The endoscopic radiofrequency procedure (Stretta) has been used for more than a decade to treat patients with gastroesophageal reflux disease (GERD). However, the efficacy of the procedure in improving objective and subjective clinical endpoints needs to be further established.

Lens autofluorescence is increased in patients with diabetes mellitus, but clinical application has been limited by the lack of an instrument suitable for routine clinical use. We investigate possible uses of a new scanning confocal biomicroscope (1) to identify subjects with undiagnosed type 2 diabetes and (2) as a marker for the progression of diabetes. One hundred seventy-eight subjects self-reported as normal and 53 subjects physician-diagnosed with diabetes or prediabetes were recruited. Measurements were collected using a ClearPath DS-120 Lens Fluorescence Biomicroscope calibrated with standards traceable to National Institute of Standards and Technology (NIST). Fluorescence intensities were corrected for age by subtracting the value expected from a regression of intensity versus age for normal subjects. This "fluorescence deviation" showed progressively higher values for normal, prediabetes, type 2 diabetes, and type 1 diabetes and a high degree of predictability of diabetes diagnosis. A receiver operating characteristics curve was used to determine sensitivity and specificity for prediction of diabetes type 2. At a fluorescence deviation of 2500, a sensitivity of 67% at 94% specificity was observed detection of type 2 diabetes. The progressively higher fluorescence deviations are consistent with the physiological mechanisms of accumulation of fluorescent advanced glycation end products as the subject ages. The sensitivity and specificity performance of the lens autofluorescence test for type 2 diabetes is comparable to the performance of glucose threshold tests. The statistically significant difference between fluorescence deviations of normal and type 2 diabetes supports the feasibility of lens autofluorescence to screen subjects for undiagnosed type 2 diabetes. Ophthalmic practices are points of care at which there may be a public health benefit for screening patients for undiagnosed diabetes.

BACKGROUND:Lens autofluorescence increases with the age of the subject, and the fluorophores responsible are associated with cataract, retinopathy, and other complications of diabetes. We built a scanning confocal lens fluorescence biomicroscope suitable for routine clinical measurement of lens autofluorescence and light scattering and report data from 127 healthy subjects.METHOD:The fluorescence biomicroscope focuses a beam of light from a blue light-emitting diode on the lens and measures fluorescent green light and blue scattered light using a sensitive silicon photomultiplier. The system includes a target fixation light and a video camera for alignment and automatic pupil tracking. Under software control, a volume of measurement is scanned from behind the posterior lens capsule, through the lens to the aqueous humor, and then back again. Software computes the average ratio of lens autofluorescence to scattered light in the central portion of the lens. Self-reported healthy nondiabetic subjects were examined by an optometrist; if their eyes were healthy and without significant cataract, they were entered into the study.RESULTS:Valid lens autofluorescence data were collected from 127 subjects between 21 and 70 years of age. A linear model for lens autofluorescence intensity with age was highly statistically significant, and the improvement in fit for higher-order polynomial models was not statistically significant. The ratio of lens autofluorescence to light scatter was also calculated; regression analysis showed significant curvature for the relationship of the fluorescence ratio to age, so a nonlinear model was used to estimate the mean ratio of autofluorescence to scatter and its prediction intervals as a function of age.CONCLUSIONS:Our observation of a strongly significant linear regression of fluorescence intensity with age of the subjects agrees with the results from previous studies, as does a nonlinear model for the fluorescence ratio. The fluorescence biomicroscope enables the clinician to identify patients with fluorescence ratio significantly higher than expected for their age.

