Stem cells respond to nanoscale surface features, with changes in cell growth and differentiation mediated by alterations in cell adhesion. The interaction of nanotopographical features with integrin receptors in the cells' focal adhesions alters how the cells adhere to materials surfaces, and defines cell fate through changes in both cell biochemistry and cell morphology. In this Review, we discuss how cell adhesions interact with nanotopography, and we provide insight as to how materials scientists can exploit these interactions to direct stem cell fate and to understand how the behaviour of stem cells in their niche can be controlled. We expect knowledge gained from the study of cell-nanotopography interactions to accelerate the development of next-generation stem cell culture materials and implant interfaces, and to fuel discovery of stem cell therapeutics to support regenerative therapies.

Harnessing nanotopography and integrin–matrix interactions to influence stem cell fate

Prelude to corneal tissue engineering – Gaining control of collagen organization

Purpose. To investigate whether structural changes to the sclera during form-deprivation myopia are caused by active tissue remodeling, the gelatinase activity of tree shrew scleras was studied in normal animals, form-vision deprived animals, and animals recovering from myopia. Methods. Infant tree shrews were monocularly deprived (MD) of form vision with translucent occluders for 5 days. Recovery animals were allowed 3 days of binocularly unoccluded vision after the period of form deprivation. Eyes were removed and dissected to provide scleral samples corresponding to equatorial and posterior regions. Gelatinase activity was assessed by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis or SDS-PAGE, gelatin, zymography of scleral matrix metalloproteinase (MMP) extracts. Results. The major gelatinolytic species present in tree shrew sclera was found to be MMP-2 (gelatinase A). In normal (nondeprived) animals, most of the MMP-2 was found to be in the latent form (the ratio of active-to-latent MMP-2 was 0.23 ± 0.05 and 0.34 ± 0.06 in equatorial and posterior samples, respectively; n = 10 eyes from five animals). After 5 days of MD, there was a threefold increase in the amount of active scleral MMP-2 in myopic eyes compared to contralateral control eyes, whereas latent MMP-2 activity levels were not altered significantly. This increase in active MMP2 was seen in both the equatorial and posterior sclera of myopic eyes (active-to-latent MMP-2 ratios were 0.53 ± 0.10 and 0.81 ± 0.09 in equatorial and posterior regions, respectively; n = 6 animals). Contralateral control eyes had levels of both active and latent MMP-2 not significantly different from normal eyes. After only 3 days of unoccluded vision, previously deprived eyes that were now recovering from myopia had a fivefold lower level of active MMP-2 than that seen in deprived eyes after 5 days of MD. In fact, active (and latent) MMP-2 levels were reduced in recovering eyes even below the levels found in their contralateral control eyes. Active-to-latent ratios for recovering eyes were 0.11 ± 0.03 and 0.15 ± 0.03 in equatorial and posterior sclera, respectively (rc = 5 animals). Conclusions. These results demonstrate that form-deprivation myopia and recovery from myopia alter scleral catabolism and provide further support for the theory that changes in eye size during mammalian refractive development are the result of active tissue remodeling rather than passive scleral stretching alone. Invest Ophthalmol Vis Sci. 1996; 37:1380-1395.

Form-Deprivation Myopia Induces Activation of Scleral Matrix Metalloproteinase-2 in Tree Shrew

There has been great interest in understanding the methods by which collagen-based load-bearing tissue is constructed, grown and maintained in vertebrate animals. To date, the responsibility for this process has largely been placed with mesenchymal fibroblastic cells that are thought to fully control the morphology of load-bearing extracellular matrix (ECM). However, given clear limitations in the ability of fibroblastic cells to precisely place or remove single collagen molecules to sculpt tissue, we have hypothesized that the material itself must play a critical role in the determination of the form of structural ECM. We here demonstrate directly, using live, dynamic, differential interference contrast imaging, that mechanically strained networks of collagen fibrils, exposed to collagenase (Clostridium histolyticum), degrade preferentially. Specifically, unstrained fibrils are removed ‘quickly’, while strained fibrils persist significantly longer. The demonstration supports the idea that collagen networks are mechanosensitive in that they are stabilized by mechanical strain. Thus, collagen molecules (together with their complement enzymes) may comprise the basis of a smart, load-adaptive, structural material system. This concept has the potential to drastically simplify the assumed role of the fibroblast, which would need only to provide ECM molecules and mechanical force to sculpt collagenous tissue.

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Mechanical strain enhances survivability of collagen micronetworks in the presence of collagenase: implications for load-bearing matrix growth and stability

The extracellular environment is an essential mediator of cell health and provides both chemical and mechanical stimuli to influence single and collective cell behaviors. While historically there has been significant emphasis placed on chemical regulators within the extracellular matrix, the role of the mechanical environment is less well known. Here, we review the role of matrix mechanics on cell function and tissue integrity. Cellular responses to mechanical signals include differentiation, migration, proliferation, and alterations in cell–cell and cell–matrix adhesion. Interestingly, the mechanical properties of tissues are altered in many disease states, leading to cellular dysfunction and further disease progression. Successful regenerative medicine strategies must consider the native mechanical environment so that they are able to elicit a favorable cellular response and integrate into the native tissue structure.

