Langevin dynamics modeling of the water diffusion tensor in partially aligned collagen networks

In this work, a Langevin dynamics model of the diffusion of water in articular cartilage was developed. Numerical simulations of the translational dynamics of water molecules and their interaction with collagen fibers were used to study the quantitative relationship between the organization of the collagen fiber network and the diffusion tensor of water in model cartilage. Langevin dynamics was used to simulate water diffusion in both ordered and partially disordered cartilage models. In addition, an analytical approach was developed to estimate the diffusion tensor for a network comprising a given distribution of fiber orientations. The key findings are that (1) an approximately linear relationship was observed between collagen volume fraction and the fractional anisotropy of the diffusion tensor in fiber networks of a given degree of alignment, (2) for any given fiber volume fraction, fractional anisotropy follows a fiber alignment dependency similar to the square of the second Legendre polynomial of cos(θ), with the minimum anisotropy occurring at approximately the magic angle (θMA), and (3) a decrease in the principal eigenvalue and an increase in the transverse eigenvalues is observed as the fiber orientation angle θ progresses from 0◦ to 90◦. The corresponding diffusion ellipsoids are prolate for θ < θMA, spherical for θ ≈ θMA, and oblate for θ > θMA. Expansion of the model to include discrimination between the combined effects of alignment disorder and collagen fiber volume fraction on the diffusion tensor is discussed.

Cohesive zone modelling of mineralized collagen fibril arrays in bending

Hard biological materials such as bone possess superior material properties of high stiffness and toughness. Two unique characteristics of bone microstructure are a large aspect ratio of mineralized collagen fibrils (MCF), and an extremely thin and large area of extrafibrillar protein matrix located between the MCF. The objective of this study is to investigate the effects of: (1) MCF aspect ratio, and (2) energy dissipation in extrafibrillar protein matrix on the mechanical behaviour of MCF arrays. In this study, notched specimens of MCF arrays in extrafibrillar protein matrix are subjected to bending. Cohesive zone model was implemented to simulate the failure of extrafibrillar protein matrix. The study reveals that the MCF array with a higher MCF aspect ratio and the MCF array with a higher protein energy dissipation in the interface direction are able to sustain a higher bending force and dissipate higher energy.

Incorporation of bioactive polyvinylpyrrolidone–iodine within bilayered collagen scaffolds enhances the differentiation and subchondral osteogenesis of mesenchymal stem cells

Polyvinylpyrrolidone–iodine (Povidone-iodine, PVP-I) is widely used as an antiseptic agent for lavation during joint surgery; however, the biological effects of PVP–I on cells from joint tissue are unknown. This study examined the biocompatibility and biological effects of PVP–I on cells from joint tissue, with the aim of optimizing cell-scaffold based joint repair. Cells from joint tissue, including cartilage derived progenitor cells (CPC), subchondral bone derived osteoblast and bone marrow derived mesenchymal stem cells (BM-MSC) were isolated. The concentration-dependent effects of PVP–I on cell proliferation, migration and differentiation were evaluated. Additionally, the efficacy and mechanism of a PVP–I loaded bilayer collagen scaffold for osteochondral defect repair was investigated in a rabbit model. A micromolar concentration of PVP–I was found not to affect cell proliferation, CPC migration or extracellular matrix production. Interestingly, micromolar concentrations of PVP–I promote osteogenic differentiation of BM-MSC, as evidenced by up-regulation of RUNX2 and Osteocalcin gene expression, as well as increased mineralization on the three-dimensional scaffold. PVP–I treatment of collagen scaffolds significantly increased fibronectin binding onto the scaffold surface and collagen type I protein synthesis of cultured BM-MSC. Implantation of PVP–I treated collagen scaffolds into rabbit osteochondral defect significantly enhanced subchondral bone regeneration at 6 weeks post-surgery compared with the scaffold alone (subchondral bone histological score of 8.80 ± 1.64 vs. 3.8 ± 2.19, p < 0.05). The biocompatibility and pro-osteogenic activity of PVP–I on the cells from joint tissue and the enhanced subchondral bone formation in PVP–I treated scaffolds would thus indicate the potential of PVP–I for osteochondral defect repair.

