hydroxylethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, a nonvolatile zwitterionic chemical buffering agent, could prevent the time-dependent degradation of the bioactivity of titanium. Commercially pure titanium samples, prepared as disks and cylinders, were acid-etched with H(2)SO(4). A third of the samples were used for experiments immediately after processing (new surfaces), while another third were stored under dark ambient conditions for 3 months (3-month-old surfaces). The remaining third were coated with HEPES after acid-etching and were stored for 3 months (HEPES-coated 3-month-old surfaces). The 3-month-old surfaces were hydrophobic, while new and HEPES-coated 3-month-old surfaces were superhydrophilic. Protein adsorption and the number of osteoblasts attached during an initial culture period were substantially lower for 3-month-old surfaces than for new and HEPES-coated 3-month-old surfaces. Alkaline phosphatase activity and calcium deposition in osteoblast cultures were reduced by more than 50% on 3-month-old surfaces compared to new surfaces, whereas such degradation was not found on HEPES-coated 3-month-old surfaces. The strength of in vivo bone-implant integration for 3-month-old implants, evaluated by the push-in test, was 60% lower than that for new implants. The push-in value of HEPES-coated 3-month-old implants was equivalent to that of new implants. Coating titanium surfaces with HEPES containing an antioxidant amino acid derivative, N-acetyl cysteine (NAC), further enhanced osteoblast attachment to the surfaces, along with the increase level of intracellular glutathione reserves as a result of cellular uptake of NAC. These results suggest that HEPES coating of titanium surfaces maintained their superhydrophilicity for at least 3 months and resulted in a continuous retention of bioactivity and osteoconductivity similar to freshly prepared surfaces. This coating technology may be useful for preventing biological aging of titanium and delivering biological molecules for synergistic enhancement of bone-titanium integration.

N-Acetyl cysteine restores viability and function of rat odontoblast-like cells impaired by polymethylmethacrylate dental resin extract.

There is concern that dental-resin materials directly loaded on a prepared tooth adversely affect dental pulp tissue by releasing the resin chemicals through dentinal tubes. This study determined whether self-curing polymethyl methacrylate (PMMA)-based dental resin extract adversely affected the viability and function of odontoblast-like cells and whether the cytotoxicity of this resin, if any, could be eliminated by N-acetyl cysteine, an antioxidant amino acid derivative. Odontoblast-like cells isolated from rat maxillary incisor dental pulp tissue were exposed to a PMMA resin extract with or without N-acetyl cysteine for 1 h and then cultured in osteoblastic media. The percentage of viable cells 24 h after seeding was 20% in cells exposed to the resin extract without N-acetyl cysteine, whereas 45% of cells were viable after exposure to the N-acetyl cysteine-supplemented extract. The cells that had been exposed to the extract showed a strong tendency for apoptosis associated with the increased reactive oxygen species production and decreased intracellular glutathione level, which was improved by the addition of N-acetyl cysteine. N-Acetyl cysteine supplementation almost completely restored the significantly reduced alkaline phosphatase activity and matrix mineralization by the resin extract. These results conclusively demonstrated that exposure of odontoblast-like cells to the resin extract impaired the cell viability and function and, more intriguingly, N-acetyl cysteine supplementation to the extract significantly prevented these toxic effects.

N-Acetyl cysteine prevents suppression of oral fibroblast function on poly(methylmethacrylate) resin.

Despite the proven cytotoxicity, poly(methylmethacrylate) (PMMA) resin is one of the most frequently and extensively used materials in medical and dental fields. The study examined the potential detoxification of the resin material and restoration of the resin-induced suppression of cellular function using an antioxidant amino acid derivative, N-acetyl cysteine (NAC). Oral fibroblasts extracted from rat oral mucosa were cultured on the resin material with or without incorporation of NAC into the material. Twenty-four hour after incubation, less than 2% of the cells were viable on the untreated control resin, while up to 35% of the cells were viable on the resin with incorporation of NAC. At day 7 of culture, the expression of collagen I and III genes was downregulated on the untreated resin, while the cells on NAC-supplemented resin showed the expression levels similar to those in polystyrene culture. The cells produced three times greater amount of collagen on the NAC-supplemented resin than on the untreated resin. The data demonstrated that the cytotoxicity of PMMA resin was substantially lower when the material contains NAC. The potential usefulness of this principle should be explored with a view of developing biocompatible polymer-based materials in a broad range of dental and medical resin materials and tissue engineering scaffolds.

High-throughput gene expression analysis in bone healing around titanium implants by DNA microarray.

OBJECTIVE: Bone generation occurs around titanium implants; however, its underlying mechanisms are relatively unknown. We attempt to identify gene transcripts specifically upregulated in in vivo bone healing with titanium implants using DNA microarray. MATERIAL AND METHODS: Titanium implants were placed into rat femurs, and total RNA was extracted from the implant-associated tissue at weeks 1, 2 and 4 of healing. As a control, RNA was extracted from the tissue undergoing osteotomy healing. The RNA samples were hybridized onto oligo DNA microarray. RESULTS: Most of the 20,000 genes tested were expressed similarly in both the implant- and osteotomy-healing groups. Eighty-six genes were upregulated (>2-fold) in the implant-healing group compared with the osteotomy-healing group in at least one time point of healing. Twelve genes were upregulated in the implant healing at week 2 and earlier, while 31 genes were upregulated at week 2 and later. Only one gene was upregulated specifically at week 1, while three genes were consistently upregulated from weeks 1 to 4. The upregulated genes included collagenous and non-collagenous extracellular matrix (ECM)-related genes, proteoglycans and bone resorption-related genes. Pathway analysis revealed the involvement of ECM and receptor interaction in implant healing. CONCLUSIONS: This study provides evidence that a set of gene transcripts is upregulated in the implant healing over the osteotomy healing, which seems to represent the coordinated biological events of long-lasting osteogenesis and bone remodeling required for osseointegration. Further studies are needed to identify the significance and biological roles of the transcripts in osseointegration. Proven reliability and usefulness of microarray technology should encourage future approaches to develop a high-throughput molecular assessment for osseointegration capacity of new implant surfaces.

The effect of superficial chemistry of titanium on osteoblastic function.

The surface topography and chemistry of titanium are postulated to be two major factors that affect the osseointegration capacity of titanium implants. However, it is extremely difficult to control one factor without changing the other, which prevents the isolation of the genuine effect of one factor. This study aimed to determine whether surface chemistry of titanium alone affects osteoblastic function. Two different titanium surfaces were prepared by sputter depositioning of titanium (Ti; 99.99% purity) or titanium dioxide (TiO2; 99.99% purity) (50-nm thick for each) onto machined commercially pure titanium disks. Rat bone marrow-derived osteoblastic cells were cultured on each of the two surfaces. TiO2 surface showed 4.4 times higher elemental oxygen concentration and higher water wettability than Ti surface. Scanning electron microscopic and atomic force microscopic examination revealed no differences in surface topography and roughness values between the two surfaces. The cell proliferated more on TiO2 than on Ti by up to 60%. Although the expression of collagen I gene increased more rapidly on TiO2 at early culture stage of day 3, the late stage marker genes for osteoblastic differentiation, including osteopontin and osteocalcin, were not modulated between the two cultures. The alkaline phosphatase positive area and mineralized nodule area were approximately two times larger on TiO2 than on Ti. In conclusion, titanium materials having different superficial chemistry, that is, titanium or titanium dioxide, may exert different biological capacity of osteoblasts; titanium dioxide may induce superior osteoconduction, primarily because of the increased osteoblastic proliferation.