Generation of a stable interface between soft tissues and biomaterials could improve the function of transcutaneous prostheses, primarily by minimizing chronic infections. We hypothesized that inclusion of non-biodegradable biomaterials in an artificial skin substrate would improve integration of the neodermis. In the present study, we compared the biocompatibility of an experimental substrate, consisting of collagen and glycosylaminoglycans, with commercially available artificial skin of similar composition. By utilizing a mouse excisional wound model, we found that the source of collagen (bovine tendon versus hide), extent of injury and wound contraction were critical determinants of inflammation and neodermis formation. Reducing the extent of injury to underlying muscle reduced inflammation and improved remodeling; the improved conditions allowed the detection of a pro-inflammatory effect of hide-derived collagen. To eliminate the complication of wound contraction, subsequent grafts were performed in guinea pigs and showed that inclusion of carbon fibers or non-degradable sutures resulted in increased foreign body response (FBR) and altered remodeling. On the other hand, inclusion of a polyester multi-stranded mesh induced a mild FBR and allowed normal neodermis formation. Taken together, our observations suggest that non-degradable biomaterials can be embedded in an artificial skin construct without compromising its ability to induce neodermis formation.

e-biomed: The Journal of Regenerative MedicineVol. 1, No. 12 Review: Technologies and Characteristics of Tissue-Engineered Skin SubstitutesFrederick CahnFrederick CahnSearch for more papers by this authorPublished Online:6 Jul 2004https://doi.org/10.1089/152489000750018361AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetails Volume 1Issue 12Oct 2000 To cite this article:Frederick Cahn.Review: Technologies and Characteristics of Tissue-Engineered Skin Substitutes.e-biomed: The Journal of Regenerative Medicine.Oct 2000.145-155.http://doi.org/10.1089/152489000750018361Published in Volume: 1 Issue 12: July 6, 2004PDF download

Artificial skin is a bilayer skin replacement system designed to regenerate dermal tissue. Prior to the commercial availability of artificial skin, surgically created wounds that cannot be closed by primary means (such as excising deep partial or full-thickness burns), had to be treated with autograft; a graft of the patient’s own skin harvested from a healthy donor site. This is because the dermal layer of skin cannot regenerate functional tissue spontaneously; instead, scar tissue forms. When applied surgically to a clean, excised wound bed, autograft becomes permanently engrafted, that is, it becomes permanently affixed to the underlying tissue and vascularized. However, autograft has serious drawbacks, including the creation of a donor wound, which has its own significant morbidity, and its unavailability in sufficient quantity in patients with large wounds.

Fourier-transform infrared (FT-IR) spectra of malignant and dysplastic cervical scrapings were abnormal, as first described in our study of a limited number of samples, where the spectra were evaluated by visual inspection and peak intensity ratios. We have expanded our study to evaluate more cervical conditions, and to analyze the spectra by a chemometric approach (principal component analysis [PCA]). Cervical samples from 436 females were evaluated by FT-IR and Papanicolaou testing; 40/436 spectra were nonanalyzable. The remaining were as follows: normal, 174; malignant, 19; dysplasia, 8; atypia, 113; atrophy, 19; inflammatory, 47; bloody smear, 12; hypocellular, 4. PCA analysis followed by chi2 test revealed that statistically significant frequencies of being predicted malignant by FT-IR were associated with samples diagnosed as malignant (P < 0.0001), and also those diagnosed as "atrophy" (P < 0.001), "atypical with bloody smear" (P < 0.05), "atypical with atrophic pattern" (P < 0.05), and "dysplasia" (P < 0.05). Based on these findings, for the diagnosis of cervical cancer by FT-IR, as defined here, the sensitivity is 79%, the specificity is 77%, the positive predictive value is 15%, and the negative predictive value is 98.6%. Our findings (a) demonstrate the application of a chemometric approach to the study of cervical FT-IR spectra; (b) assess its potential diagnostic role; (c) suggest that atrophic and neoplastic samples share structural features; and (d) suggest that blood may interfere with such spectroscopic evaluation. These findings warrant further evaluation of FT- IR spectroscopy in cervical and other malignancies.