Chitosan-collagen scaffolds with nano/microfibrous architecture for skin tissue engineering

In this study, a hierarchical nano/microfibrous chitosan/collagen scaffold that approximates structural and functional attributes of native extracellular matrix (ECM), has been developed for applicability in skin tissue engineering. Scaffolds were produced by electrospinning of chitosan followed by imbibing of collagen solution, freeze-drying and subsequent cross-linking of two polymers. Scanning electron microscopy showed formation of layered scaffolds with nano/microfibrous architechture. Physico-chemical properties of scaffolds including tensile strength, swelling behavior and biodegradability were found satisfactory for intended application. 3T3 fibroblasts and HaCaT keratinocytes showed good in vitro cellular response on scaffolds thereby indicating the matrices′ cytocompatible nature. Scaffolds tested in an ex vivo human skin equivalent (HSE) wound model, as a preliminary alternative to animal testing, showed keratinocyte migration and wound re-epithelization — a pre-requisite for healing and regeneration. Taken together, the herein proposed chitosan/collagen scaffold, shows good potential for skin tissue engineering.

Stimulation of MMP-11 (stromelysin-3) expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

Background Matrix metalloproteinases (MMPs) are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14) and stromelysin-3 (MMP-11) are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. Methods To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs) were: a) treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b) grown on collagens I, IV and V; c) treated with fibronectin, con-A and matrigel; and d) co-cultured with a range of HBC (human breast cancer) cell lines of varied invasive and metastatic potential. Results Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. Conclusion We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

Fine tuning of elasticity via crosslinking collagen-based materials to mediate mechanotransduction and stability using corona treatment

The common goal of tissue engineering is to develop substitutes that can closely mimic the structure of extracellular matrix (ECM). However, similarly important is the intensive material properties which have often been overlooked, in particular, for soft tissues that are not to bear load assumingly. The mechanostructural properties determine not only the structural stability of biomaterials but also their physiological functionality by directing cellular activity and regulating cell fate decision. The aim here is to emphasize that cells could sense intensive material properties like elasticity and reside, proliferate, migrate and differentiate accordinglyno matter if the construct is from a natural source like cartilage, skin etc. or of synthetic one. Meanwhile, the very objective of this work is to provide a tunable scheme for manipulating the elasticity of collagen-based constructs to be used to demonstrate how to engineer cell behavior and regulate mechanotransduction. Articular cartilage was chosen as it represents one of the most complex hierarchical arrangements of collagen meshwork in both connective tissues and ECM-like biomaterials. Corona discharge treatment was used to produce constructs with varying density of crosslinked collagen and stiffness accordingly. The results demonstrated that elastic modulus increased up to 33% for samples treated up to one minute as crosslink density was found to increase with exposure time. According to the thermal analysis, longer exposure to corona increased crosslink density as the denaturation enthalpy increased. However the spectroscopy results suggested that despite the stabilization of the collagen structure the integrity of the triple helical structure remained intact. The in vitro superficial culture of heterologous chondrocytes also determined that the corona treatment can modulate migration with increased focal adhesion of cells due to enhanced stiffness, without cytotoxicity effects, and providing the basis for reinforcing three-dimensional collagen-based biomaterials in order to direct cell function and mediate mechanotransduction.

Second harmonic generation and multiphoton microscopic detection of collagen without the need for species specific antibodies

