Cell therapy for medication-related osteonecrosis of the jaw: update on treatment strategies.

Bioactive glasses (BAG) are used as bone-graft substitutes in orthopaedic surgery. A specific BAG scaffold was developed by sintering BAG-S53P4 granules. It is hypothesised that this scaffold can be used as a bone substitute to fill bone defects and induce a bioactive membrane (IM) around the defect site. Beyond providing the scaffold increased mechanical strength, that the initial inflammatory reaction and subsequent IM formation can be enhanced by coating the scaffolds with poly(DL-lactide-co-glycolide) (PLGA) is also hypothesised. To study the immunomodulatory effects, BAG-S53P4 (± PLGA) scaffolds were placed on monolayers of primary human macrophage cultures and the production of various pro- and anti-inflammatory cytokines was assessed using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and ELISA. To study the osteogenic effects, BAG-S53P4 (± PLGA) scaffolds were cultured with rabbit mesenchymal stem cells and osteogenic differentiation was evaluated by RT-qPCR and matrix mineralisation assays. The scaffold ion release was quantified and the BAG surface reactivity visualised. Furthermore, the pH of culture media was measured. BAG-S53P4 scaffolds had both anti-inflammatory and osteogenic properties that were likely attributable to alkalinisation of the media and ion release from the scaffold. pH change, ion release, and immunomodulatory properties of the scaffold could be modulated by the PLGA coating. Contrary to the hypothesis, the coating functioned by attenuating the BAG surface reactions and subsequent anti-inflammatory properties, rather than inducing an elevated inflammatory response compared to BAG-S53P4 alone. These results further validated the use of BAG-S53P4 (± PLGA) scaffolds as bone substitutes and indicate that scaffold properties can be tailored to a specific clinical need.

Great Ears: Low-Frequency Sensitivity Correlates in Land and Marine Leviathans.

Like elephants, baleen whales produce low-frequency (LF) and even infrasonic (IF) signals, suggesting they may be particularly susceptible to underwater anthropogenic sound impacts. Analyses of computerized tomography scans and histologies of the ears in five baleen whale and two elephant species revealed that LF thresholds correlate with basilar membrane thickness/width and cochlear radii ratios. These factors are consistent with high-mass, low-stiffness membranes and broad spiral curvatures, suggesting that Mysticeti and Proboscidea evolved common inner ear adaptations over similar time scales for processing IF/LF sounds despite operating in different media.

Brain imaging in methamphetamine-treated mice using a nitroxide contrast agent for EPR imaging of the redox status and a gadolinium contrast agent for MRI observation of blood-brain barrier function.

Methamphetamine (METH)-induced neurotoxicity is associated with mitochondrial dysfunction and enhanced oxidative stress. The aims of the present study conducted in the mouse brain repetitively treated with METH were to (1) examine the redox status using the redox-sensitive imaging probe 3-methoxycarbonyl-2,2,5,5-tetramethylpiperidine-1-oxyl (MCP) and (2) non-invasively visualize the brain redox status with electron paramagnetic resonance (EPR) imaging. The rate of reduction of MCP was measured from a series of temporal EPR images of mouse heads, and this rate was used to construct a two-dimensional map of rate constants called a "redox map." The obtained redox map clearly illustrated the change in redox balance in the METH-treated mouse brain that is a known result of oxidative damage. Biochemical assays also showed that the level of thiobarbituric acid-reactive substance, an index of lipid peroxidation, was increased in mouse brains by METH. The enhanced reduction in MCP observed in mouse brains was remarkably suppressed by treatment with the dopamine synthase inhibitor, α-methyl-p-tyrosine, suggesting that enhancement of the reduction reaction of MCP resulted from enzymatic reduction in the mitochondrial respiratory chain. Furthermore, magnetic resonance imaging (MRI) of METH-treated mice using a blood-brain barrier (BBB)-impermeable paramagnetic contrast agent revealed BBB dysfunction after treatment with METH for 7 days. MRI also indicated that the impaired BBB recovered after withdrawal of METH. EPR imaging and MRI are useful tools not only for following changes in the redox status and BBB dysfunction in mouse brains repeatedly administered METH, but also for tracing the drug effect after withdrawal of METH.

