Analysis of the aqueous humor outflow pathway
Elevated intraocular pressure (IOP) is a major risk factor for the development of glaucoma and all existing glaucoma therapies aim to lower IOP. Despite the importance of IOP, the molecular mechanisms of aqueous humor outflow and their abnormalities in glaucoma are very poorly understood. In collaboration with investigators at The Jackson Laboratory, we have discovered a novel cellular and molecular architecture at the inner wall of Schlemm's canal (SC), the location thought to be critical for IOP regulation. We observe a network of tubes and channels, primarily associated with the inner wall. These tubes contain extracellular matrix components yet also permit fluid flow. This unexpected finding suggests these tubes may provide a mechanism for outflow resistance and thus implies novel locations for IOP disruption in glaucoma.
Figure 1. The inner wall of SC has a complex architecture. Panel A shows 3D reconstructions of an en face view of the inner wall of SC (green) showing interconnected tubes (open arrows) running mostly parallel to the endothelium of the inner wall (asterisks). Type IV collagen can be seen within the tube lumens (red). Panels B and C show the tubes also contain elastin (magenta) and that they also permit fluid flow as visualized by lectin perfusion (Panel D, red).
Figure 2. High-resolution 4pi microscopy reveals the tubes contain type IV collagen in an unusual composition. Panels A and B show 2D and 3D reconstruction views of tubes within the inner wall of SC. Type IV collagen can be visualized lining the tubes (green in Panel A; red in Panel B). 4pi confocal microscopy reveals that type IV collagen not only lines the tubes (solid arrowheads) but fills their lumens with a lattice (asterisks) (Panels C-E).
Development of cornea replacements through tissue engineering
Corneal damage causes significant vision loss, second only to cataracts. Corneal replacement is a developing technology that is becoming a necessity for many patients due to disease, complications from LASIK, hereditary problems and complications from related surgeries. Through a collaboration with Dr. David Kaplan in the Tufts Bioengineering Department, we use silk protein-biomaterial lamellar systems coupled with cornea-specific stem cells to match in vivo corneal properties and meet functional requirements. The strategy exploits the novel material and biological features of silk, including surface micropatterning to guide cells and extracellular matrix deposition, slow degradation, biocompatibility, optical transparency and mechanical durability for handling, suturing and tolerating ocular pressures. A cornea tissue system that slowly degrades to allow for host native tissue replacement offers a significant and novel advancement in corneal transplantation technology. The approach to addressing this need is unique and offers novel methodology to meet the ever-growing demands for corneal
Figure 3. A. Grooved silk films promote ordered deposition of collagens (COL), decorin (DCR) and biglycan (BGN), mimicking the ordered deposition in vivo. B. By layering cell-populated films such that matrix deposition between layers is organized in an orthogonal manner, we can reproduce the in vivo lamellar morphology. By modifying the beta sheet content of the silk films, they can be tailored to degrade over time thereby leaving no trace in the reconstructed corneal implant.
Figure 4. Surgical implantation of silk constructs into rabbit corneas. Different surgical approaches yield different outcomes. Approach C-D, in which pockets were created by using air flushes to separate corneal lamellae, resulted in clear implants with no evidence of immune or neovascular responses. Approaches P-M and C-S, used surgically-created pockets or flaps, respectively, and led to either neovascularization or film degradation.