Anatomic Approximation Approach to Bilateral Macrostomia Repair.

ABSTRACT: In principle, reconstruction in macrostomia requires symmetry and accurate positioning of the newly reconstructed commissure. The proper position of the new commissure can be determined by several methods. In the determination of the new commissure of bilateral cases, generally the average length of the lips or the distance from anatomical landmarks other than the lips, such as the pupils or tragi, has been used. A new approach was reported by Tse et al in 2018: the anatomic approximation approach. Macrostomia repair refers to anatomical landmarks in the lips to determine the new commissure. The authors performed surgery using this anatomic approximation approach for bilateral macrostomia and achieved the good results.

Farnesoid X receptor and liver X receptors regulate Oct3/4 expression by multiple feedback regulating system in normal renal-derived cells and renal adenocarcinoma cells.

In this study, we found that nuclear receptors FXR and LXR (originally characterized as regulatory factors involved in cholesterol/bile acid homeostasis) regulate the expression of Oct3/4, a marker for cell differentiation, in both normal renal-derived cell line HK-2 and renal adenocarcinoma cell line ACHN. Down-regulation of Oct3/4 expression by activating FXR and LXR occurs only in normal renal cell-derived HK-2 cells. We also found that the RNA-binding protein, ELAVL2, oppositely regulates Oct3/4 expressions in HK-2 and ACHN cells. Moreover, we revealed that LXR-alpha and LXR-beta regulate each other's expression. Although an LXR-beta-specific agonist is assumed to be the basis for an anti-arteriosclerotic drug that only stimulates reverse cholesterol transport, our findings show that the development of such an anti-arteriosclerotic drug would require further elucidation of the complex mechanism of LXR-alpha and LXR-beta regulation.

SIRT1 knockdown up-regulates p53 and p21/Cip1 expression in renal adenocarcinoma cells but not in normal renal-derived cells in a deacetylase-independent manner.

SIRT1, an NAD+-dependent deacetylase, causes deacetylation and down-regulation of its target p53. Given that p53 is an upstream regulator of the transcription of the cyclin-dependent kinase inhibitor p21/Cip1, SIRT1 is hypothesized to play a stimulatory role in carcinoma cell proliferation. We previously reported that down-regulation of SIRT1 caused the increase in p21/Cip1 in a post-transcriptional manner, suggesting that p53 is not involved in the p21/Cip1 increase and raising the question whether SIRT1 exhibits the activity other than deacetylase. In the present study, we examined whether SIRT1 down-regulation and the inhibitor for SIRT1 deacetylase activity affects p21/Cip1 and p53 expression in renal adenocarcinoma cells and normal renal cells. SIRT1 knockdown caused an increase in p53 and p21/Cip1 protein levels in renal adenocarcinoma ACHN cells but not normal renal-derived HK-2 cells. The increase in p53 in ACHN cells is unlikely to contribute to the upregulation of p21/Cip1 expression, given that SIRT1 knockdown did not increase p21/Cip1 mRNA levels in these cells. In contrast to the SIRT1-knock down assay, SIRT1 deacetylase inhibitor did not affect p53 or p21/Cip1 protein levels in ACHN cells. Therefore, SIRT1-knockdown likely stimulates p53 and p21/Cip1 protein expression in a deacetylase-independent manner.

AU-1 from Agavaceae plants downregulates the expression of glycolytic enzyme phosphoglycerate mutase.

The spirostanol saponin AU-1 from Agavaceae plants stimulates the expression of the glycolytic enzyme phosphoglycerate mutase (PGAM) in ACHN cells. We hypothesized that this may arise from the downregulation of the NAD+-dependent deacetylase SIRT1. In this article, we showed that, unlike in renal adenocarcinoma cells, AU-1 does not affect the expression of SIRT1 in the normal renal cell-derived cell line HK-2. Consistent with the lack of a downregulation of SIRT1, AU-1 did not upregulate, but rather decreased PGAM expression. Moreover, AU-1 inhibited the increase in PGAM levels that results from the knock-down of SIRT1. Our results suggest that AU-1 may prevent carcinogenesis caused by increased cellular PGAM.

