Multi-pin contact drawing enables production of anisotropic collagen fiber substrates for alignment of fibroblasts and monocytes.

Type I collagen is the most abundant protein in the human body and is known to play important roles in numerous biological processes including tissue morphogenesis and wound healing. As such, it is one of the most frequently used substrates for cell culture, and there have been considerable efforts to develop collagen-based cell culture substrates that mimic the structural organization of collagen as it is found in native tissues, i.e., collagen fibers. However, producing collagen fibers from extracted collagen has been notoriously difficult, with existing methods providing only low throughput production of collagen fibers. In this study, we prepared collagen fibers using a highly efficient, bio-friendly, and cost-effective approach termed contact drawing, which uses an entangled polymer fluid to aid in fiber formation. Contact drawing technology has been demonstrated previously for collagen using highly concentrated dextran solutions with low concentrations of collagen. Here, we show that by replacing dextran with polyethylene oxide (PEO), high collagen content fibers may be readily formed from mixtures of soluble collagen and PEO, a polymer that readily forms fibers by contact drawing at concentrations as low as 0.5%wt. The presence of collagen and the formation of well-ordered collagen structures in the resulting fibers were characterized by attenuated total reflectance Fourier-transform infrared spectromicroscopy, Raman spectromicroscopy, and fluorescence microscopy. Corresponding to well-ordered collagen, the mechanical properties of the PEO-collagen fibers approximated those observed for native collagen fibers. Growth of cells on aligned PEO-collagen fibers attached to a polydimethyl siloxane support was examined for human dermal fibroblast (WS1) and human peripheral leukemia blood monocyte (THP-1) cell lines. WS1 and THP-1 cells readily attached, displayed alignment through migration and spreading, and proliferated on the collagen fiber substrate over the course of several days. We also demonstrated the retrieval of viable cells from the PEO-collagen fiber substrates through enzymatic digestion of the collagen substrate with collagenase IV.

Material properties of disulfide-crosslinked hyaluronic acid hydrogels influence prostate cancer cell growth and metabolism.

Cells reside in vivo within three dimensional environments in which they interact with extracellular matrices (ECMs) that play an integral role in maintaining tissue homeostasis and preventing tumour growth. Thus, tissue culture approaches that more faithfully reproduce these interactions with the ECM are needed to study cancer development and progression. Many materials exist for modeling tissue environments, and the effects of differing mechanical, physical, and biochemical properties of such materials on cell behaviour are often intricately coupled and difficult to tease apart. Here, an optimized protocol was developed to generate low reaction volume disulfide-crosslinked hyaluronic acid (HA) hydrogels for use in cell culture applications to relate the properties of ECM materials to cell signalling and behaviour. Mechanically, HA hydrogels are comparable to other soft hydrogel materials such as Matrigel and agarose or to tissues lacking type I collagen and other fibrillar ECM components. The diffusion of soluble materials in these hydrogels is affected by unique mass transfer properties. Specifically, HA hydrogel concentration affects the diffusion of anionic particles above 500 kDa, whereas diffusion of smaller particles appears unimpeded by HA content, likely reflecting hydrogel pore size. The HA hydrogels have a strong exclusion effect that limits the movement of proteins into and out of the material once fully formed. Such mass transfer properties have interesting implications for cell culture, as they ultimately affect access to nutrients and the distribution of signalling molecules, affecting nutrient sensing and metabolic activity. The use of disulfide-crosslinked HA hydrogels for the culture of the model prostate cancer cell lines PC3 and LNCaP reveals correlations of protein activation linked to metabolic flux, which parallel and can thus potentially provide insights into cell survival mechanisms in response to starvation that occurs in cancer cell microenvironments.

A new longitudinal variation in the structure of collagen fibrils and its relationship to locations of mechanical damage susceptibility.

The hierarchical architecture of the collagen fibril is well understood, involving non-integer staggering of collagen molecules which results in a 67 nm periodic molecular density variation termed D-banding. Other than this variation, collagen fibrils are considered to be homogeneous at the micro-scale and beyond. Interestingly, serial kink structures have been shown to form at discrete locations along the length of collagen fibrils from some mechanically overloaded tendons. The formation of these kinks at discrete locations along the length of fibrils (discrete plasticity) may indicate pre-existing structural variations at a length scale greater than that of the D-banding. Using a high velocity nanomechanical mapping technique, 25 tendon collagen fibrils, were mechanically and structurally mapped along 10 µm of their length in dehydrated and hydrated states with resolutions of 20 nm and 8 nm respectively. Analysis of the variation in hydrated indentation modulus along individual collagen fibrils revealed a micro-scale structural variation not observed in the hydrated or dehydrated structural maps. The spacing distribution of this variation was similar to that observed for inter-kink distances seen in SEM images of discrete plasticity type damage. We propose that longitudinal variation in collagen fibril structure leads to localized mechanical susceptibility to damage under overload. Furthermore, we suggest that this variation has its origins in heterogeneous crosslink density along the length of collagen fibrils. The presence of pre-existing sites of mechanical vulnerability along the length of collagen fibrils may be important to biological remodeling of tendon, with mechanically-activated sites having distinct protein binding capabilities and enzyme susceptibility.

