Expression of p75(NGFR), a Proliferative and Basal Cell Marker, in the Buccal Mucosa Epithelium during Re-epithelialization.

We investigated the expression of p75(NGFR), a proliferative and basal cell marker, in the mouse buccal mucosa epithelium during wound healing in order to elucidate the role of epithelial stem cells. Epithelial defects were generated in the epithelium of the buccal mucosa of 6-week-old mice using CO2 laser irradiation. BrdU was immediately administered to mice following laser irradiation. They were then sacrificed after 1, 3, 7, and 14 days. Paraffin sections were prepared and the irradiated areas were analyzed using immunohistochemistry with anti-p75(NGFR), BrdU, PCNA, and CK14 antibodies. During re-epithelialization, PCNA (-)/p75(NGFR) (+) cells extended to the wound, which then closed, whereas PCNA (+)/p75(NGFR) (+) cells were not observed at the edge of the wound. In addition, p75(NGFR) (-)/CK14 (+), which reflected the presence of post-mitotic differentiating cells, was observed in the supra-basal layers of the extended epithelium. BrdU (+)/p75(NGFR) (+), which reflected the presence of epithelial stem cells, was detected sparsely in buccal basal epithelial cells after healing, and disappeared after 7 days. These results suggest that p75(NGFR) (+) keratinocytes are localized in the basal layer, which contains oral epithelial stem cells, and retain the ability to proliferate in order to regenerate the buccal mucosal epithelium.

Re-epithelialization of the Buccal Mucosa after Alkaline Chemical Injury.

Alkaline conditions in the oral cavity may be caused by a variety of stimuli, including tobacco products, antacids, alkaline drinking water and bicarbonate toothpaste. However, the effects of an alkaline pH on the oral mucosa had not been elucidated. The purpose of this study was to investigate how basal keratinocytes are actively involved in re-epithelialization after alkaline chemical injury. We generated epithelial defects in the oral mucosa of mice by applying an alkaline chemical, and the localization of cytokeratin 13, cytokeratin 14, PCNA and p63 was investigated during the re-epithelialization process. PCNA- and p63-positive staining was seen in basal cells covering the wound surface at 1 day after the chemical injury. Cytokeratin 14-positive and PCNA-negative basal keratinocytes were localized in a few layers of the wound epithelium during epithelial outgrowth. Cytokeratin 14-positive and PCNA-positive basal keratinocytes, indicating proliferation, were localized over the entire layer of the epithelium at the wound margin. These results imply that basal keratinocytes at the wound margin migrate to the wound surface, provoke differentiation and keratinization during epithelial outgrowth and that epithelial cells are supplied from the wound margin to the epithelial outgrowth after alkaline chemical injury.

Influence of the precutting and overseas transportation of corneal grafts for Descemet stripping automated endothelial keratoplasty on donor endothelial cell loss.

PURPOSE: The authors evaluated the changes in donor endothelial cell density (ECD) caused by the precutting and long-distance transportation of corneal grafts for Descemet stripping automated endothelial keratoplasty (DSAEK) from overseas eye banks. METHODS: A total of 124 consecutive precut donor tissues for DSAEK were obtained from overseas eye banks. The ECD was examined before precutting, after precutting, and after overseas transportation. Changes in ECD secondary to the precutting procedure and after long-distance transportation were evaluated. RESULTS: The average ECD before and after precutting was 2637 ± 336 and 2578 ± 305, respectively (P = 0.0063). The ECD after overseas transportation was 2468 ± 272, which is a significant decrease compared with the ECD after the precutting procedure (P < 0.001). The average rate of ECD loss was 1.75% for precutting and 3.79% for overseas transportation, with a total ECD loss of 5.68%. The ECD before surgery was >2000 cells per square millimeter in all cases, and all grafts were available for DSAEK. CONCLUSIONS: Imported precut donor corneas form overseas eye banks are a valuable source of donor corneas for DSAEK. The cell loss associated with precutting and the overseas transportation of corneal grafts on donor endothelial cell loss is acceptable.

ADAMTS5 is a biomarker for prediction of response to infliximab in patients with rheumatoid arthritis.