Surgical procedure of extracting teeth for obtaining dental pulp for regenerative medicine in swine.

Dental pulp is a potential source of cells that can be used in cell replacement therapy for various nerve disorders, including stroke, spinal cord injury, and peripheral nerve defect. However, the validation of an animal model closely related to humans is needed in translational research. The miniature pig is a suitable experimental model in maxillofacial surgery, because its anatomical structure and size are similar to those of humans. However, the swine tooth is extremely long. The routine closed extraction procedure for harvesting dental pulp tissue causes root fracture. This report describes the details of a surgical procedure for tooth extraction. Four healthy 7-8-month-old male NIBS miniature pigs were used. Two mandibular deciduous right incisors (Di1 and Di2) were extracted in order to obtain dental pulp tissue. Gingival envelope incision with vertical-releasing incision was performed, and a full-thickness mucoperiosteal flap was made. The buccal alveolar bone was exposed and removed by osteotomy. Di1 and Di2 were extracted. Dental pulp tissue was obtained from these extracted teeth by splitting hard tissue. In this procedure, 9.8 ± 2.5 × 10(5) cells were obtained from the mandibular Di1 and Di2 (n = 4).

Studies of the humoral factors produced by layered chondrocyte sheets.

The authors aimed to repair and regenerate articular cartilage with layered chondrocyte sheets, produced using temperature-responsive culture dishes. The purpose of this study was to investigate the humoral factors produced by layered chondrocyte sheets. Articular chondrocytes and synovial cells were harvested during total knee arthroplasty. After co-culture, the samples were divided into three groups: a monolayer, 7 day culture sheet group (group M); a triple-layered, 7 day culture sheet group (group L); and a monolayer culture group with a cell count identical to that of group L (group C). The secretion of collagen type 1 (COL1), collagen type 2 (COL2), matrix metalloproteinase-13 (MMP13), transforming growth factor-ß (TGFß), melanoma inhibitory activity (MIA) and prostaglandin E2 (PGE2) were measured by enzyme-linked immunosorbent assay (ELISA). Layered chondrocyte sheets produced the most humoral factors. PGE2 expression declined over time in group C but was significantly higher in groups M and L. TGFß expression was low in group C but was significantly higher in groups M and L (p<0.05). Our results suggest that the humoral factors produced by layered chondrocyte sheets may contribute to cartilaginous tissue repair and regeneration.

Topographical arrangement of α- and ß-cells within neo-islet tissues engineered by islet cell sheet transplantation in mice.

BACKGROUND: We established a procedure to engineer therapeutic neo-islets in subcutaneous spaces in mice by transplanting contiguous layers of islet cell sheets. In this study, we investigated the cellular arrangements of α and ß within these engineered neo-islets in vivo as a function of time after sheet transplantation. METHODS AND RESULTS: Temperature-responsive culture dishes optimized for dispersed islet cell culture were prepared by covalently immobilizing a temperature-responsive polymer poly(N-isopropylacrylamide) (PIPAAm) on plastic dishes followed by laminin-5 coating. Dispersed islet cells obtained from Lewis rats were plated onto the PIPAAm dishes. After reaching confluence at day 2, islet cells were harvested as uniformly spread islet cell sheets by lowering the culture temperature from 37°C to 20°C for 20 minutes. Islet sheet transplantation was performed to creat neo-islet tissues in the subcutaneous spaces of SCID mice with streptozotocin-induced diabetes. This neo-islet engineering approach successfully lowered mouse blood glucose levels, achieving euglycemia at day 5 and thereafter. Histologic analyses of samples obtained at day 4 revealed that neo-islet tissues in the subcutaneous spaces showed heterogeneous cellular alignment of α and ß cells. In contrast, analyses of samples at days 14 and 60 revealed α and ß cells predominantly located at the peripheral and central parts of the engineered tissues, respectively. CONCLUSIONS: Reassembly of α and ß cells occurred in neo-islet tissues engineered by sheet transplantation. The unique cellular arrangements in neo-islet tissues, which were similar to those in naïve pancreatic islets, may contribute to their longevity and long-term function.

Requirement of integrin ß3 for iron transportation during enamel formation.

