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.

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.

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.

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.

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.

Urothelium regeneration using viable cultured urothelial cell sheets grafted on demucosalized gastric flaps.

OBJECTIVE: To evaluate urothelium regeneration by grafting viable cultured urothelial cell sheets, harvested from temperature-responsive culture surfaces, on demucosalized gastric flaps in a dog model. MATERIALS AND METHODS: Viable urothelium was obtained from eight beagle dogs by partial cystectomy. Harvested urothelial cells were seeded on temperature-responsive culture dishes modified with the thermally sensitive polymer, poly(N-isopropylacrylamide). Urothelial cells cultured for 3 weeks generated contiguous urothelial cell sheets that were noninvasively harvested with no enzymatic treatment from these dishes, by reducing culture temperature. Urothelial cell sheets were autografted onto surgically demucosalized gastric flaps. Three weeks after autografting the dogs were killed and the gastric flaps with the urothelial cell sheets were examined. Cell and tissue characteristics were compared between these urothelial cell sheet-grafted gastric flaps and native urothelium. Ultrafine structures were also examined by electron microscopy. RESULTS: Five of the eight urothelial cell sheet-grafted flaps showed viable urothelial regeneration. Urothelial cell sheets attached spontaneously to demucosalized tissue surfaces completely, with no suture or fixing, and developed into a stratified viable epithelium very similar to native urothelium. Regenerated urothelium remained unstained by antiproton pump antibody, which typically stains epithelial cells positively in gastric mucosal layers. On three of the eight flaps where there were severe haematomas, grafted cell sheets were not adherent and there was no urothelial regeneration. CONCLUSIONS: Urothelial cell sheets were autografted onto dog demucosalized gastric flaps successfully, with no suture or fixation, generating a multilayered urothelium in vivo. The novel intact cell-sheet grafting method rapidly produces native-like epithelium in vivo. This versatile technology should prove useful in urinary tract tissue engineering and surgical reconstruction.

Alteration of collagen IV in acutely deteriorated renal allografts.

BACKGROUND: The changes in the basement membrane occurring in acutely deteriorated renal allografts (ADR) have not been extensively investigated. Our purpose is to elucidate the alteration of collagen IV, a main constituent of the basement membrane in ADR. METHODS: Fifty biopsy specimens of ADR and 10 of chronic transplant nephropathy (CTN) were examined with two monoclonal antibodies specific for collagen IV. JK199 and JK132 are monoclonal antibodies that recognize triple helical collagen IV containing the alpha1 chain. JK199 recognizes all the basement membrane containing [alpha1 (IV)]2alpha2(IV), although JK132 reacts only with a limited portion of it. In the normal kidney, JK199 reacts with the mesangial matrix, the basement membrane of Bowman's capsule (BBM), and the tubular basement membrane, as well as with the glomelular basement membrane (GBM). JK132 reacts with the mesangial matrix, BBM, and the tubular basement membrane. RESULTS: In ADR, increased intensity of JK199 was observed in GBM, the mesangial matrix, BBM, the tubular basement membrane, and the interstitium. Increased intensity of JK132 was observed in the mesangial matrix, BBM, and the tubular basement membrane, but was not remarkable in GBM or the interstitium. In contrast, biopsy specimens of CTN showed increased intensity of JK132 in GBM, the mesangial matrix, BBM, the tubular basement membrane and the interstitium. CONCLUSION: These results suggest that collagen IV is up-regulated in ADR. Differential staining of collagen IV with JK199 and JK132 in GBM and the interstitium may contribute to diagnose CTN.

Detection of malignant thymoma in primary tumor and metastatic lesions using 99mTc-tetrofosmin scintigraphy.

99mTc-tetrofosmin was developed as a myocardial perfusion imaging agent and can also be used to depict tumors. We have experienced five cases of malignant thymoma delineated on 99mTc-tetrofosmin SPECT. In one case significant activity was clearly detected in the primary tumor and metastatic lesions. In quantitative analysis, similar 99mTc-tetrofosmin and 201Tl-chloride uptake ratios were obtained (1.95+/-0.57 versus 2.27+/-0.85, respectively; n.s.). The ability of 99mTc-tetrofosmin to detect malignant thymoma was comparable to that of 201Tl-chloride. Therefore, 99mTc-tetrofosmin might be a useful tracer for the detection of malignant thymoma, although more studies will be required to evaluate its diagnostic accuracy.

Intact microglia are cultured and non-invasively harvested without pathological activation using a novel cultured cell recovery method.

Because spontaneous host regeneration of damaged tissues is limited, novel therapeutics utilizing cultured cells with the aid of tissue engineering methods are promising alternatives for tissue replacement. One critical shortcoming is current requirement for invasive cell harvest from culture to fabricate cell-based devices. Although microglia that secrete neurotrophic factors are attractive candidates for novel cell transplantation therapy for damaged central nervous system tissue, the intact harvest of cultured microglia is presently not achievable. Therefore, primary microglia were plated onto culture surfaces grafted with the temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm). This surface undergoes rapid, reversible temperature-dependent changes in its hydration state and surface hydrophilicity. Microglia attached and proliferated on PIPAAm-grafted dishes at 37 degrees C. By reducing culture temperature, more than 90% of the cells spontaneously detached from the dishes within several minutes without trypsin or EDTA treatment. Recovered and replated microglia exhibited phenotypic properties comparable to those of primary microglia freshly isolated from brain. By contrast, less than 60% of the cells were harvested by trypsin digestion, and exhibited significant alteration of characteristic cellular properties as monitored by pathological states in vivo. This new technology exhibits utility for the preparation of cell sources required for cell transplantation as well as microglial function analysis.

Mut-Test to detect substances suppressing spontaneous mutation due to oxidative damage.

Since it has been considered that suppression of spontaneous mutation in cells is related to suppression of spontaneous carcinogenesis, it is significant to detect substances which suppress spontaneous mutation in bacterial cells such as Escherichia coli and Salmonella typhimurium in the environment. However, since the frequency of spontaneous mutation in bacteria is usually very low, generally 10(-8)-10(-10),it is difficult to determine significant suppressive ability of such substances on spontaneous mutation. A new method, Mut-Test, was developed by us, applying Luria & Delbruck fluctuation test, to detect substances which suppress spontaneous mutation using E. coli mutT mutant in which spontaneous mutation frequency due to oxidative damage is enhanced to approximately 500-1000 times of the wild type strain. Suppressive abilities of two hydroxyl radical scavengers: D(-)-mannitol and thiourea, were examined and clear positive results were obtained, suggesting that the radical scavengers are suitable as the positive control for the test. Using Mut-Test, suppressive abilities of four vitamins: L-ascorbic acid, beta-carotene, folic acid and riboflavin; 10 polyphenols: caffeic acid, ellagic acid, (-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin, gallic acid, pyrocatechol, pyrogallol, quercetin and tannic acid which are recognized as antimutagens, were examined. Furthermore, the concentrations for 50% of suppressive abilities of five positive samples, L-ascorbic acid, folic acid, caffeic acid, pyrocatechol and pyrogallol were compared. Negative results were obtained in nine samples, riboflavin, tannic acid, etc. suggesting that their antimutagenic effect on cells may not be related to oxidative damage in cells.