OBJECTIVE: To identify a biomarker for prediction of the response to infliximab (IFX) in patients with rheumatoid arthritis (RA), we focused on a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) that seems to play a key role in aggrecan degradation in cartilage. METHODS: Seventy-three randomly selected patients with active RA were treated with IFX. Peripheral blood samples were collected at baseline and ADAMTS5 messenger RNA (mRNA) was quantified using real-time polymerase chain reaction. RESULTS: Baseline ADAMTS5 mRNA levels in the good responder group were significantly lower (1.84 +/- 1.56; p = 0.0408) than those in the moderate and nonresponder groups (2.54 +/- 1.70) at 38 weeks of treatment with IFX. The 28-joint count Disease Activity Score (DAS28) at 38 weeks of treatment was significantly lower in the low ADAMTS5 group (2.30 +/- 1.28; p = 0.0038) than in the high ADAMTS5 group (3.90 +/- 1.61). The percentage reduction of the DAS28 was significantly higher in the low ADAMTS5 group (52.5% +/- 28.8%; p = 0.0156) than in the high ADAMTS5 group (29.4% +/- 27.2%). Further, the Delta Health Assessment Questionnaire (DeltaHAQ) score, an estimate of the improvement in the HAQ score, at 38 weeks of treatment was significantly higher in the low ADAMTS5 group (1.18 +/- 0.60; p = 0.0102) than in the high ADAMTS5 group (0.21 +/- 0.78). The positive predictive value of a low baseline ADAMTS5 level for predicting good response and remission (DAS28 < 2.6 at 38 weeks) was 90.0% and 70.0%, respectively. CONCLUSION: The baseline ADAMTS5 mRNA level is a candidate biomarker for prediction of the response to IFX in patients with RA.

Coding and traceability for cells, tissues and organs for transplantation.

Modern transplantation of cells, tissues and organs has been practiced within the last century achieving both life saving and enhancing results. Associated risks have been recognized including infectious disease transmission, malignancy, immune mediated disease and graft failure. This has resulted in establishment of government regulation, professional standard setting and establishment of vigilance and surveillance systems for early detection and prevention and to improve patient safety. The increased transportation of grafts across national boundaries has made traceability difficult and sometimes impossible. Experience during the first Gulf War with mis-identification of blood units coming from multiple countries without standardized coding and labeling has led international organizations to develop standardized nomenclature and coding for blood. Following this example, cell therapy and tissue transplant practitioners have also moved to standardization of coding systems. Establishment of an international coding system has progressed rapidly and implementation for blood has demonstrated multiple advantages. WHO has held two global consultations on human cells and tissues for transplantation, which recognized the global circulation of cells and tissues and growing commercialization and the need for means of coding to identify tissues and cells used in transplantation, are essential for full traceability. There is currently a wide diversity in the identification and coding of tissue and cell products. For tissues, with a few exceptions, product terminology has not been standardized even at the national level. Progress has been made in blood and cell therapies with a slow and steady trend towards implementation of the international code ISBT 128. Across all fields, there are now 3,700 licensed facilities in 66 countries. Efforts are necessary to encourage the introduction of a standardized international coding system for donation identification numbers, such as ISBT 128, for all donated biologic products.

Collagen-phosphorylcholine interpenetrating network hydrogels as corneal substitutes.

A biointeractive collagen-phospholipid corneal substitute was fabricated from interpenetrating polymeric networks comprising 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide and N-hydroxysuccinimide crosslinked porcine atelocollagen, and poly(ethylene glycol) diacrylate crosslinked 2-methacryloyloxyethyl phosphorylcholine (MPC). The resulting hydrogels showed an overall increase in mechanical strength beyond that of either original component and enhanced stability against enzymatic digestion (by collagenase) or UV degradation. More strikingly, these hydrogels retained the full biointeractive, cell friendly properties of collagen in promoting corneal cell and nerve in-growth and regeneration (despite MPC's known anti-adhesive properties). Measurements of refractive indices, white light transmission and backscatter showed the optical properties of collagen-MPC are comparable or superior to those of the human cornea. In addition, the glucose and albumin permeability were comparable to those of human corneas. Twelve-month post-implantation results of collagen-MPC hydrogels into mini-pigs showed regeneration of corneal tissue (epithelium, stroma) as well as the tear film and sensory nerves. We also show that porcine collagen can be substituted with recombinant human collagen, resulting in a fully-synthetic implant that is free from the potential risks of disease transmission (e.g. prions) present in animal source materials.