Rodent incisors exhibit pigmentation on their labial surfaces. Although previous studies have shown that this pigment is composed of iron, the existence of other elements has not been investigated. This study found that the lower incisors of CD61, also known as integrin ß3, null mice (CD61(-/-)) lacked pigmentation. Although ameloblasts differentiated and formed enamel normally, no ferric ion accumulation was observed in maturation-stage ameloblasts in CD61(-/-) mice. Surface elements of control and CD61-/- lower incisors were compared by x-ray photoelectron spectroscopy (XPS). XPS analysis detected C, Ca, N, O, and P on the labial surfaces of lower incisors of both mice, whereas Fe was detected only in control samples. No peak of non-ferrous metal or other element was detected in either group. Quantitative RT-PCR analysis of 18 iron-transportation-related genes with mRNA from maturation-stage ameloblasts and ALC, a pre-ameloblastic cell line, was performed. The results suggested that CD61 regulates the expressions of Slc11a2 and Slc40a1, both of which are involved in iron transportation in epithelial tissues. These results suggested that the pigment on the labial surface of mouse incisors is composed of Fe and that both anemia and reduction of iron-transporting proteins may cause the loss of pigmentation in CD61(-/-) mice.

Cell sheet engineering: a unique nanotechnology for scaffold-free tissue reconstruction with clinical applications in regenerative medicine.

Cell sheet technology (CST) is based on the use of thermoresponsive polymers, poly(N-isopropylacrylamide) (PIPAAm). The surface of PIPAAms is formulated in such a way as to make its typical thickness <100 nm. In this review, we first focus on how the methods of PIPAAm-grafted surface preparations and functionalization are important to be able to harvest a functional cell sheet, to be further transplanted. Then, we present aspects of tissue mimics and three-dimensional reconstruction of a tissue in vitro. Finally, we give an overview of clinical applications and clinically relevant animal experimentations of the technology, such as cardiomyopathy, visual acuity, periodonty, oesophageal ulcerations and type 1 diabetes.

Analysis of soluble vascular endothelial growth factor receptor-1 secreted from cultured corneal and oral mucosal epithelial cell sheets in vitro.

BACKGROUND: In clinical trials, eyes transplanted with cultured oral mucosal epithelial cell sheets have shown increased neovascularisation compared with eyes treated with cultured corneal epithelial cell sheets. As reported recently, soluble vascular endothelial growth factor receptor-1 (soluble VEGFr-1) is a main factor to maintain a corneal avascularity. AIM: To investigate soluble VEGFr-1 of cultured corneal epithelial cells (CCE) and cultured oral mucosal epithelial cells (COE) in vitro. METHODS: Rabbit corneal and oral mucosal epithelial cells were co-cultured with mitomycin C-treated NIH/3T3 cells on culture plates. After CCE and COE were multilayered, culture medium was replaced by basal medium and incubated. Protein secretion of soluble VEGFr-1 was assessed in conditioned medium from CCE and COE by ELISA. Angiogenic potential was examined by invasion, migration assays with human umbilical vein endothelial cells (HUVECs) in addition to recombinant soluble VEGFr-1. RESULTS: CCE secreted a significantly higher amount of soluble VEGFr-1 than did COE. Recombinant soluble VEGFr-1 significantly suppressed HUVEC migration induced by COE, without suppression in CCE. In conclusion, these findings suggest that low protein levels of soluble VEGFr-1 may lead to corneal neovascularisation after COE sheet transplantation.

Touch imprint cytology with cytokeratin immunostaining versus Papanicolau staining for intraoperative evaluation of sentinel lymph node metastasis in clinically node-negative breast cancer.