The role of permeable substrates on the proliferation and differentiation of rat spermatogenic cells in co-culture with Sertoli cells was evaluated. Co-cultures were prepared on substrate discs consisting of a thin, stable analog of extracellular matrix attached to a polyester mesh to facilitate handling. Substrate discs were used alone or mounted within a polysulfone reusable holder. Substrates included collagen type I alone or crosslinked with the glycosaminoglycans (GAG) chondroitin-6-sulfate (8%) or heparin (5%). Chemical crosslinking immobilizes collagen and GAG components, preserves the native triple-helical configuration of collagen type I, and renders the copolymer more resistant to degradation by cellular enzymes. Cell attachment and growth properties of Sertoli and spermatogenic cells on these substrates were compared with another permeable substrate (HATF, a mixture of cellulose esters) uncoated or coated with Matrigel (extracellular matrix material derived from EHS tumors) and with the more conventional nonpermeable glass or plastic substrates. We have found that Sertoli-spermatogenic cell co-cultures prepared from pubertal rats readily attach to collagen or collagen-GAG substrate discs as well as to HATF. However, the optical transparency of collagen or collagen-GAG, as compared to the opaque HATF substrate, facilitates monitoring cell attachment and growth by phase contrast microscopy. Substrate discs processed for light and electron microscopy using standard procedures demonstrate the organization of a structurally polarized epithelial layer with basally located Sertoli cells and spermatogenic cells associated by lateral and apical Sertoli cell surfaces. The permeable nature of the collagen-GAG substrate, its optical transparency, and the formation of an electrical-resistant, polarized Sertoli-spermatogenic cell epithelial layer opens new in vitro experimental possibilities for testing agents that may favor or disrupt the spermatogenic process.

A novel collagen-glycosaminoglycan (C-GAG) substrate was developed to overcome the optical opacity of a HATF nitrocellulose substrate and to provide a more physiological permeable substrate for cocultured Sertoli and spermatogenic cells. Cocultures were prepared on optically transparent C-GAG discs attached to a polyester mesh to facilitate handling. Sertoli cells displayed a cuboidal-to-columnar shape; a large number of spermatogonia and primary spermatocytes connected by intercellular bridges were associated with basolateral and apical surfaces of Sertoli cells up to 12 days after plating. Rat Sertoli-spermatogenic cell cocultures have been used for testing the effect of toxicants on rat spermatogenesis in vitro. In our initial studies, we tested the effects of the toxicant gossypol on spermatogenic cells cocultured with Sertoli cells on nonpermeable (plastic) and permeable substrates (HATF nitrocellulose) under both standard culture conditions and during perifusion after achieving a continuous electrical-resistant cell monolayer. A selective mitochondrial structural damage was observed in spermatogenic cells (spermatogonia and spermatocytes) but not in the coexisting Sertoli cells. This damage was time- (15–60 min) and dose-dependent (0.1–10µM) and developed more rapidly under perifusion conditions. Similar mitochondrial damage was reported in the intact animal but required higher concentrations (mg) and longer administration time (months) for detection. Studies are in progress to evaluate the effect of additional toxic chemical agents on functional properties of Sertoli and spermatogenic cells in cocultures prepared on various classes of C-GAG substrates.

Spherical porous microcarriers (PMCs) made from collagen-glycosaminoglycan crosslinked copolymers have exhibited considerable promise as growth surfaces for the proliferation of anchorage-dependent mammalian cell lines and have demonstrated the ability to entrap anchorage-independent cells. However, quantification of cell growth on PMCs has proved difficult. A method of measuring the proliferation of PMCs, based on image analysis, is presented. Using CV1 and CHO cell lines, samples of PMCs were removed from culture at various times, fixed, embedded and sectioned. The 2 microns sections were stained, photographed and digitized in three colors. A computer program was developed to evaluate digitized PMC cross-sections and to classify pixels as conforming to either background, cytoplasmic, matrix or nuclear parameters, based on a set of classification rules determined by statistical analysis. Growth curves were generated by relating the number of pixels occupied by cellular material to the total number of pixels in the PMC cross-section. The PMCs were found to foster cell proliferation, with cell densities approaching 100% occupancy.