Long-Term in vivo Evaluation of Orthotypical and Heterotypical Bioengineered Human Corneas.

PURPOSE: Human cornea substitutes generated by tissue engineering currently require limbal stem cells for the generation of orthotypical epithelial cell cultures. We recently reported that bioengineered corneas can be fabricated in vitro from a heterotypical source obtained from Wharton's jelly in the human umbilical cord (HWJSC). METHODS: Here, we generated a partial thickness cornea model based on plastic compression nanostructured fibrin-agarose biomaterials with cornea epithelial cells on top, as an orthotypical model (HOC), or with HWJSC, as a heterotypical model (HHC), and determined their potential in vivo usefulness by implantation in an animal model. RESULTS: No major side effects were seen 3 and 12 months after implantation of either bioengineered partial cornea model in rabbit corneas. Clinical results determined by slit lamp and optical coherence tomography were positive after 12 months. Histological and immunohistochemical findings demonstrated that in vitro HOC and HHC had moderate levels of stromal and epithelial cell marker expression, whereas in vivo grafted corneas were more similar to control corneas. CONCLUSION: These results suggest that both models are potentially useful to treat diseases requiring anterior cornea replacement, and that HHC may be an efficient alternative to the use of HOC which circumvents the need to generate cornea epithelial cell cultures.

Photographic-Based Optical Evaluation of Tissues and Biomaterials Used for Corneal Surface Repair: A New Easy-Applied Method.

PURPOSE: Tissues and biomaterials used for corneal surface repair require fulfilling specific optical standards prior to implantation in the patient. However, there is not a feasible evaluation method to be applied in clinical or Good Manufacturing Practice settings. In this study, we describe and assess an innovative easy-applied photographic-based method (PBM) for measuring functional optical blurring and transparency in corneal surface grafts. METHODS: Plastic compressed collagen scaffolds (PCCS) and multilayered amniotic membranes (AM) samples were optically and histologically evaluated. Transparency and image blurring measures were obtained by PBM, analyzing photographic images of a standardized band pattern taken through the samples. These measures were compared and correlated to those obtained applying the Inverse Adding-Doubling (IAD) technique, which is the gold standard method. RESULTS: All the samples used for optical evaluation by PBM or IAD were histological suitable. PCCS samples presented transmittance values higher than 60%, values that increased with increasing wavelength as determined by IAD. The PBM indicated that PCCS had a transparency ratio (TR) value of 80.3 ± 2.8%, with a blurring index (BI) of 50.6 ± 4.2%. TR and BI obtained from the PBM showed a high correlation (ρ>|0.6|) with the diffuse transmittance and the diffuse reflectance, both determined using the IAD (p<0.005). The AM optical properties showed that there was a largely linear relationship between the blurring and the number of amnion layers, with more layers producing greater blurring. CONCLUSIONS: This innovative proposed method represents an easy-applied technique for evaluating transparency and blurriness of tissues and biomaterials used for corneal surface repair.

Predictive algorithms for determination of reflectance data from quantity of pigments within experimental dental resin composites.

BACKGROUND: Being able to estimate (predict) the final spectrum of reflectance of a biomaterial, especially when the final color and appearance are fundamental for their clinical success (as is the case of dental resin composites), could be a very useful tool for the industrial development of these type of materials. The main objective of this study was the development of predictive models which enable the determination of the reflectance spectrum of experimental dental resin composites based on type and quantity of pigments used in their chemical formulation. METHODS: 49 types of experimental dental resin composites were formulated as a mixture of organic matrix, inorganic filler, photo activator and other components in minor quantities (accelerator, inhibitor, fluorescent agent and 4 types of pigments). Spectral reflectance of all samples were measured, before and after artificial chromatic aging, using a spectroradiometer. A Multiple Nonlinear Regression Model (MNLR) was used to predict the values of the Reflectance Factors values in the visible range (380 nm-780 nm), before and after aging, from % Pigment (%P1, %P2, %P3 and %P4) within the formulation. RESULTS: The average value of the prediction error of the model was 3.46% (SD: 1.82) across all wavelengths for samples before aging and 3.54% (SD: 1.17) for samples after aging. The differences found between the predicted and measured values of the chromatic coordinates are smaller than the acceptability threshold and, in some cases, are even below the perceptibility threshold. CONCLUSIONS: Within the framework of this pilot study, the nonlinear predictive models developed allow the prediction, with a high degree of accuracy, of the reflectance spectrum of the experimental dental resin composites.

Evaluation of small intestine grafts decellularization methods for corneal tissue engineering.

