Molecular mechanochemistry: low force switch slows enzymatic cleavage of human type I collagen monomer.

In vertebrate animals, fibrillar collagen accumulates, organizes, and persists in structures which resist mechanical force. This antidissipative behavior is possibly due to a mechanochemical force-switch which converts collagen from enzyme-susceptible to enzyme-resistant. Degradation experiments on native tissue and reconstituted fibrils suggest that collagen/enzyme kinetics favor the retention of loaded collagen. We used a massively parallel, single molecule, mechanochemical reaction assay to demonstrate that the effect is derivative of molecular mechanics. Tensile loads higher than 3 pN dramatically reduced (10×) the enzymatic degradation rate of recombinant human type I collagen monomers by Clostridium histolyticum compared to unloaded controls. Because bacterial collagenase accesses collagen at multiple sites and is an aggressive cleaver of the collagen triple helical domain, the results suggest that collagen molecular architecture is generally more stable when mechanically strained in tension. Thus the tensile mechanical state of collagen monomers is likely to be correlated to their longevity in tissues. Further, strain-actuated molecular stability of collagen may constitute the fundamental basis of a smart structural mechanism which enhances the ability of animals to place, retain, and load-optimize material in the path of mechanical forces.

Keratan sulfate glycosaminoglycan and the association with collagen fibrils in rudimentary lamellae in the developing avian cornea.

PURPOSE: Keratan sulfate (KS), through its association with fibrillar collagen as KS-substituted proteoglycan (KS PG), is thought to be instrumental in the structural development of the corneal stroma. The authors used two different sulfate motif-specific antibodies to identify the sequence of appearance, and the association with collagen, of sulfated KS during avian corneal morphogenesis. METHODS: Corneas from chicken embryos throughout the developmental period, from day 8 through day 18 of incubation, were examined by immunofluorescence and immunoelectron microscopy using monoclonal antibodies 5D4 and 1B4, which react with high- and low-sulfated epitopes on KS, respectively. RESULTS: KS was identified as punctate labeling at incubation day 8, the earliest stage examined, suggesting a cell-associated distribution. By day 10, labeling was more homogeneous, indicating that KS sulfation motifs were present in the stromal extracellular matrix. At day 12 through day 14, immunopositive sites were concentrated primarily in the anterior stroma but became more uniform throughout the full stromal thickness by day 18. From day 10 on, electron microscopy revealed a high-sulfated KS epitope closely associated with bundles of regularly arranged collagen fibrils, initially near cell surfaces in rudimentary lamellae. Individual cells, associated with collagen bundles with different fibril orientations, imply the potential for simultaneous deposition of multiple lamellae. CONCLUSIONS: During chick corneal morphogenesis, significant matrix deposition of high-sulfated KS epitope occurs by day 10, with accumulation subsequently proceeding in an anterior-to-posterior manner. High-sulfated KS likely serves to help define the regular spatial organization of collagen fibrils in bundles newly extruded into the extracellular milieu.

Alterations in the biomolecular signatures of developing chick corneas as determined by biospectroscopy and multivariate analysis.

PURPOSE: Biospectroscopy tools are increasingly being recognized as novel approaches toward interrogating complex biological structures in a nondestructive fashion. This study was conducted to apply these tools to interrogate alterations in the molecular signatures of developing chick corneas during the onset and development of transparency. METHODS: Embryonic chick corneas (n = 46) were obtained at 2-day intervals from embryonic day (E)10 to E18 of incubation and investigated with attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy and Raman microspectroscopy. Resultant spectra were analyzed for variance by using principal component analysis and linear discriminant analysis (PCA-LDA). RESULTS: Mean spectra after ATR-FTIR spectroscopy or Raman microspectroscopy derived from corneas at each developmental stage showed some overlap; however, in PCA-LDA scores plots, a clear segregation of spectra was evident, and two-category discrimination indicated that significant molecular alterations occur during tissue morphogenesis. Notable by both techniques was the increasing intensity of DNA signal (1080 cm⁻¹) from E10 onward. Major segregating biomarkers identified by ATR-FTIR spectroscopy between E10 and E18 were in the DNA/RNA (1126 cm⁻¹), glycogen (1045 cm⁻¹), protein (1470 cm⁻¹), and amide II (1512 cm⁻¹ and 1524 cm⁻¹) spectral regions. Raman spectroscopy also identified major distinguishing vibrational modes that included proteins, amino acids (tyrosine, proline phenylalanine, and valine), and secondary structures of proteins (amide I and amide II). CONCLUSIONS: The developing chick cornea undergoes significant changes in its biomolecular composition in the E10 to E18 developmental period, with the major changes occurring in the spectral regions associated with DNA/RNA, proteins, glycogen, and secondary protein structures.