MMP-9 selectively cleaves non-D-banded material on collagen fibrils with discrete plasticity damage in mechanically-overloaded tendon.

The mechanical properties of tendon are due to the properties and arrangement of its collagen fibril content. Collagen fibrils are highly-organized supermolecular structures with a periodic banding pattern (D-band) indicative of the geometry of molecular organization. Following mechanical overload of whole tendon, collagen fibrils may plastically deform at discrete sites along their length, forming kinks, and acquiring a fuzzy, non-D-banded, outer layer (shell). Termed discrete plasticity, such non-uniform damage to collagen fibrils suggests localized cellular response at the fibril level during subsequent repair/replacement. Matrix metallo-proteinases (MMPs) are enzymes which act upon the extracellular matrix, facilitating cell mobility and playing important roles in wound healing. A sub-group within this family are the gelatinases, MMP-2 and MMP-9, which selectively cleave denatured collagen molecules. Of these two, MMP-9 is specifically upregulated during the initial stages of tendon repair. This suggests a singular function in damage debridement. Using atomic force microscopy (AFM), a novel fibril-level enzymatic assay was employed to assess enzymatic removal of material by trypsin and MMP-9 from individual fibrils which were: (i) untreated, (ii) partially heat denatured, (iii) or displaying discrete plasticity damaged after repeated mechanical overload. Both enzymes removed material from heat denatured and discrete plasticity-damaged fibrils; however, only MMP-9 demonstrated the selective removal of non-D-banded material, with greater removal from more damaged fibrils. The selectivity of MMP-9, coupled with documented upregulation, suggests a likely mechanism for the in vivo debridement of individual collagen fibrils, following tendon overload injury, and prior to deposition of new collagen.

Characterization via atomic force microscopy of discrete plasticity in collagen fibrils from mechanically overloaded tendons: Nano-scale structural changes mimic rope failure.

Tendons exposed to tensile overload show a structural alteration at the fibril scale termed discrete plasticity. Serial kinks appear along individual collagen fibrils that are susceptible to enzymatic digestion and are thermally unstable. Using atomic force microscopy we mapped the topography and mechanical properties in dehydrated and hydrated states of 25 control fibrils and 25 fibrils displaying periodic kinks, extracted from overloaded bovine tail tendons. Using the measured modulus of the hydrated fibrils as a probe of molecular density, we observed a non-linear negative correlation between molecular density and kink density of individual fibrils. This is accompanied by an increase in water uptake with kink density and a doubling of the coefficient of variation of the modulus between kinked, and control fibrils. The mechanical property maps of kinked collagen fibrils show radial heterogeneity that can be modeled as a high-density core surrounded by a low-density shell. The core of the fibril contains the kink structures characteristic of discrete plasticity; separated by inter-kink regions, which often retain the D-banding structure. We propose that the shell and kink structures mimic characteristic damage motifs observed in laid rope strands.

Nanomechanical mapping of hydrated rat tail tendon collagen I fibrils.

Collagen fibrils play an important role in the human body, providing tensile strength to connective tissues. These fibrils are characterized by a banding pattern with a D-period of 67 nm. The proposed origin of the D-period is the internal staggering of tropocollagen molecules within the fibril, leading to gap and overlap regions and a corresponding periodic density fluctuation. Using an atomic force microscope high-resolution modulus maps of collagen fibril segments, up to 80 µm in length, were acquired at indentation speeds around 10(5) nm/s. The maps revealed a periodic modulation corresponding to the D-period as well as previously undocumented micrometer scale fluctuations. Further analysis revealed a 4/5, gap/overlap, ratio in the measured modulus providing further support for the quarter-staggered model of collagen fibril axial structure. The modulus values obtained at indentation speeds around 10(5) nm/s are significantly larger than those previously reported. Probing the effect of indentation speed over four decades reveals two distinct logarithmic regimes of the measured modulus and point to the existence of a characteristic molecular relaxation time around 0.1 ms. Furthermore, collagen fibrils exposed to temperatures between 50 and 62°C and cooled back to room temperature show a sharp decrease in modulus and a sharp increase in fibril diameter. This is also associated with a disappearance of the D-period and the appearance of twisted subfibrils with a pitch in the micrometer range. Based on all these data and a similar behavior observed for cross-linked polymer networks below the glass transition temperature, we propose that collagen I fibrils may be in a glassy state while hydrated.