Farnesoid X receptor regulates the growth of renal adenocarcinoma cells without affecting that of a normal renal cell-derived cell line.

The farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor which is abundant in the liver, intestine, and kidney. FXR is a pivotal factor in cholesterol/bile acid homeostasis but is involved in the growth of hepatocellular carcinoma cells. In the present study, we investigated whether FXR is also involved in the growth of renal adenocarcinoma cells. The cell growth of renal adenocarcinoma cell line ACHN was inhibited by FXR knockdown and stimulated by FXR ligand, while that of a normal renal cell-derived cell line, HK-2, was not affected. The carcinoma-specific stimulation of cell growth by FXR was found to arise from down-regulation of p53 and p21/Cip1 mRNA expression. Our study showed that FXR stimulates proliferation of renal adenocarcinoma cells and that FXR knockdown is useful for growth suppression of renal adenocarcinoma without cytotoxicity to normal renal cells.

Differentiation with elaidate tends to impair insulin-dependent glucose uptake and GLUT4 translocation in 3T3-L1 adipocytes.

Development of type 2 diabetes mellitus and insulin resistance is associated with a quality of dietary fatty acids such as saturated and unsaturated fatty acids. Dietary fatty acids also include transform of unsaturated fatty acids and intake of transform of oleate (elaidate) is associated with cardiovascular disease. However, little is known about the roles of elaidate in insulin responsiveness. We show here that elaidate impairs insulin-dependent glucose uptake in adipocytes. Differentiation with 10 µM elaidate, which is close to physiological plasma concentration, reduces insulin-dependent glucose uptake. Furthermore, insulin-dependent GLUT4 translocation is disturbed in adipocytes differentiated with elaidate. In addition, analysis of lipolysis and gene expression shows that deteriorative effects of elaidate on insulin responsiveness are limited but not general. Thus, our findings reveal that differentiation with elaidate tends to affect insulin-dependent glucose uptake through alternation of GLUT4 translocation from cytosol to the plasma membrane.

Can mesenchymal stem cells and novel gabapentin-lactam enhance maxillary bone formation?

PURPOSE: Novel gabapentin-lactam (GBP-L) has shown its potency in enhancing new bone formation (NBF) in vitro. The objective of the present preclinical trial was to investigate the in vivo performance of GBP-L. MATERIALS AND METHODS: Bilateral sinus floor augmentations in 10 adult sheep were conducted. Bovine bone mineral (BBM) and mesenchymal stem cells (MSCs) combined with novel GBP-L were placed into the test sinus of each sheep. The BBM and MSCs alone served as the control on the contralateral side. Simultaneously, 3 dental implants were inserted in each maxillary sinus. The animals were sacrificed after 8 and 16 weeks, and the amount of NBF was analyzed using histomorphometry. The osteogenic potency of the MSCs was demonstrated using the colony-forming unit and differentiation assay. Statistical evaluation was performed using the Wilcoxon signed rank test and 3-factorial nonparametric analysis of variance. RESULTS: The histologic examination showed NBF in tight contact with the original bone in the control and test groups. The NBF was not significantly different between the test and control sites (P > .05). However, a highly significant difference in NBF between the apical and coronal sites in the specimens from the control and test groups was detected (P < .05). GBP-L did not alter the multipotency of the MSCs or impair NBF. CONCLUSIONS: Bone formation is initiated from the residual alveolar crest and along the implant. The elected mode of GBP-L application did not induce faster NBF. Alternate forms of application (eg, slow release or systemic administration) might clarify the controversial in vitro findings.

The effect of gabapentin-lactam hydroxamic acid derivatives on ovine osteoblast proliferation and phenotype: perspectives for tissue engineering application.