High-resolution, high-contrast, three-dimensional images of live cell and tissue architecture can be obtained using second harmonic generation (SHG), which comprises non-absorptive frequency changes in an excitation laser line. SHG does not require any exogenous antibody or fluorophore labeling, and can generate images from unstained sections of several key endogenous biomolecules, in a wide variety of species and from different types of processed tissue. Here, we examined normal control human skin sections and human burn scar tissues using SHG on a multi-photon microscope (MPM). Examination and comparison of normal human skin and burn scar tissue demonstrated a clear arrangement of fibers in the dermis, similar to dermal collagen fiber signals. Fluorescence-staining confirmed the MPM-SHG collagen colocalization with antibody staining for dermal collagen type-I but not fibronectin or elastin. Furthermore, we were able to detect collagen MPM-SHG signal in human frozen sections as well as in unstained paraffin embedded tissue sections that were then compared with hematoxylin and eosin staining in the identical sections. This same approach was also successful in localizing collagen in porcine and ovine skin samples, and may be particularly important when species-specific antibodies may not be available. Collectively, our results demonstrate that MPM SHG-detection is a useful tool for high resolution examination of collagen architecture in both normal and wounded human, porcine and ovine dermal tissue.

Collagen in the scarless fetal skin wound : detection with picrosirius-polarization

Our group has developed an ovine model of deep dermal, partial-thickness burn where the fetus heals scarlessly and the lamb heals with scar. The comparison of collagen structure between these two different mechanisms of healing may elucidate the process of scarless wound healing. Picrosirius staining followed by polarized light microscopy was used to visualize collagen fibers, with digital capture and analysis. Collagen deposition increased with fetal age and the fibers became thicker, changing from green (type III collagen) to yellow/red (type I collagen). The ratio of type III collagen to type I was high in the fetus (166), whereas the lamb had a much lower ratio (0.2). After burn, the ratios of type III to type I collagen did not differ from those in control skin for either fetus or lamb. The fetal tissue maintained normal tissue architecture after burn while the lamb tissue showed irregular collagen organization. In conclusion, the type or amount of collagen does not alter significantly after injury. Tissue architecture differed between fetal and lamb tissue, suggesting that scar development is related to collagen cross-linking or arrangement. This study indicates that healing in the scarless fetal wound is representative of the normal fetal growth pattern, rather than a "response" to burn injury.

Collagen induced MMP-2 activation in human breast cancer

Matrix metalloproteinase-2 (MMP-2), a zymogen requiring proteolytic activation for catalytic activity, has been implicated broadly in the invasion and metastasis of many cancer model systems, including human breast cancer (HBC). MMP-2 has been immunolocalized to carcinomatous human breast, where the degree of activation of MMP-2 correlates well with tumor grade and patient prognosis. Using Matrigel assays, we have stratified HBC cell lines for invasiveness in vitro, and compared this to their potential for metastatic spread in nude mice. HBC cell lines expressing the mesenchymal marker protein vimentin were found to be highly invasive in vitro, and tended to form metastases in nude mice. We have further discovered that culture on collagen-I gels (Vitrogen(TM): Vg) induces MMP-2-activator in highly invasive but not poorly invasive HBC cell lines. As seen for other MMP-2-activator inducing regimens, this induction requires protein synthesis and an intact MMP-2 hemopexin-like domain, appears to be mediated by a cell surface activity, and can be inhibited by metalloproteinase inhibitors. The induction is highly specific to collagen I, and is not seen with thin coatings of collagen I, collagen IV, laminin, or fibronectin, or with 3-dimensional gels of laminin, Matrigel, or gelatin. This review focuses on collagen I and MMP- 2, their localization and source in HBC, and their relationship(s) to MMP-2 activation and HBC metastasis. The relevance of collagen I in activation of MMP-2 in vivo is discussed in terms of stromal cell: tumor cell interaction for collagen I deposition, MMP-2 production and MMP-2-activation. Such cooperativity may exist in vivo for MMP-2 participation in HBC dissemination. A more complete understanding of the regulation of MMP-2-activator by type I collagen may provide new avenues for improved diagnosis and prognosis of human breast cancer.