AIM: This study investigated whether intraoperative assessment of SLN status in patients with clinically node-negative breast cancer was improved using touch imprint immunohistochemistry. MATERIAL AND METHODS: Each SLN was cut into slices 2mm thick and evaluated intraoperatively by touch imprint cytology with Papanicolaou staining until the end of 2005, or by a combination of Papanicolaou staining and immunostaining with an anti-cytokeratin antibody from early 2006. RESULTS: When intraoperative cytology of SLN in 85 patients who were clinically node-negative was evaluated with Papanicolaou staining, 81 patients were diagnosed as negative and four were positive. Intraoperative cytology with Papanicolaou staining had a sensitivity of 30%, specificity of 99%, false-negative rate of 70%, false-positive rate of 1.3%, and accuracy of 90.6%. When intraoperative cytology was done with immunohistochemistry plus Papanicolaou staining for SLN evaluation, 92 patients were diagnosed as negative and 17 patients were positive. Intraoperative cytology with immunohistochemistry had a sensitivity of 79%, specificity of 98%, false-negative rate of 21%, false-positive rate of 2.2%, and accuracy of 94.5%. Compared with intraoperative cytology using Papanicolaou staining alone, the combination of immunohistochemistry and Papanicolaou staining achieved a significant increase in sensitivity and a significant decrease in the false-negative rate. CONCLUSION: Intraoperative SLN evaluation by imprint cytology with immunohistochemistry achieves a more accurate diagnosis of metastasis than imprint cytology alone. This combined method is considered useful for deciding whether to perform axillary lymph node dissection.

Differential expression of MUC16 in human oral mucosal epithelium and cultivated epithelial sheets.

Cultivated oral mucosal epithelial sheet transplantation is a new surgical strategy to treat severe ocular surface disorders such as chemical burns, ocular cicatricial pemphigoid, and Stevens-Johnson syndrome. MUC16 is thought to be the most important membrane-associated mucin on the ocular surface because it forms a protective barrier on the epithelial cell surface. In this study, we studied MUC16 expression in mRNA and protein levels and compared the expression patterns between cultivated oral mucosal epithelial cell sheet and oral mucosal tissue. Specimens (5x5 mm) of oral mucosal tissue harvested from healthy volunteers were used. The oral mucosal epithelial cells were cultured on temperature-responsive culture dishes to generate stratified cell sheets. Cultivated oral mucosal epithelial cells formed three- to five-cell thick stratified sheets for 2 weeks. Scanning electron micrographs revealed that the apical surfaces of the oral mucosal tissue and the oral mucosal sheets were covered with dense microvilli/microplicae. Real-time PCR showed significantly more MUC16 transcripts in the cultivated oral mucosal sheets and corneal epithelial sheets than in the oral mucosal tissue (P=0.023 and 0.008, respectively, Mann-Whitney rank sum test). These findings were confirmed by immunohistochemical examination using an MUC16 antibody to the protein. MUC16 protein was localized to the apical cells of the oral mucosal sheets, but the human oral mucosal tissue did not express MUC16 protein in any cell layers. In this study, interestingly, the expression of membrane-associated mucin MUC16 differs between human oral mucosal epithelia and cultivated epithelial sheets. MUC16 expressed in the oral mucosal sheets may contribute to ocular surface reconstruction after oral mucosal sheet transplantation.

Abnormal keratocytes and stromal inflammation in chronic phase of severe ocular surface diseases with stem cell deficiency.

BACKGROUND/AIMS: Stevens-Johnson syndrome (SJS), ocular cicatricial pemphigoid (OCP) and alkali burns are associated with chronic, severe inflammation of the ocular surface that occasionally lead to corneal stem cell deficiencies. The corneal stroma in these diseases has not been studied comprehensively. The purpose of this study was to determine whether the keratocytes in the stroma were normal and whether the stroma remained inflamed in the chronic phase of these diseases. METHODS: Five pathological corneas, two with SJS, two with OCP and one with an alkali burn were examined. Corneal specimens were obtained during lamellar keratoplasty and the histological sections were immunostained with antibodies against CD34 and several cell surface antigens. The level of expression of proteoglycans (lumican, keratocan, biglycan) and chemokines (monocyte chemoattractant protein 1, macrophage inflammatory protein (MIP) 1alpha, MIP1beta) were examined by quantitative real-time RT-PCR. RESULTS: The number of CD34-positive cells in the stroma was decreased and the expression level of biglycan increased in all of the pathological corneas. The numbers of CD45-positive and CD14-positive cells were increased in four of the five pathological corneas. The expression level of MIP1alpha and MIP1beta were markedly increased in all of the pathological corneas. CONCLUSIONS: These findings indicate that the keratocytes are abnormal and inflammation is still present in the corneal stroma in the chronic phase of SJS, OCP and alkali burns.

Cementum-periodontal ligament complex regeneration using the cell sheet technique.

