George E. Marshall

Immunogold fine structural localization of extracellular matrix components in aged human cornea

Using the immunogold technique combined with cryoultramicrotomy and London Resin white (LR white) embedding, we studied the fine structural distribution of types I–IV collagen and laminin in corneal tissue from seven enucleated human eyes (age range, 63–78 years). Type II collagen was not identified in any corneal layer. Type I and type III collagen were distributed in a similar fashion in striated collagen fibrils in Bowmans layer and in the stroma. Type IV collagen was located only in the posterior non-banded region of Descemets membrane. Laminin was identified in subepithelial anchoring plaques and the sub-endothelial region of Descemets membrane in accordance with its recognized adhesive function.

Type IV collagen and laminin in Bruch's membrane and basal linear deposit in the human macula.

Tissue obtained from the macula in 10 human eyes (53-77 years) was used for an investigation into the extracellular matrices of the retinal pigment epithelium (RPE), Bruchs membrane, and the choriocapillaris. The ultrastructural distribution of type IV collagen and laminin was documented using immunogold labelling. Labelling for type IV collagen was strongly positive in all the specimens in the basement membranes of the choriocapillaris but not that of the RPE where labelling was either weak or absent. Laminin was localised to deposits of granular material in Bruchs membrane but was absent from the basement membrane of the RPE and the choriocapillaris. Basal linear deposit, observed in three cases, demonstrated labelling for laminin but not for type IV collagen. The series was too small for correlation of these morphological changes with age.

Immunogold fine structural localization of extracellular matrix components in aged human cornea II. Collagen types V and VI

Using immunogold immunocytochemical techniques we studied the distribution of collagen types V and VI in corneal tissue from seven enucleated human eyes (age range, 63–78 years). Results obtained by cryoultramicrotomy were marginally more intense than those obtained using London Resin white (LR white) embedding. Type V collagen was present in the striated collagen fibrils in Bowmans layer, in the stroma and in a thin, non-banded anterior zone of Descemets membrane. Our results suggest that types I, IIl and V collagen co-distribute in striated collagen fibrils. By contrast, type VI collagen was located in fine filaments in the interfibrillar matrix of the stroma, in Bowmans layer and in the anchoring plaques of the sub-epithelial basement-membrane complex. This implies an importance in epithelial adhesion which was previously unsuspected. Keratocyte bodies were electron-dense, amorphous extracellular deposits of matrix-like material, and these were labelled with types III, V and VI collagen antibodies. Long-spacing collagen was observed in the corneal stroma, and this deposit did not contain any of the collagen types studied.

Immunogold ultrastructural localization of collagens in the aged human outflow system

Immunogold labeling was applied at the ultrastructural level to the identification of collagen types I, II, III, V, and VI in the human trabecular meshwork obtained from 11 surgically enucleated globes. Both London resin white embedding and cryoultramicrotomy were used, and for types V and VI, the latter provided more sensitive localization. Type II collagen was not identified. Types I and III were localized to the striated collagen fibrils of the trabecular core, the basement membrane of the trabecular beams, and loose aggregates in the juxtacanalicular tissue. Types V and VI formed a fine network around the striated fibrils in the trabecular cores and linkage strands to the basement membranes. The outer wall of Schlemms canal contained collagens I, III, V, and VI. Long-spacing collagen and elastin-like material did not label with any of the antibodies used in this study.

Immunogold localisation of laminin in normal and exfoliative iris.

Immunoelectron microscopic studies of exfoliative iris tissue (seven specimens) revealed the presence of laminin in the fibrillar component of exfoliation material. The immunogold label was uniformly distributed on the exfoliation fibres. Deposition of laminin labelled exfoliation material in the dilator muscle was a noteworthy feature, as was an apparent depletion of laminin in the basement membranes of ostensibly unaffected vessels. In control iris tissue (five enucleated eyes) laminin was identified in the basement membrane round vascular contractile cells, but not beneath the endothelium.