Collagen-induced activation of the Mr. 72,000 type IV collagenase in normal and malignant human fibroblastoid cells

Although the Mr. 72,000 type IV collagenase (matrix metalloproteinase 2) has been implicated in a variety of normal and pathogenic processes, its activation mechanism in vivo is unclear. We have found that fibroblasts from normal and neoplastic human breast, as well as the sarcomatous human Hs578T and HT1080 cell lines, activate endogenous matrix metalloprotease 2 when cultured on type I collagen gels, but not on plastic, fibronectin, collagen IV, gelatin, matrigel, or basement membrane-like HR9 cell matrix. This activation is monitored by the zymographic detection of Mr 59,000 and/or Mr 62,000 species, requires 2-3 days of culture on vitrogen to manifest, is cycloheximide inhibitable, and correlates with an arborized morphology. A similar activation pattern was seen in these cells in response to Concanavalin A but not transforming growth factor β or 12-O-tetradecanoylphorbol-13-acetate. The interstitial matrix may thus play an important role in regulating matrix degradation in vivo.

MT1-MMP-dependent and -independent regulation of gelatinase A activation in long-term, ascorbate-treated fibroblast cultures: Regulation by fibrillar collagen

Human skin fibroblasts were cultured long-term in the presence of ascorbic acid to allow formation of a three-dimensional collagen matrix, and the effects of this on activation of secreted matrix metalloproteinase-2 (MMP-2) were examined. Accumulation of collagen over time correlated with increased levels of both mature MMP-2 and cell-associated membrane type 1-MMP (MT1-MMP), and subsequently increased mRNA levels for MT1-MMP, providing temporal resolution of the "nontranscriptional" and "transcriptional" effects of collagen on MT-1MMP functionality. MMP-2 activation by these cultures was blocked by inhibitors of prolyl-4-hydroxylase, or when fibroblasts derived from the collagen α1(I) gene-deficient Mov-13 mouse were used. MMP-2 activation by the Mov-13 fibroblasts was rescued by transfection of a full-length α1(I) collagen cDNA, and to our surprise, also by transfection with an α1(I) collagen cDNA carrying a mutation at the C-proteinase cleavage, which almost abrogated fibrillogenesis. Although studies with ascorbate-cultured MT1-MMP-/- fibroblasts showed that MT1-MMP played a significant role in the collagen-induced MMP-2 activation, a residual MT1-MMP-independent activation of MMP-2 was seen which resembled the level of MMP-2 activation persisting when wild-type fibroblasts were cultured in the presence of both ascorbic acid and MMP inhibitors. We were also unable to block this residual activation with inhibitors specific for serinyl, aspartyl, or cysteinyl enzymes.

A collagen prolyl 4-hydroxylase inhibitor reduces adhesions after tendon injury

Collagen synthesis inhibition potentially can reduce adhesion formation after tendon injury but also may affect cutaneous wound healing. We hypothesized that a novel orally administered collagen synthesis inhibitor (CPHI-I) would substantially reduce flexor tendon adhesions after injury, without any clinically important effect on cutaneous wound healing. The experiments were performed in a rat model with an in-continuity crush injury model in the rat hindfoot flexor tendon to provoke adhesion formation. Assays of dermal collagen production and the rate of healing of an excised wound were performed to assess cutaneous wound healing. Animals in the treatment groups received CPHI-I for 1, 2, or 6 weeks and were assessed at either 2 or 6 weeks. The work of flexion in the injured digit was reduced in the CPHI-I-treated animals compared with control animals, (0.188 J versus 0.0307 J at 2 weeks, and 0.0231 J versus 0.0331 J at 6 weeks) The cutaneous wound healing rate was similar in all animals, but dermal collagen synthesis was reduced in the treated animals. The CPHI-I seems to reduce tendon adhesion, and although collagen synthesis was reduced in cutaneous wounds, CPHI-I did not retard wound healing.