BACKGROUND AND OBJECTIVE: In the present study we evaluated if a multilayered human periodontal ligament cell sheet could reconstruct the physiological architecture of a periodontal ligament-cementum complex. MATERIAL AND METHODS: Human periodontal ligament cells were isolated and then cultured in dishes coated with a temperature-responsive polymer to allow cell detachment as a cell sheet. In the control group, human periodontal ligament cells were cultured in Dulbecco's modified Eagle's minimal essential medium containing 10% fetal bovine serum and 1% antibiotics. In the experimental group, human periodontal ligament cells were cultured in Dulbecco's modified Eagle's minimal essential medium and osteodifferentiation medium containing dexamethasone, ascorbic acid and beta-glycerophosphate. After 3 wk, scanning electron microscopy was carried out, in addition to staining for alkaline phosphatase activity and for calcium (using the Von Kossa stain). Then human periodontal ligament cell sheets were multilayered and placed onto dentin blocks. The constructs were transplanted subcutaneously into the back of immunodeficient rats. At 1 and 6 wk after transplantation, the animals were killed. Demineralized tissue sections were stained using hematoxylin and eosin, and Azan, and then analyzed. RESULTS: After 3 wk of culture in osteodifferentiation medium, human periodontal ligament cells produced mineral-like nodules and also showed positive staining for alkaline phosphatase, calcium (Von Kossa) and mRNA expression of type I collagen. By contrast, in the control group only weak alkaline phosphatase staining was observed, the Von Kossa stain was negative and there was no mRNA expression of type I collagen. Six weeks after transplantation with human periodontal ligament cells cultured in osteodifferentiation medium, most of the dentin surfaces showed a newly immature cementum-like tissue formation and periodontal ligament with perpendicular orientation inserted into the newly deposited cementum-like tissue. CONCLUSION: This study suggests that the multilayered temperature-responsive culture system can be used as a novel strategy for periodontal regeneration. The human periodontal ligament cell sheet technique may be applicable for regeneration of the clinical periodontal ligament-cementum complex.

Mesothelial cells from tunica vaginalis, a practical source for mesothelial transplantation.

Transplantation of mesothelial cells is used to repair peritoneum that is damaged by surgery, peritonitis, and peritoneal dialysis. The largest obstacle for clinical application of mesothelial cell transplantation is the lack of a reliable source of mesothelial cells. So far, they are isolated from omentum, mesentery, parietal wall and ascites. Procedures used to obtain mesothelial cells from the omentum or mesentery are invasive, however, especially in pre-operative situations. Sufficient amounts of ascites for aspiration can not be obtained under physiological conditions. We have developed a novel method of isolating mesothelial cells from the tunica vaginalis. The tunica vaginalis originates from the peritoneum and descends into the scrotum along with the testis during fetal development. This region provides a source of mesothelial cells that is convenient to approach and free from abdominal complications. Transplantation of autologous mesothelial cells that were isolated from tunica vaginalis was effective in preventing post-operative adhesions. In this review, we summarize mesothelial cell transplantation trials and describe the method of isolating mesothelial cells form the tunica vaginalis. Mesothelial cell transplantation might be widely accepted for clinical use in the near future.

Treatment of oesophageal ulcerations using endoscopic transplantation of tissue-engineered autologous oral mucosal epithelial cell sheets in a canine model.

BACKGROUND: With the recent development of endoscopic submucosal dissection (ESD), large oesophageal cancers can be removed with a single procedure, with few limits on the resectable range. However, after aggressive ESD, a major complication that arises is postoperative inflammation and stenosis that can considerably affect the patient's quality of life. AIMS: To examine a novel treatment combining ESD and the endoscopic transplantation of tissue-engineered cell sheets created using autologous oral mucosal epithelial cells, in a clinically relevant large animal model. METHODS: Oral mucosal epithelial cells, harvested from beagle dogs, were cultured under normal conditions at 37 degrees C, on temperature-responsive dishes. After ESD (5 cm in length, 180 degrees in range), cell sheets were harvested by a simple reduction in temperature to 20 degrees C, and transplanted by endoscopy. RESULTS: The transplanted cell sheets were able to adhere to and survive on the underlying muscle layers in the ulcer sites, providing an intact, stratified epithelium. Four weeks after surgery, complete wound healing, with no observable stenosis, was seen in the animals receiving autologous cell sheet transplantation. By contrast, noticeable fibrin mesh and host inflammation, consistent with the intermediate stages of wound healing, were observed in the control animals that received only ESD. CONCLUSIONS: These findings in a clinically relevant canine model show the effectiveness of a novel combined endoscopic approach for the potential treatment of oesophageal cancers that can effectively enhance wound healing and possibly prevent postoperative oesophageal stenosis.