PURPOSE: Modern bone tissue engineering associated with mesenchymal stem cells (MSCs) provides promising treatment alternatives for the loss of bone, one of the foremost challenges in oral and craniofacial surgery today. The effect of gabapentin-lactam (GBP-L) and its analogs on osteogenic differentiated MSCs has not yet been deciphered. Consequently, this study investigates the effect of novel trans-8-tertbutylgabapentin-lactam (trans-8-TB-GBP-L) hydroxamic acid derivatives on metabolism, proliferation, and physiologic mineralization characteristics of ovine osteoblast cells. MATERIALS AND METHODS: Osteoblasts were extracted and prepared from sheep femoral heads and cultured in medium enriched with hydroxamic acid derivatives of trans-8-TB-GBP-L. The cell proliferation rate, cell metabolism, cell viability, and basic osteoblastic function were assessed. RESULTS: After 3 and 5 days of incubation, no significant increase in DNA content was detected in any of 12 test groups versus the control group. However, after 8 days of incubation, a significant increase of DNA contents in the test groups containing nanomolar concentrations of trans-8-TB-GBP-L hydroxamic acid derivatives was found. No significant aberration in metabolic activity was detected when any of the test substances were applied. ALP displayed similar activity rates among the test groups and the control at all time points. Calcification of osteoblastic cells occurred solely when nanomolar concentrations were used. CONCLUSION: Trans-8-TB-GBP-L hydroxamic acid derivatives do not interfere with physiologic function and phenotype of ovine osteoblasts. However, when applied at nanomolar concentrations, the assessed GBP-L derivatives significantly increased the cell proliferation rate after 8 days of incubation, indicating a dose-response curve with the maximum peak at nanomolar concentration and a retarded drug response between 5 and 8 days.

Bone marrow aspirate concentrate used with bovine bone mineral to reconstruct vertical and horizontal mandibular defects: report of two techniques.

PURPOSE: Following initial positive reports of the use of bone marrow aspirate concentrate (BMAC) in combination with bovine bone mineral (BBM) in augmentation procedures, the technique was evaluated in patients with mandibular deficiency. MATERIALS AND METHODS: Two adult patients required surgical correction of a deficient alveolar ridge (one patient showed horizontal deficiency only, and the other patient presented with horizontal and vertical deficiency) prior to dental implant placement. In both patients, the reconstruction was performed with BBM in combination with mononuclear cells concentrated by the BMAC method using different techniques. RESULTS: The patients recovered well from all surgical procedures. Histologically, there was uniform bone formation, which allowed placement of dental implants. CONCLUSION: The results suggest that the use of BMAC in combination with BBM, without autogenous bone, has the potential to restore horizontal and vertical mandibular alveolar defects, providing a functional bone structure and allowing dental implant placement for subsequent prosthetic rehabilitation.

Bone marrow concentrate and bovine bone mineral for sinus floor augmentation: a controlled, randomized, single-blinded clinical and histological trial--per-protocol analysis.

PURPOSE: The purpose of this work was to evaluate the potential of substituting autogenous bone (AB) by bone marrow aspirate concentrate (BMAC). Both AB and BMAC were tested in combination with a bovine bone mineral (BBM) for their ability of new bone formation (NBF) in a multicentric, randomized, controlled, clinical and histological noninferiority trial. MATERIALS AND METHODS: Forty-five severely atrophied maxillary sinus from 26 patients were evaluated in a partial cross-over design. As test arm, 34 sinus of 25 patients were augmented with BBM and BMAC containing mesenchymal stem cells. Eleven control sinus from 11 patients were augmented with a mixture of 70% BBM and 30% AB. Biopsies were obtained after a 3-4-month healing period at time of implant placement and histomorphometrically analyzed for NBF. RESULTS: NBF was 14.3%±1.8% for the control and nonsignificantly lower (12.6%±1.7%) for the test (90% confidence interval: -4.6 to 1.2). Values for BBM (31.3%±2.7%) were significantly higher for the test compared with control (19.3%±2.5%) (p<0.0001). Nonmineralized tissue was lower by 3.3% in the test compared with control (57.6%; p=0.137). CONCLUSIONS: NBF after 3-4 months is equivalent in sinus, augmented with BMAC and BBM or a mixture of AB and BBM. This technique could be an alternative for using autografts to stimulate bone formation.