Adipose tissue engineering based on the controlled release of fibroblast growth factor-2 in a collagen matrix

Adipose tissue forms when basement membrane extract (Matrigel™) and fibroblast growth factor-2 (FGF-2) are added to our mouse tissue engineering chamber model. A mouse tumor extract, Matrigel is unsuitable for human clinical application, and finding an alternative to Matrigel is essential. In this study we generated adipose tissue in the chamber model without using Matrigel by controlled release of FGF-2 in a type I collagen matrix. FGF-2 was impregnated into biodegradable gelatin microspheres for its slow release. The chambers were filled with these microspheres suspended in 60 μL collagen gel. Injection of collagen containing free FGF-2 or collagen containing gelatin microspheres with buffer alone served as controls. When chambers were harvested 6 weeks after implantation, the volume and weight of the tissue obtained were higher in the group that received collagen and FGF-2 impregnated microspheres than in controls. Histologic analysis of tissue constructs showed the formation of de novo adipose tissue accompanied by angiogenesis. In contrast, control groups did not show extensive adipose tissue formation. In conclusion, this study has shown that de novo formation of adipose tissue can be achieved through controlled release of FGF-2 in collagen type I in the absence of Matrigel.

Type I collagen abrogates the clathrin-mediated internalization of membrane type 1 matrix metalloproteinase (MT1-MMP) via the MT1-MMP hemopexin domain

Type I collagen (Col I)-stimulated matrix metalloproteinase-2 (MMP-2) activation via membrane type 1 MMP (MT1-MMP) involves both a transcriptional increase in MT1-MMP expression and a nontranscriptional response mediated by preexisting MT1-MMP. In order to identify which MT1-MMP domains were required for the nontranscriptional response, MCF-7 cells that lack endogenous MT1-MMP were transfected with either wild type or domain mutant MT1-MMP constructs. We observed that mutant constructs lacking the MT1-MMP cytoplasmic tail were able to activate MMP-2 in response to Col I but not a construct lacking the MT1-MMP hemopexin domain. Col I did not alter total MT1-MMP protein levels; nor did it appear to directly induce MT1-MMP oligomerization. Col I did, however, redistribute preexisting MT1-MMP to the cell periphery compared with unstimulated cells that displayed amore diffuse staining pattern. In addition, Col I blocked the internalization of MT1-MMP in a dynamin-dependent manner via clathrin-coated pit-mediated endocytosis. This mechanism of impaired internalization is different from that reported for concanavalin A, since it is not mediated by the cytoplasmic tail of MT1-MMP but rather by the hemopexin domain. In summary, upon Col I binding to its cell surface receptor, MT1-MMP internalization via clathrin-coated pit-mediated endocytosis is impaired through interactions with the hemopexin domain, thereby regulating its function and ability to activate MMP-2.

Implication of collagen type I-induced membrane-type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in the metastatic progression of breast carcinoma

We have previously demonstrated that fibroblasts and invasive human breast carcinoma (HBC) cells specifically activate matrix metalloproteinase- 2 (MMP-2) when cultured on 3-dimensional gels of type I collagen but not a range of other substrates. We show here the constitutive expression of membrane-type 1 (MT1)-MMP in both fibroblasts, and invasive HBC cell lines, that have fibroblastic attributes presumably acquired through an epithelial- to-mesenchymal transition (EMT). Treatment with collagen type I increased the steady-state MT1-MMP mRNA levels in these cells but did not induce either MT1-MMP expression or MMP-2 activation in noninvasive breast carcinoma cell lines, which retain epithelial features. Basal MT3-MMP mRNA expression had a pattern similar to that of MT1-MMP but was not up-regulated by collagen. MT4- MMP mRNA was seen in both invasive and noninvasive HBC cell lines and was also not collagen-regulated, and MT2-MMP mRNA was not detected in any of the HBC cell lines tested. These data support a role for MT1-MMP in the collagen- induced MMP-2-activation seen in these cells. In situ hybridization analysis of archival breast cancer specimens revealed a close parallel in expression of both collagen type I and MT1-MMP mRNA in peritumoral fibroblasts, which was correlated with aggressiveness of the lesion. Relatively high levels of expression of both mRNA species were seen in fibroblasts close to invasive tumor nests and, although only focally, in certain areas close to preinvasive tumors. These foci may represent hot spots for local degradation and invasive progression. Collectively, these results implicate MT1-MMP in collagen- stimulated MMP-2 activation and suggest that this mechanism may be employed in vivo by both tumor-associated fibroblasts and EMT-derived carcinoma cells to facilitate increased invasion and/or metastasis.