Subretinal Implantation of a Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium Monolayer in a Porcine Model.

The goal of this study was to quantitatively assess retinal thickness using spectral domain optical coherence tomography (SD-OCT) after subretinal implantation of human embryonic stem cell-derived retinal pigment epithelium in a porcine model. The implant is called CPCB-RPE1 for the California Project to Cure Blindness-Retinal Pigment Epithelium 1. Data were derived from previous experiments on 14 minipigs that received either subretinal implantation of CPCB-RPE1 (n = 11) or subretinal bleb formation alone (sham; n = 3) using previously described methods and procedures (Brant Fernandes et al. Ophthalmic Surg Lasers Imaging Retina 47:342-51, 2016; Martynova et al. (2016) Koss et al. Graefes Arch Clin Exp Ophthalmol 254:1553-65, 2016; Hu et al. Ophthalmic Res 48:186-91, 2016; Martynova et al. ARVO Abstract 2016. SD-OCT retinal thickness (RT) and sublayer thickness over the implant were compared with topographically similar preimplantation regions as described previously Martynova et al. ARVO Abstract 2016. Imaging results were compared to postmortem histology using hematoxylin-eosin staining. RT overlying the CPCB-RPE1 postimplantation was not significantly different from preimplantation (308 ± 72 µm vs 292 ± 41 µm; p = 0.44). RT was not significantly different before and after implantation in any retinal sublayer at 1 month. Histology demonstrated grossly normal retinal anatomy as well as photoreceptor interdigitation with RPE.

Functional domains for amelogenin revealed by compound genetic defects.

We have previously used the yeast two-hybrid assay and multiple in vitro methodologies to show that amelogenin undergoes self-assembly involving two domains (A and B). Using transgenic animals, we show that unique enamel phenotypes result from disruptions to either the A- or B-domain, supporting the role of amelogenin in influencing enamel structural organization. By crossbreeding, animals bearing two defective amelogenin gene products have a more extreme enamel phenotype than the sum of the defects evident in the individual parental lines. At the nanoscale level, the forming matrix shows alteration in the size of the amelogenin nanospheres. At the mesoscale level of enamel structural hierarchy, 6-week-old enamel exhibits defects in enamel rod organization caused by perturbed organization of the precursor organic matrix. These studies reflect the critical dependency of amelogenin self-assembly to form a highly organized enamel organic matrix, and that amelogenins engineered to be defective in self-assembly produce compound defects in the structural organization of enamel.

A simplified genetic design for mammalian enamel.

A biomimetic replacement for tooth enamel is urgently needed because dental caries is the most prevalent infectious disease to affect man. Here, design specifications for an enamel replacement material inspired by Nature are deployed for testing in an animal model. Using genetic engineering we created a simplified enamel protein matrix precursor where only one, rather than dozens of amelogenin isoforms, contributed to enamel formation. Enamel function and architecture were unaltered, but the balance between the competing materials properties of hardness and toughness was modulated. While the other amelogenin isoforms make a modest contribution to optimal biomechanical design, the enamel made with only one amelogenin isoform served as a functional substitute. Where enamel has been lost to caries or trauma a suitable biomimetic replacement material could be fabricated using only one amelogenin isoform, thereby simplifying the protein matrix parameters by one order of magnitude.

Dentin sialoprotein and dentin phosphoprotein overexpression during amelogenesis.

The gene for dentin sialophosphoprotein produces a single protein that is post-translationally modified to generate two distinct extracellular proteins: dentin sialoprotein and dentin phosphoprotein. In teeth, dentin sialophosphoprotein is expressed primarily by odontoblast cells, but is also transiently expressed by presecretory ameloblasts. Because of this expression profile it appears that dentin sialophosphoprotein contributes to the early events of amelogenesis, and in particular to those events that result in the formation of the dentino-enamel junction and the adjacent "aprismatic" enamel. Using a transgenic animal approach we have extended dentin sialoprotein or dentin phosphoprotein expression throughout the developmental stages of amelogenesis. Overexpression of dentin sialoprotein results in an increased rate of enamel mineralization, however, the enamel morphology is not significantly altered. In wild-type animals, the inclusion of dentin sialoprotein in the forming aprismatic enamel may account for its increased hardness properties, when compared with bulk enamel. In contrast, the overexpression of dentin phosphoprotein creates "pitted" and "chalky" enamel of non-uniform thickness that is more prone to wear. Disruptions to the prismatic enamel structure are also a characteristic of the dentin phosphoprotein overexpressing animals. These data support the previous suggestion that dentin sialoprotein and dentin phosphoprotein have distinct functions related to tooth formation, and that the dentino-enamel junction should be viewed as a unique transition zone between enamel and the underlying dentin. These results support the notion that the dentin proteins expressed by presecretory ameloblasts contribute to the unique properties of the dentino-enamel junction.

Overexpression of TRAP in the enamel matrix does not alter the enamel structural hierarchy.

The secreted, full-length amelogenin is the dominant protein of the forming enamel organ. As enamel mineralization progresses, amelogenin is quickly subjected to proteolytic activity, and eliminated from the enamel environment. Mature enamel contains only traces of structural proteins, including enamelin and the sheath protein ameloblastin. In addition, a proteolytic fragment of amelogenin, known as the tyrosine-rich amelogenin peptide or TRAP, is present in low but isolatable quantities. By overexpressing TRAP during enamel development we sought to determine if such overexpression would result in structural alterations to the mature enamel. We reasoned that overexpressing a protein associated with enamel maturation, at an inappropriate developmental stage, would result in alterations to the enamel protein assembly and hence, alterations in enamel structure and morphology. As judged by transmission and scanning electron microscopy, the enamel formed by overexpressing TRAP showed little morphological differences when compared to the enamel of normal nontransgenic animals. Based on scanning electron-microscopic images, there was modest hypomineralization evident in the interrod enamel of the TRAP-overexpressing animals. However, this finding was inconsistent and inconsequential from a structural and functional perspective. From these results it appears that additional amounts of TRAP protein in the immature enamel matrix are not sufficient to alter the properties of the enamel extracellular matrix to an extent that the hierarchical structure of mature enamel is altered.