Role of FGD1, a Cdc42 guanine nucleotide exchange factor, in epidermal growth factor-stimulated c-Jun NH2-terminal kinase activation and cell migration.

FGD1 encodes a guanine nucleotide exchange factor for Cdc42. Mutations in the FGD1 gene are responsible for an X-linked disorder known as Aarskog-Scott syndrome (AAS). While most mutations were found in the catalytic region, which consists of Dbl homology (DH) domain and adjacent pleckstrin homology (PH) domain, a missense mutation in the proline-rich domain is also found in a patient with typical clinical features as AAS. In this mutant FGD1, the serine residue at 205 is replaced with isoleucine. We recently demonstrated that FGD1 translocated to the membrane in response to extracellular stimuli such as epidermal growth factor (EGF) whereas FGD1 with S(205)/I substitution did not. Here we show that the proline-rich domain is critical for FGD1-induced directionally persistent cell migration. When inducibly expressed in HeLa Tet-Off cells, FGD1 stimulates directional migration whereas FGD1 with S(205)/I substitution does not affect it. We further demonstrate that FGD1 augments EGF-stimulated c-Jun NH(2)-terminal kinase (JNK) activation. In the presence of JNK inhibitor SP600125, motility of FGD1-expressing cells is significantly impaired, indicating a critical role of JNK in cell migration. However, FGD3, an FGD1 homologue lacking the proline-rich domain, and FGD1 with S(205)/I substitution augment EGF-stimulated JNK activation similarly to FGD1, suggesting that the proline-rich domain is not involved in the regulation of JNK. Finally, we show that FGD1, but not FGD1 with S(205)/I substitution, is phosphorylated in response to EGF, suggesting that the phosphorylation of S(205) may trigger the FGD1 translocation to the leading edge membrane and enable cells to undergo directional migration.

Making bone II: maxillary sinus augmentation with mononuclear cells--case report with a new clinical method.

We report a simplified method of using bone marrow aspirate concentrate (BMAC™) to regenerate hard tissue. The results suggest that BMAC™ combined with a suitable biomaterial can form sufficient bone within 3 months for further implants to be inserted, and at the same time minimise morbidity at the donor site.

Influence of rhBMP-2 on bone formation and osseointegration in different implant systems after sinus-floor elevation. An in vivo study on sheep.

BACKGROUND: Several studies have reported certain bone morphogenic proteins (BMPs) to have positive effects on bone generation. Although some investigators have studied the effects of human recombinant BMP (rhBMP-2) in sinus augmentation in sheep, none of these studies looked at the placement of implants at the time of sinus augmentation. Furthermore, no literature could be found to report on the impact that different implant systems, as well as the positioning of the implants had on bone formation if rhBMP-2 was utilized in sinus-lift procedures. PURPOSE: The aim of this study was to compare sinus augmentation with rhBMP-2 on a poly-d, l-lactic-co-glycolic acid gelatine (PLPG) sponge with sinus augmentation with autologous pelvic cancellous bone in the maxillary sinus during the placement of different dental implants. MATERIALS AND METHODS: Nine adult female sheep were submitted to bilateral sinus-floor elevation. In one side (test group) the sinus lift was performed with rhBMP-2 on a PLPG-sponge, while the contralateral side served as the control by using cancellous bone from the iliac crest. Three different implants (Bränemark(®), 3i(®) and Straumann(®)) were inserted either simultaneously with the sinus augmentation or as a two staged procedure 6 weeks later. The animals were sacrificed at 6 and 12 weeks for histological and histomorphometrical evaluations during which bone-to-implant contact (BIC) and bone density (BD) were evaluated. RESULTS: BD and BIC were significantly higher at 12 weeks in the test group if the implants were placed at the time of the sinus lift (p<0.05). No difference was observed between the different implant systems or positions. CONCLUSIONS: The use of rhBMP-2 with PLPG-sponge increased BIC as well as BD in the augmented sinuses if compared to autologous bone. Different implant systems and positions of the implants had no effect on BIC or BD.

Mesenchymal stem cells and bovine bone mineral in sinus lift procedures--an experimental study in sheep.