Advances in the development of cornea substitutes by tissue engineering techniques have focused on the use of decellularized tissue scaffolds. In this work, we evaluated different chemical and physical decellularization methods on small intestine tissues to determine the most appropriate decellularization protocols for corneal applications. Our results revealed that the most efficient decellularization agents were the SDS and triton X-100 detergents, which were able to efficiently remove most cell nuclei and residual DNA. Histological and histochemical analyses revealed that collagen fibers were preserved upon decellularization with triton X-100, NaCl and sonication, whereas reticular fibers were properly preserved by decellularization with UV exposure. Extracellular matrix glycoproteins were preserved after decellularization with SDS, triton X-100 and sonication, whereas proteoglycans were not affected by any of the decellularization protocols. Tissue transparency was significantly higher than control non-decellularized tissues for all protocols, although the best light transmittance results were found in tissues decellularized with SDS and triton X-100. In conclusion, our results suggest that decellularized intestinal grafts could be used as biological scaffolds for cornea tissue engineering. Decellularization with triton X-100 was able to efficiently remove all cells from the tissues while preserving tissue structure and most fibrillar and non-fibrillar extracellular matrix components, suggesting that this specific decellularization agent could be safely used for efficient decellularization of SI tissues for cornea TE applications.

Color and translucency of zirconia ceramics, human dentine and bovine dentine.

OBJECTIVES: Evaluate color and translucency of both non-colored and colored zirconia ceramics and compare them with corresponding properties of human dentine. METHODS: 0.5mm thick samples of human (MSHD) and bovine (MSBD) dentine were obtained from 5 extracted human anterior maxillary teeth and 5 bovine incisors, respectively. The zirconia systems evaluated (0.5mm samples) were: (1) IPS e.max(®) ZirCAD sintered samples: (a) regular (ZC1); (b) colored - coloring liquid CL2 (ZC2); (2) LAVA™ Zirconia sintered samples: (a) regular (LV1); (b) colored - coloring liquid FS3 (LV2). Translucency parameter (TP) and ΔE(ab)(*) and ΔE(00) color differences (with respect to the MSHD) were calculated. Spectral reflectance curves were compared using the VAF coefficient. RESULTS: Reflectance spectral behaviour of MSBD and MSHD were similar (VAF=99.3%). All zirconia samples showed higher spectral reflectance values than the MSHD. Lightness values obtained for all zirconia ceramics (especially LV) were higher than MSHD and MSBD. The range of color differences, with respect to MSHD, was 6.9-20.5 for ΔE(ab)(*) and 4.9-15.6 for ΔE(00). Colored samples showed lower color differences with respect to MSHD. The highest TP values were found for IPS e.max(®) ZirCAD system and the lowest for LAVA™ Zirconia system. In terms of translucency, MSHD, MSBD and zirconia ceramics showed no statistically significant differences. CONCLUSIONS: In terms of translucency, the studied zirconia systems could satisfactorily replace the human dentine within a dental restoration but, in order to produce a clinically acceptable match, it is necessary to carefully adjust the color of these systems.

Transparency in a fibrin and fibrin-agarose corneal stroma substitute generated by tissue engineering.

PURPOSE: To examine the transparency characteristics at different times of development in the culture of 2 different types of human corneal stroma substitutes generated by tissue engineering using human fibrin or human fibrin and 0.1% agarose, with human keratocytes entrapped within. METHODS: The transparency of these artificial corneal stromas was analyzed after 1, 7, 14, 21, and 28 days of development in culture by determining the scattering and absorption coefficients from the spectral reflectance data of each sample using the Kubelka-Munk equations. RESULTS: The scattering coefficient of both types of bioengineered tissues tended to increase with culture time and wavelength until 550 nm, whereby a slight decrease was observed for longer wavelengths. In general, the spectral distribution of the Kubelka-Munk scattering coefficient of the fibrin-agarose corneal constructs was more stable than that of the fibrin constructs. The absorption coefficient of the human fibrin and fibrin-agarose corneal substitutes tended to decrease with increasing wavelength, and their absolute values were higher for fibrin-agarose than for fibrin scaffolds, especially for short wavelengths. In addition, the spectral transmittance behavior of both types of tissue analyzed was similar to the ones of the theoretical and control corneas, with absolute values above 90% for all wavelengths at 28 days of development. CONCLUSIONS: The transparency, scattering, and absorption of both fibrin and fibrin-agarose corneal stroma substitutes indicate that these new biomaterials could be adequate for clinical use.