In vivo engineering of metabolically active hepatic tissues in a neovascularized subcutaneous cavity.

Recent success in clinical hepatocyte transplantation therapy has encouraged further investigation into bioengineering hepatic tissues in vivo. Engineering tissues in the subcutaneous space is an attractive method; however, hepatocyte survival has been transient due to insufficient vascular network formation. To establish a vascularized cavity, we created a polyethylene terephthalate mesh device coated with poly(vinylalcohol) that allowed for the gradual release of basic fibroblast growth factor (bFGF), a potent angiogenic factor. The efficacy of the bFGF-releasing device in inducing vascular network formation in the subcutaneous space was observed in mouse and rat studies. Isolated mouse hepatocytes transplanted into newly vascularized subcutaneous cavities allowed for persistent survival up to 120 days. In the absence of a vascularized compartment, the survival of the transplanted hepatocytes was markedly diminished. Functional maintenance of the engineered hepatic tissues was confirmed by high expression of liver-specific mRNAs and proteins. These engineered hepatic tissues have the ability to take up inoculated compounds and express strong induction of drug-metabolizing enzymes, demonstrating functional relevance as a metabolic tissue. In conclusion, we have created a novel technology to engineer functionally active hepatic tissues in the subcutaneous space, which will likely facilitate hepatocyte-based therapies.

Serological diversity demonstrable by a set of monoclonal antibodies to eight serotypes of the mutans streptococci.

A set of monoclonal antibodies were prepared by the conventional cell fusion of myeloma cells (SP2/0-Ag14) with spleen cells from BALB/c mice immunised with whole cells of a strain of mutans streptococci. Their specificities were examined against 35 reference strains of mutans streptococci, 34 reference strains of other oral streptococci and 8 reference strains of other microorganisms often inhabiting the oral cavity. Specificity was examined by enzyme immunoassay using whole cells. A total of 52 strains, consisting of 19 strains isolated in Japan, 19 strains isolated in Italy and 14 strains isolated in England, were characterised by conventional physiological and biochemical tests and then serotyped by the use of 8 monoclonal antibodies with different specificities. They were also confirmed by guanine-plus-cytosine contents of their nucleic acid and DNA-DNA hybridisation test. The results indicated that all monoclonal antibodies are useful for identification of 8 serotypes of the mutans streptococci responsible for dental caries. They also suggest the existence of more serological varieties among mutans species.

A noninvasive transfer system for polarized renal tubule epithelial cell sheets using temperature-responsive culture dishes.

We used temperature-responsive culture dishes onto which the temperature-responsive polymer, poly(Nisopropylacrylamide), was covalently grafted for tissue engineering. Confluent cells harvested as intact sheets from these surfaces by simple temperature reduction can be transferred to various surfaces including additional culture dishes, other cell sheets, and tissues. In order to examine the maintenance of cell polarity, Madin-Darby canine kidney cells and human primary renal proximal tubule epithelial cells which had developed apical-basal cell polarity in culture, were subjected to cell sheet transfer. This functional and structural cell polarity, which is susceptible to treatment with trypsin, was examined by immunohistochemistry and transmission electron microscopy. Using our cell-sheet method, the noninvasive transfer of these cell sheets retaining typical distributions of Na+/K+-ATPase, GLUT-1, SGLT-1, aquaporin-1, neutral endopeptidase and dipeptidylendopeptidase IV, could be achieved. The transferred cell sheets also developed numerous microvilli and tight junctions at the apical and lateral membranes, respectively. For biochemical analysis, immunoblotting of occludin, a transmembrane protein that composes tight junctions, was conducted and results confirmed that occludin remained intact after cell sheet transfer. This two-dimensional cell sheet manipulation method promises to be useful for tissue engineering as well as in the investigation of epithelial cell polarity.