INTRODUCTION: New reconstructive and less invasive methods have been searched to optimize bone formation and osseointegration of dental implants in maxillary sinus augmentation. PURPOSE: The aim of the presented ovine split-mouth study was to compare bovine bone mineral (BBM) alone and in combination with mesenchymal stem cells (MSCs) regarding their potential in sinus augmentation. MATERIAL AND METHODS: Bilateral sinus floor augmentations were performed in six adult sheep. BBM and MSCs were placed into the test side and only BBM in the contra-lateral control side of each sheep. Animals were sacrificed after 8 and 16 weeks. Augmentation sites were analyzed by computed tomography, histology, and histomorphometry. RESULTS: The initial volumes of both sides were similar and did not change significantly with time. A tight connection between the particles of BBM and the new bone was observed histologically. Bone formation was significantly (p = 0.027) faster by 49% in the test sides. CONCLUSION: The combination of BBM and MSCs accelerated new bone formation in this model of maxillary sinus augmentation. This could allow early placement of implants.

Proline-rich domain plays a crucial role in extracellular stimuli-responsive translocation of a Cdc42 guanine nucleotide exchange factor, FGD1.

We previously demonstrated that FGD1, the Cdc42 guanine nucleotide exchange factor (GEF) responsible for faciogenital dysplasia, and its homologue FGD3 are targeted by the ubiquitin ligase SCF(FWD1) upon phosphorylation of two serine residues in their DSGIDS motif and subsequently degraded by the proteasome. FGD1 and FGD3 share highly homologous Dbl homology (DH) and adjacent pleckstrin homology (PH) domains, both of which are responsible for GEF activity. However, their function and regulation are remarkably different. Here we demonstrate extracellular signal-responsive translocation of FGD1, but not FGD3. During the wound-healing process, translocation of FGD1 to the leading edge membrane occurs in cells facing to the wound. Furthermore, epidermal growth factor (EGF) stimulates the membrane translocation of FGD1, but not FGD3. As the most striking difference, FGD3 lacks the N-terminal proline-rich domain that is conserved in FGD1, indicating that proline-rich domain may play a crucial role in signal-responsive translocation of FGD1. Indeed, there is a faciogenital dysplasia patient who has a missense mutation in proline-rich domain of FGD1, by which the serine residue at position 205 is substituted with isoleucine. When expressed in cells, the mutant FGD1 with S(205)/I substitution fails to translocate to the membrane in response to the mitogenic stimuli. Thus we present a novel mechanism by which the activity of FGD1, a GEF for Cdc42, is temporally and spatially regulated in cells.

Mesenchymal stem cells and inorganic bovine bone mineral in sinus augmentation: comparison with augmentation by autologous bone in adult sheep.

Our aim was to compare the osteogenic potential of mononuclear cells harvested from the iliac crest combined with bovine bone mineral (BBM) (experimental group) with that of autogenous cancellous bone alone (control group). We studied bilateral augmentations of the sinus floor in 6 adult sheep. BBM and mononuclear cells (MNC) were mixed and placed into one side and autogenous bone in the other side. Animals were killed after 8 and 16 weeks. Sites of augmentation were analysed radiographically and histologically. The mean (SD) augmentation volume was 3.0 (1.0) cm(3) and 2.7 (0.3) cm(3) after 8 and 16 weeks in the test group, and 2.8 (0.3) cm(3) (8 weeks) and 2.8 (1.2) cm(3) (16 weeks) in the control group, respectively. After 8 weeks, histomorphometric analysis showed 24 (3)% BBM, and 19 (11)% of newly formed bone in the test group. The control group had 20 (13%) of newly formed bone. Specimens after 16 weeks showed 29 (12%) of newly formed bone and 19 (3%) BBM in the test group. The amount of newly formed bone in the control group was 16 (6%). The results show that mononuclear cells, including mesenchymal stem cells, in combination with BBM as the biomaterial, have the potential to form bone.

In vivo comparison of hard tissue regeneration with human mesenchymal stem cells processed with either the FICOLL method or the BMAC method.