Morphological evidence of basal keratinocyte migration during the re-epithelialization process.

The regeneration of wounded stratified epithelium is accomplished via the migration of keratinocytes from the margins of the wound. However, the process of keratinocyte migration on the wound surface and the role of epithelial stem cells during re-epithelialization remain to be elucidated. Therefore, we administered BrdU to embryonic mice and generated epithelial defects on the buccal mucosa of these mice at two weeks after birth, using CO(2) laser irradiation, with which we removed the entire thickness of the epithelium. In the unwounded epithelium, cytokeratin 14, p63, and BrdU were localized within the basal layer of the epithelium, but the majority of cells within the regenerated epithelium were immunopositive for these proteins. PCNA-negative and BrdU-positive basal keratinocytes, which evidence a slow cell cycle, were localized solely within the basal layer of the unwound epithelium facing the tips of dermal papillae. After laser irradiation, these basal keratinocytes facing the tips of the papillae evidenced positive immunoreactivity for PCNA, in addition to BrdU. These results indicate that epithelial stem cells of oral mucosa may be localized in the basal layer of the epithelium facing the tips of dermal papillae, and may migrate laterally with other basal keratinocytes in response to external stimuli.

Comparative analysis of ABCG2-expressing and label-retaining cells in mouse submandibular gland.

The submandibular gland (SMG) is a tissue that can be regenerated in a tissue injury model and that has adult stem cells capable of self-renewal and differentiation into functional cells. We have analyzed the localization of label-retaining cells (LRCs), which are putative progenitor cells, by using the BrdU-labeling method. 5-Bromo-2'-deoxyuridine (BrdU) injection followed by a long chasing period permitted the identification of LRCs based on the slow-cycling characteristic. In order to confirm the accurate localization of LRCs, BrdU and SMG-specific markers, including aquaporin5, cytokeratin, and smooth muscle actin, were examined by double-immunofluorescence staining. We found that LRCs were distributed in the acinus, duct, myoepithelium, and connective tissue. Moreover, ABCG2 (a known stem cell marker) was used for the characterization of LRCs and the localization of cells as putative stem/progenitor cells. ABCG2-expressing cells were distributed in various regions of the SMG but did not co-localize with LRCs. We suggest that putative progenitor cells exist in various regions of the SMG and have diverse capacities to differentiate into specific cells.

Creutzfeldt-Jakob disease in recipients of corneal transplants.

PURPOSE: Creutzfeldt-Jakob disease (CJD) transmission has been documented to occur from the use of corneal grafts. We report 4 cases of CJD with a history of corneal transplantation and assess the frequency of coincidental CJD among corneal transplant recipients. METHODS: Medical records and eye bank documents were reviewed. Genetic and neuropathologic tests on available specimens were performed at the National Prion Disease Pathology Surveillance Center. Statistical analyses were used to determine the expected number of coincidental CJD cases among the US population with a history of corneal transplantation. RESULTS: Four CJD decedents with histories of corneal transplantation were identified: 3 from the United States and 1 from Japan. The time from transplant to onset of CJD symptoms ranged from 2 years, 11 months to 18 years. Available eye bank records did not suggest evidence of neurologic illness in the donors. Using corneal transplantation and CJD death data from 1990 through 2006, statistical analyses suggest that a case of coincidental sporadic CJD will occur among the population of corneal transplant recipients approximately every 1.5 years. CONCLUSIONS: It is likely that these 4 recipients of transplanted corneas had sporadic CJD. Because of the many corneal transplantations performed each year in the United States, occasional cases of sporadic CJD in this population are expected.

Regeneration of corneal cells and nerves in an implanted collagen corneal substitute.