Mechanical strain stabilizes reconstituted collagen fibrils against enzymatic degradation by mammalian collagenase matrix metalloproteinase 8 (MMP-8).

BACKGROUND: Collagen, a triple-helical, self-organizing protein, is the predominant structural protein in mammals. It is found in bone, ligament, tendon, cartilage, intervertebral disc, skin, blood vessel, and cornea. We have recently postulated that fibrillar collagens (and their complementary enzymes) comprise the basis of a smart structural system which appears to support the retention of molecules in fibrils which are under tensile mechanical strain. The theory suggests that the mechanisms which drive the preferential accumulation of collagen in loaded tissue operate at the molecular level and are not solely cell-driven. The concept reduces control of matrix morphology to an interaction between molecules and the most relevant, physical, and persistent signal: mechanical strain. METHODOLOGY/PRINCIPAL FINDINGS: The investigation was carried out in an environmentally-controlled microbioreactor in which reconstituted type I collagen micronetworks were gently strained between micropipettes. The strained micronetworks were exposed to active matrix metalloproteinase 8 (MMP-8) and relative degradation rates for loaded and unloaded fibrils were tracked simultaneously using label-free differential interference contrast (DIC) imaging. It was found that applied tensile mechanical strain significantly increased degradation time of loaded fibrils compared to unloaded, paired controls. In many cases, strained fibrils were detectable long after unstrained fibrils were degraded. CONCLUSIONS/SIGNIFICANCE: In this investigation we demonstrate for the first time that applied mechanical strain preferentially preserves collagen fibrils in the presence of a physiologically-important mammalian enzyme: MMP-8. These results have the potential to contribute to our understanding of many collagen matrix phenomena including development, adaptation, remodeling and disease. Additionally, tissue engineering could benefit from the ability to sculpt desired structures from physiologically compatible and mutable collagen.

Differential relative sulfation of Keratan sulfate glycosaminoglycan in the chick cornea during embryonic development.

PURPOSE: To investigate structural remodeling of the developing corneal stroma concomitant with changing sulfation patterns of keratan sulfate (KS) glycosaminoglycan (GAG) epitopes during embryogenesis and the onset of corneal transparency. METHODS: Developing chick corneas were obtained from embryonic day (E)12 to E18 of incubation. Extracellular matrix composition and collagen fibril spacing were evaluated by synchrotron x-ray diffraction, hydroxyproline assay, ELISA (with antibodies against lesser and more highly sulfated KS), and transmission electron microscopy with specific proteoglycan staining. RESULTS: A significant relative increase in highly sulfated KS epitope labeling occurred with respect to hydroxyproline content in the final week of chick development, as mean collagen interfibrillar distance decreased. Small KS PG filaments increased in frequency with development and were predominantly fibril associated. CONCLUSIONS: The accumulation of highly sulfated KS during the E12 to E18 timeframe could serve to fine tune local matrix hydration and collagen fibril spacing during corneal growth, as gross dehydration and compaction of the stroma progress through the action of the nascent endothelial pump.

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

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.

Gene expression and immunolocalisation of a calcium-activated chloride channel during the stratification of cultivated and developing corneal epithelium.

The spatial and temporal localisation of a calcium-activated chloride channel (CLCA) and its mRNA was investigated, during the in vivo and in vitro development of stratified epithelia, by fluorescence immunohistochemistry and quantitative polymerase chain reaction in embryonic chicken corneas and the expansion of excised human corneal stem cells on amniotic membrane. Single-layered human epithelial cultures on amniotic membrane and early day embryonic chicken corneas expressed relatively little human CLCA2 or its chicken homologue. However, as the epithelium in both models matured and the number of cell-layers increased, the gene expression level and protein staining intensity increased, primarily within the basal cells of both the cultured and embryonic tissues. These results demonstrate that human CLCA2 protein and mRNA expression are elevated during epithelial stratification, suggesting that this protein plays a role in the growth of multi-layered corneal epithelia during both natural development and tissue cultivation.