OBJECTIVE: To compare new bone formation in maxillary sinus augmentation procedures using biomaterial associated with mesenchymal stem cells (MSCs) separated by two different isolation methods. BACKGROUND: In regenerative medicine open cell concentration systems are only allowed for clinical application under good manufacturing practice conditions. METHODS: Mononuclear cells, including MSCs, were concentrated with either the synthetic polysaccharide (FICOLL) method (classic open system--control group, n = 6 sinus) or the bone marrow aspirate concentrate (BMAC) method (closed system--test group, n = 12 sinus) and transplanted in combination with biomaterial. A sample of the cells was characterized by their ability to differentiate. After 4.1 months (SD +/- 1.0) bone biopsies were obtained and analyzed. RESULTS: The new bone formation in the BMAC group was 19.9% (90% confidence interval [CI], 10.9-29), and in the FICOLL group was 15.5% (90% CI, 8.6-22.4). The 4.4% difference was not significant (90% CI, -4.6-13.5; p = 0.39). MSCs could be differentiated into osteogenic, chondrogenic, and adipogenic lineages. CONCLUSION: MSCs harvested from bone marrow aspirate in combination with bovine bone matrix particles can form lamellar bone and provide a reliable base for dental implants. The closed BMAC system is suited to substitute the open FICOLL system in bone regeneration procedures.

Novel insights into FGD3, a putative GEF for Cdc42, that undergoes SCF(FWD1/beta-TrCP)-mediated proteasomal degradation analogous to that of its homologue FGD1 but regulates cell morphology and motility differently from FGD1.

We previously demonstrated that FGD1, the Cdc42 guanine nucleotide exchange factor (GEF) responsible for faciogenital dysplasia, is targeted by the ubiquitin ligase SCF(FWD1/beta-TrCP) upon phosphorylation of two serine residues in its DSGIDS motif and subsequently degraded by the proteasome. Here we show that FGD3, which was identified as a homologue of FGD1 but has been poorly characterized, has conserved the same motif and is down-regulated similarly by SCF(FWD1/beta-TrCP). Although FGD3 and FGD1 share strikingly similar Dbl homology (DH) domains and adjacent pleckstrin homology (PH) domains, both of which are responsible for guanine nucleotide exchange, there also exist remarkable differences in their structures. Indeed, FGD1 and FGD3 induced significantly different morphological changes in HeLa Tet-Off cells: whereas FGD1 induced long finger-like protrusions, FGD3 induced broad sheet-like protrusions when the level of GTP-bound Cdc42 was significantly increased by the inducible expression of FGD3. Furthermore, FGD1 and FGD3 reciprocally regulated cell motility: when inducibly expressed in HeLa Tet-Off cells, FGD1 stimulated cell migration whereas FGD3 inhibited it. Thus we demonstrate that the highly homologous GEFs, FGD1 and FGD3 play different roles to regulate cellular functions but that their intracellular levels are tightly controlled by the same destruction pathway through SCF(FWD1/beta-TrCP).

Identification and functional analyses of two cDNAs that encode fatty acid 9-/13-hydroperoxide lyase (CYP74C) in rice.

Fatty acid hydroperoxide lyase (HPL), a member of cytochrome P450 (CYP74), produces aldehydes and oxo-acids involved in plant defensive reactions. In monocots, HPL that cleaves 13-hydroperoxides of fatty acids has been reported, but HPL that cleaves 9-hydroperoxides is still unknown. To find this type of HPL, in silico screening of candidate cDNA clones and subsequent functional analyses of recombinant proteins were performed. We found that AK105964 and AK107161 (Genbank accession numbers), cDNAs previously annotated as allene oxide synthase (AOS) in rice, are distinctively grouped from AOS and 13-HPL. Recombinant proteins of these cDNAs produced in Escherichia. coli cleaved both 9- and 13-hydroperoxide of linoleic and linolenic into aldehydes, while having only a trace level of AOS activity and no divinyl ether synthase activity. Hence we designated AK105964 and AK107161 OsHPL1 and OsHPL2 respectively. They are the first CYP74C family cDNAs to be found in monocots.