PURPOSE: Our objective was to evaluate promotion of tissue regeneration by extracellular matrix (ECM) mimics, by using corneal implantation as a model system. METHODS: Carbodiimide cross-linked porcine type I collagen was molded into appropriate corneal dimensions to serve as substitutes for natural corneal ECM. These were implanted into corneas of mini-pigs after removal of the host tissue, and tracked over 12 months, by clinical examination, slit-lamp biomicroscopy, in vivo confocal microscopy, topography, and esthesiometry. Histopathology and tensile strength testing were performed at the end of 12 months. Other samples were biotin labeled and implanted into mice to evaluate matrix remodeling. RESULTS: The implants promoted regeneration of corneal cells, nerves, and the tear film while retaining optical clarity. Mechanical testing data were consistent with stable, seamless host-graft integration in regenerated corneas, which were as robust as the untreated fellow corneas. Biotin conjugation is an effective method for tracking the implant within the host tissue. CONCLUSIONS: We show that a simple ECM mimetic can promote regeneration of corneal cells and nerves. Gradual turnover of matrix material as part of the natural remodeling process allowed for stable integration with host tissue and restoration of mechanical properties of the organ. The simplicity in fabrication and shown functionality shows potential for ECM substitutes in future clinical applications.

Tissue-engineered recombinant human collagen-based corneal substitutes for implantation: performance of type I versus type III collagen.

PURPOSE: To compare the efficacies of recombinant human collagens types I and III as corneal substitutes for implantation. METHODS: Recombinant human collagen (13.7%) type I or III was thoroughly mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. The final homogenous solution was either molded into sheets for in vitro studies or into implants with the appropriate corneal dimensions for transplantation into minipigs. Animals with implants were observed for up to 12 months after surgery. Clinical examinations of the cornea included detailed slit lamp biomicroscopy, in vivo confocal microscopy, and fundus examination. Histopathologic examinations were also performed on corneas harvested after 12 months. RESULTS: Both cross-linked recombinant collagens had refractive indices of 1.35, with optical clarity similar to that in human corneas. Their chemical and mechanical properties were similar, although RHC-III implants showed superior optical clarity. Implants into pig corneas over 12 months show comparably stable integration, with regeneration of corneal cells, tear film, and nerves. Optical clarity was also maintained in both implants, as evidenced by fundus examination. CONCLUSIONS: Both RHC-I and -III implants can be safely and stably integrated into host corneas. The simple cross-linking methodology and recombinant source of materials makes them potentially safe and effective future corneal matrix substitutes.

Recombinant human collagen for tissue engineered corneal substitutes.

We successfully fabricated transparent, robust hydrogels as corneal substitutes from concentrated recombinant human type I and type III collagen solutions crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). White light transmission through these gels is comparable or superior to that of human corneas. Hydrogels from both type I and type III collagens supported in vitro epithelium and nerve over-growth. While both these biocompatible hydrogels have adequate tensile strength and elasticity for surgical manipulation, type III collagen hydrogels tended to be mechanically superior. Twelve-month post-implantation results of type I recombinant collagen-based corneal substitutes into mini-pigs showed retention of optical clarity, along with regeneration of corneal cells, nerves and tear film. For clinical use, implants based on fully characterized, recombinant human collagen eliminate the risk of pathogen transfer or xenogeneic immuno-responses posed by animal collagens.

Renal L-type fatty acid--binding protein in acute ischemic injury.

Fatty acid-binding proteins (FABPs) bind unsaturated fatty acids and lipid peroxidation products during tissue injury from hypoxia. We evaluated the potential role of L-type FABP (L-FABP) as a biomarker of renal ischemia in both human kidney transplant patients and animal models. Urinary L-FABP levels were measured in the first urine produced from 12 living-related kidney transplant patients immediately after reperfusion of their transplanted organs, and intravital video analysis of peritubular capillary blood flow was performed simultaneously. A significant direct correlation was found between urinary L-FABP level and both peritubular capillary blood flow and the ischemic time of the transplanted kidney (both P < 0.0001), as well as hospital stay (P < 0.05). In human-L-FABP transgenic mice subjected to ischemia-reperfusion injury, immunohistological analyses demonstrated the transition of L-FABP from the cytoplasm of proximal tubular cells to the tubular lumen. In addition, after injury, these transgenic mice demonstrated lower blood urea nitrogen levels and less histological injury than injured wild-type mice, likely due to a reduction of tissue hypoxia. In vitro experiments using a stable cell line of mouse proximal tubule cells transfected with h-L-FABP cDNA showed reduction of oxidative stress during hypoxia compared to untransfected cells. Taken together, these data show that increased urinary L-FABP after ischemic-reperfusion injury may find future use as a biomarker of acute ischemic injury.