Immunogold localization of type IV collagen and laminin in the aging human outflow system

Tissue from the outflow system of six surgically enucleated aged eyes was used for an ultrastructural immunocytochemical study of the distribution of laminin and type IV collagen. The immunogold technique provided precise localization of laminin beneath lining endothelial cells of the inner wall of Schlemms canal. Laminin labelling was absent in the trabecular beams. Type IV collagen was found in the basement membranes of the trabecular beams and in fine filamentous basement membrane material in the cribriform layer. Electron dense plaques in the cribriform layer labelled positively for laminin in the outer coarse fibrillar zone but not in the electron dense core. Long-spacing collagen was negative for type IV collagen.

Interactions of human blood and tissue cell types with 95-nm-high nanotopography

Two of the major concerns for tissue engineering materials are inflammatory responses from blood cells and fibrous encapsulation by the body in order to shield the implant from blood reaction. A further hurdle is that of vascularization. In order to develop new tissues, or to repair parts of the vascular system, nutrients need to be carried to the basal cell layers. If a material promotes tissue formation, but not vascularization, necrosis will be observed as multilayered cells develop. In this paper, polymer demixed island topography with a 95-nm Z axis was tested using human mononuclear blood cells, platelets, fibroblasts, and endothelial cells. The results showed no difference in blood response between the islands and the flat controls, suggesting that in vivo there would be negligible immunological difference. Fibroblasts reacted by changing morphology into a rounded shape with thick processes and poorly developed cytoskeleton. Retardation of fibroblast growth may be an advantageous, as it is this cell type that forms the fibrous capsule, preventing growth of the required tissue type. Finally, endothelial cells were seen to form arcuate, or curved, morphologies in response to the islands. This is the normal, in vivo, morphology for vascular endothelium. This result suggests that the nano-features are promoting a more phenotypically correct morphology.

Collagens in the aged human macula.

Immunogold cytochemistry was used to investigate the fine structural distribution of collagen types I–VI in Bruchs membrane and choroid of the aged human macula. Macular tissue was obtained from ten eyes, and processed for cryoultramicrotomy and London Resin white embedding. Striated collagen fibrils within the inner and outer collagenous layers were found to contain collagen types I, III and V In addition, type V collagen was also present in the basement membrane of the choriocapillaris. Gross thickening of the choriocapillaris basement membrane was attributed to the deposition of type IV collagen. However, type IV collagen appeared to be absent from the basement membrane of the retinal pigment epithelium. The interesting location of type VI collagen on the choroidal side of the choriocapillaris suggested that its function is to anchor the choriocapillaris onto the choroid. The collagens studied were absent from fibrous banded material, long-spacing collagen, the elastic layer and amorphous granular material. It was concluded that, of the collagen types studied, only the deposition of type IV collagen contributes to the age-related thickening of Bruchs membrane.

An immunoelectron microscope study of the aged human lens capsule

The distribution of types I-IV collagen and laminin was studied in seven aged human lens capsules using the immunogold EM technique on LR White embedded tissue. Samples were taken from the anterior, equatorial and posterior regions. Labelling for type II collagen was not observed. Type IV collagen was evenly distributed throughout the thickness of the capsule but was absent from the zonules. However, an unexpected finding was strong labelling for types I and III collagen, again evenly distributed throughout the capsule. The presence of type III collagen makes the lens capsule unique among ocular basement membranes. Laminin was present in linear densities, zonular lamellae and zonular fibres, suggesting that linear densities are an integral part of the zonular apparatus.

Human scleral elastic system: an immunoelectron microscopic study.

An immunocytochemical study was conducted on elastic components in the sclera of seven aged human eyes. By conventional electron microscopy, elastic tissue consists of three distinct fibre types--elastic fibres, elaunin fibres, and oxytalan fibres. The distribution of six components associated with the elastic system (elastin, amyloid P component, laminin, fibronectin, gp 115, and vitronectin) were studied by immunogold transmission electron microscopy. The codistribution of amyloid P component and laminin was further studied by double immunolabelling. Both elastic and elaunin fibres contained elastin. The microfibrillar sheaths of elastic fibres labelled for amyloid P component, those of elaunin fibres for amyloid P and laminin, and those of oxytalan fibres for laminin only. No labelling was observed for fibronectin, gp 115, and vitronectin. In terms of the proteins investigated, the biochemical profile of the three fibre types was not completely identical and was manifest as different affinities in the binding of serum amyloid P component and an association with laminin.