Matrix Stiffness: A Regulator of Cellular Behavior and Tissue Formation

The pattern and relative rates of diametric growth of the avian eye and cornea are described throughout embryonic development The effect of reduced intraocular pressure on eye and corneal diametric growth also was investigated Between embryonic day 4 (E4) and 1 day posthatching, the eye undergoes two distinct phases of linear growth The first phase (E4-10) is very rapid (1193 mm/day) The second phase, after E10, is significantly slower (0346 mm/day) By contrast, over the same developmental period, the cornea undergoes three distinct and sequential phases of linear growth The second phase of corneal growth (E7-10) is the most rapid (0429 mm/day) and separates two periods of slow growth (0211 mm/day during E4-7 and 0128 mm/day after E10) After the sustained release of intraocular pressure by intubation on E4, growth of both the eye and cornea is reduced significantly Operated eyes grow at a rate of 0356 mm/day (E4-10) and 0155 mm/day (E10-16) Intubation reduces corneal growth to a single phase of 0125 mm/day (E7-16) Thus, from E4-10 both the eye and cornea possess intrinsic growth potentials that are elevated significantly by intraocular pressure After E10, the rate of growth of both the eye and the cornea is independent of intraocular pressure Because both control and intubated eyes change their growth rate on E10, this transition also is independent of intraocular pressure This contrasts with the cornea which, after intubation, shows no detectable variation in growth rate Correlation of eye with corneal growth demonstrates an exponential relationship in the presence of intraocular pressure and an almost linear relationship after intubation

Intraocular pressure-dependent and -independent phases of growth of the embryonic chick eye and cornea.

Growth of the embryonic chick cornea was directly related to, and coordinated with, overall eye growth. During normal development, the size of the embryonic chick cornea increased in three linear phases of diametric growth. Corneal diameter increased at a rate of 216 microns per day between embryonic day 4 (E4) and E7, 511 microns per day between E7 and E10, and 144 microns per day from E10 until after hatching. After the sustained release of intraocular pressure by intubation on E4, corneal diametric growth was reduced to a single phase of 122 microns per day. After intubation on E4, the mesenchyme surrounding the developing cornea was substantially thicker and the neural crest-derived corneal endothelium was established earlier. The primary corneal stroma of the intubated eye swelled and was precociously populated by neural crest-derived corneal fibroblasts. Thus, the timing of arrival of neural crest cells in the anterior segment and their contribution to the cornea were determined by the growth rate of the eye. Although the diameter of the cornea was substantially reduced after intubation, it was more densely populated by fibroblasts, resulting in a cornea that was substantially thicker than the control by E14. Prospective corneal nerves normally extend into the cornea proper on E11, concomitant with a decrease in its diametric growth rate. After intubation on E4, the perilimbal nerve ring was virtually complete by E5 and numerous nerves had extended throughout the E8 cornea. By E16, the cornea from the intubated eye contained a very high density of nerve fibers, possibly reflecting its reduced size. These data suggest that the primary corneal stroma does not permit nerve fiber extension and demonstrate that the timing of nerve fiber extension into the secondary corneal stroma is specified by the rate of oppositional diametric growth of the cornea.

Reducing intraocular pressure by intubation elicits precocious development and innervation of the embryonic chick cornea.

Purpose. During the early stages (embryonic day 3 [E3]) of avian corneal development, nerve fibers extend from the trigeminal ganglion to the corneal limbus. On E11, these nerve fibers enter the cornea and extend through the secondary stroma to begin innervation of the epithelium on E13. This process of innervation is concomitant with the cornea's dehydration and transition from opacity to transparency ; thus, suggesting a link between innervation and the attainment of corneal function. This investigation attempts to ascertain whether the developing cornea can support its innervation in vitro and whether there is a possible developmental interrelationship between corneal innervation and dehydration, with the associated transition from opacity to transparency. Methods. Isolated corneas from either E8 or E14 chicks were co-cultured with E8 dorsal root ganglia. After 4 days of culture, innervation was visualized by silver staining and immunohistochemistry. Changes in corneal composition and organization associated with this innervation in vitro were analyzed by measuring changes in specific hydration, thickness and compaction, and incorporation of [ 35 S]sulfate into glycosaminoglycans during co-culture. Results. The E8 and E14 corneas support extensive innervation in vitro. Developing nerve fibers extend through the secondary stroma to innervate the epithelium. In vitro innervation of E8, but not E14, corneas was associated with a decrease in corneal specific hydration, whereas control corneas (without dorsal root ganglia) failed to show any such changes. E8 corneas also showed a significant increase in compaction when innervated in vitro. Corneal innervation in vitro did not significantly change the overall incorporation of [ 35 S]sulfate into glycosaminoglycans. Furthermore, incorporation of [ 35 S]sulfate into corneal sulfated glycosaminoglycans (sGAG) is not influenced by either the number of nerve fibers innervating the cornea or nerve growth factor (NGF). In addition, the distribution of staining of the corneal glycosaminoglycans, chondroitin sulfate and keratan sulfate, and peanut agglutinin-binding epitopes, suggests that these molecules are not associated with inhibition of axonal development. Conclusions. The in vitro system described here is a useful model to understand the process of corneal development. Co-culture has shown that corneal innervation promotes the process of dehydration, which is dependent on the age of the cornea. However, other functionally related refinements necessary for transparency-notably proteoglycan synthesis-may not be linked to innervation or NGF production. The authors conclude that the development of transparency is dependent on corneal innervation, though not exclusively, and that other controlling factors also are required.

J A Bee

Papers

Intraocular pressure-dependent and -independent phases of growth of the embryonic chick eye and cornea.

Reducing intraocular pressure by intubation elicits precocious development and innervation of the embryonic chick cornea.

Innervation of the chick cornea analyzed in vitro.