Innervation of tissue-engineered corneal implants in a porcine model: a 1-year in vivo confocal microscopy study.

PURPOSE: To examine the pattern of nerve regeneration within tissue-engineered corneal substitutes grafted into host porcine corneas over a 1-year postoperative period. METHODS: Biodegradable corneal substitutes from cross-linked collagen were implanted into the left eyes of 12 pigs by deep lamellar keratoplasty. Regeneration of severed nerves into the central implant region was investigated with in vivo confocal microscopy. Both implant-recipient and control (right) eyes were examined before surgery and 2, 6, 10, and 12 months after surgery, to quantify the number, density, diameter, and branching of nerve fiber bundles at various corneal depths. Transmission electron microscopy was used to confirm the presence of nerve bundles. RESULTS: Two months after surgery, corneal nerve ingrowth was observed within the deep anterior stroma, with a number and density of regenerated nerves significantly higher than in nonsurgical control eyes (P < 0.01). Nerves within the superficial anterior stroma regenerated by 6 to 10 months after surgery, and the first subbasal epithelial nerves were seen 10 months after surgery. After 1 year, subbasal nerve density recovered to preoperative levels. Nerve fibers in the deep anterior stroma remained significantly thinner relative to control eyes after 1 year (P < 0.001), where both superficial anterior and subbasal nerve diameter did not change relative to control eyes. CONCLUSIONS: The pattern of reinnervation within tissue-engineered corneal substitutes has been quantified in vivo. Innervation proceeded rapidly in the deep anterior stroma, followed by repopulation of more superficial regions. One year after surgery, nerve density within the tissue-engineered cornea increased or remained unchanged relative to controls in all corneal regions examined.

Long-standing bullous keratopathy is associated with peripheral conjunctivalization and limbal deficiency.

OBJECTIVE: To investigate whether peripheral corneal neovascularization in bullous keratopathy (BK) is due to conjunctivalization, a sign of limbal stem cell deficiency. DESIGN: Observational case-control study. PARTICIPANTS: Sixteen BK patients. METHODS: Patients were divided into 2 groups: BK without peripheral neovascularization [NV(-) group; 5 patients, 5 eyes] and BK with neovascularization [NV(+) group; 11 patients, 13 eyes]. Evidence of conjunctivalization was evaluated by periodic acid-Schiff staining of impression cytology samples from the peripheral vascularized cornea. The 2 groups' durations of disease also were compared. Penetrating keratoplasty (PK) was performed in all 16 cases, and the 2 groups' durations of reepithelialization after PK were compared. MAIN OUTCOME MEASURES: Presence of goblet cells using impression cytology, duration of BK, and duration of postoperative reepithelialization. RESULTS: Goblet cells were found on the peripheral corneal surface in all eyes in the NV(+) group. However, all eyes in the NV(-) group were negative for goblet cells (P<0.0001). Duration of disease was 14.4+/-5.4 months in the NV(-) group and 66.2+/-65.5 months in the NV(+) group (P = 0.030). Duration of postoperative epithelialization was 6.2+/-2.2 days in the NV(-) group and 28.8+/-36.5 days in the NV(+) group (P = 0.046). CONCLUSION: Conjunctivalization of the peripheral cornea and delayed postoperative epithelialization in BK patients with NV suggest the presence of limbal stem cell deficiency in such patients. Patients with long-standing disease were found to be more prone to neovascularization. For this reason, early surgery may lead to a better surgical outcome.

[Donor action program].

Recent advances of organ transplantation accelerated shortage of organs. Donor Action Program (DAP) was developed to establish a proper donation process in a hospital using total quality management methodology. It has been demonstrated effective in increasing donation and has been introduced in 23 countries. In Japan preliminary study demonstrated that (1) DAP could be implemented and was likely to increase donation in Japan, (2) Japanese healthcare staffs were likely to underestimate social needs and clinical results of transplantation and to be suspicious about brain death, (3) they had limited knowledge and experience in communicating with family members of the deceased, and their needs for education were not satisfied. In order to implement DAP in Japan, an organization which is responsible for data management and development for educational program should be considered with high priority.

Serum-free spheroid culture of mouse corneal keratocytes.

PURPOSE: To develop a serum-free mass culture system for mouse keratocytes. METHODS: Corneas of C57BL6/J mice were enzyme digested after the epithelium and endothelium were removed. Stromal cells were cultured in serum-free DMEM/F12 (1:1) containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), and B27 supplement. Primary spheres were dissociated by trypsin and subcultured as suspended secondary spheres. Cells from postnatal day (P)6 to P10 spheres were subcultured onto plastic dishes or type I collagen gels for phenotype analysis. The expression of the keratocyte markers keratocan, aldehyde dehydrogenase (Aldh), and CD34, were analyzed by RT-PCR, and vimentin and alpha-smooth muscle actin (alpha-SMA) were examined by immunocytochemistry. RESULTS: Primary keratocytes formed spheres, which were cultured for over 12 passages. Suspended sphere cells expressed vimentin, keratocan, CD34, and lumican, but were negative for cytokeratin K12 (K12) and Pax6. Sphere cells subcultured on plastic exhibited a dendritic morphology characteristic of keratocytes, and maintained keratocan, Aldh, and CD34 expression in serum-free medium. Sphere cells subcultured with 10% serum became fibroblastic, and expressed alpha-SMA when stimulated by transforming growth factor (TGF)-beta. alpha-SMA-positive cells demonstrated contractile properties on collagen gels, compatible with the myofibroblast phenotype. CONCLUSIONS: The phenotype of mouse keratocytes can be maintained in vitro for more than 12 passages by the serum-free sphere culturing technique.

Efficacy and safety of international donor sharing: a single-center, case-controlled study on corneal transplantation.

BACKGROUND: To overcome the shortage of donor corneas, we are currently using donor corneas supplied by foreign eye banks. This single-center, case-controlled study was conducted to show the efficacy and safety of corneal transplantation using corneas from foreign donors. METHODS: A retrospective, case-controlled comparison of 118 corneal transplants using foreign donor corneas (foreign group) and domestic donor corneas (domestic group) was performed. The two groups were matched according to original disease, age, severity of preoperative neovascularization, and history of previous grafting. Clinical outcome and incidence of postoperative complications were compared between the two groups. RESULTS: The foreign group had a longer preservation-to-operation time than the domestic group, reflecting the longer transportation time. However, the incidence of clear grafts and postoperative complications such as immunologic rejection, infection, and glaucoma was not significantly different between the two groups. CONCLUSIONS: Corneal transplantations using foreign donor corneas are as effective and safe as those using domestic donor corneas, despite the longer preservation time.

Innervated human corneal equivalents as in vitro models for nerve-target cell interactions.

A sensory nerve supply is crucial for optimal tissue function. However, the mechanisms for successful innervation and the signaling pathways between nerves and their target tissue are not fully understood. Engineered tissue substitutes can provide controllable environments in which to study tissue innervation. We have therefore engineered human corneal substitutes that promote nerve in-growth in a pattern similar to in vivo re-innervation. We demonstrate that these nerves (a) are morphologically equivalent to natural corneal nerves; (b) make appropriate contact with target cells; (c) can generate action potentials; (d) respond to chemical and physical stimuli; and (e) play an important role in the overall functioning of the bioengineered tissue. This model can be used for studying the more general topics of nerve ingrowth or regeneration and the interaction between nerves and their target cells and, more specifically, the role of nerves in corneal function. This model could also be used as an in vitro alternative to animals for safety and efficacy testing of chemicals and drugs.