Collagen vitrigel membrane useful for paracrine assays in vitro and drug delivery systems in vivo

https://doi.org/10.1016/j.jbiotec.2007.05.033Get rights and content

Abstract

We previously succeeded in converting a soft and turbid disk of type-I collagen gel into a strong and transparent vitrigel membrane utilizing a concept for the vitrification of heat-denatured proteins and have demonstrated its protein-permeability and advantage as a scaffold for reconstructing crosstalk models between two different cell types. In this study, we observed the nano-structure of the type-I collagen vitrigel membrane and verified its utility for paracrine assays in vitro and drug delivery systems in vivo. Scanning electron microscopic observation revealed that the vitrigel membrane was a dense network architecture of typical type-I collagen fibrils. In the crosstalk model between PC-12 pheochromocytoma cells and L929 fibroblasts, nerve growth factor (NGF) secreted from L929 cells passed through the collagen vitrigel membrane and induced the neurite outgrowth of PC-12 cells by its paracrine effect. Also, the collagen vitrigel membrane containing vascular endothelial growth factor (VEGF) showed sustained-release of VEGF in vitro and its subcutaneous transplantation into a rat resulted in remarkable angiogenesis. These data suggest that the collagen vitrigel membrane is useful for paracrine assays in vitro and drug delivery systems in vivo.

Introduction

Type-I collagen has been used as a biomaterial in tissue engineering because it plays a role of cellular scaffolding in various forms such as coat, membrane, gel, etc. (Takezawa, 2003). The molecule of type-I collagen shows monomeric status in an acidic aqueous solution. The optimization of pH, temperature and salt concentration induces the polymerization of type-I collagen monomers, and consequently the gelation was completed by the formation of reticulated collagen fibril networks (Kadler et al., 1996). The three-dimensional cell culture system utilizing the collagen gel can mimic lattice architectures in interstitial soft tissues and dermis in vivo although the two-dimensional collagen-based scaffolds are impossible to reproduce the architectures (Cukierman et al., 2002). Therefore, the transition from two-dimensional to three-dimensional culture modes modulates the gene expression and signal transduction pathway, resulting in the up-regulation (or down-regulation) of cell growth, migration and differentiation (Tasaki et al., 2004, Stegemann et al., 2005, Fassett et al., 2006, Winters et al., 2006). In addition, the mechanical strength of collagen gel affects cell motility (Parkhurst and Saltzman, 1992). Thus the three-dimensional cell culture system utilizing the collagen gel is useful for extrapolating the cell behavior in vivo, however the conventional collagen gel prepared from an acid soluble type-I collagen solution is not mechanically strong as native tissues due to the low density of collagen fibrils.

To overcome the defects of the conventional collagen gel in the utilization for three-dimensional cell culture systems, we previously developed a novel technology for converting a fragile disk of the conventional type-I collagen gel into a strong and transparent vitrigel membrane utilizing a concept for the vitrification of heat-denatured proteins (Takezawa et al., 2004). To date, we have demonstrated the protein-permeability of the type-I collagen vitrigel membrane and its advantage as a scaffold for reconstructing crosstalk models between two different cell types (Takezawa et al., 2004, Takezawa et al., 2007). Also, the collagen concentration of the vitrigel membrane can be estimated to be 10 to 25 (w/v)% from the calculated volume using its thickness that is regulated by the initial collagen amount and the vitrification period in preparation process. Therefore, we attempted to clarify the nano-structure of the type-I collagen vitrigel membrane and to verify its utility for paracrine assay systems in vitro and drug delivery systems in vivo in the current study.

Growth factors are major component of the paracrine effectors by activating intracellular signaling pathway to regulate the cell proliferation, differentiation, migration and adhesion. They accomplish their activity by binding to a specific receptor on the surface of target cells. The neural differentiation of neural crest-derived cells greatly depends on growth factors present in the local environment. Nerve growth factor (NGF) is a potent neurotrophic factor that maintains the neural crest cell-derived sympathetic and sensory neurons (Greene and Tischler, 1976, Vaudry et al., 2002). PC-12 cells (a phenochromocytoma cell line derived from rat adrenal medullas) have been used as a model of NGF-induced neurite outgrowth of periphery neurons, as they can exert extensive neurites in response to NGF stimulation. In addition, several established cell lines including L929 mouse fibroblasts can secrete NGF in the culture medium (Pantazis and Jensen, 1988). Therefore, co-culture of PC-12 cells with L929 fibroblasts induces neurite extension of PC-12 cells (Brachet and Dicou, 1984). In this study, we accomplished paracrine-induced neural differentiation of PC-12 cells by L929 fibroblasts in the co-culture system via the scaffold of a type-I collagen vitrigel membrane.

Also, another usage of collagen gel as a carrier for sustained-release of growth factors has been developed for improving their delivery efficiency to the target cells (Weiner et al., 1985, Kimoto et al., 1998, Babensee et al., 2000). The activity of many growth factors can be sustained by their deposition to extracellular matrix (ECM) components (Vlodavsky et al., 1987, Rogelj et al., 1989). Therefore, the transplantation of a growth factor-containing ECM carrier into a target tissue is powerful way to accomplish its sustained-release (Babensee et al., 2000, Saltzman and Olbricht, 2002, Andreadis and Geer, 2006). Vascular endothelial growth factor (VEGF) is a mitogen consisted with 45 kDa homodimeric glycoprotein secreted from retinal cells and cardiac myocytes. VEGF promotes the proliferation and migration of endothelial cells, so that it is well known as a point inducer of angiogenesis (Ferrara et al., 2003). Angiogenesis is defined as the formation of new blood vessels from a pre-existing micro-vascular bud and is essential for the treatment of heart and peripheral artery disease (Khurana et al., 2005). In addition, VEGF-induced angiogenesis is accompanied by epithelialization and migration of mesenchymal cells into the wounded area during the wound healing process (Galiano et al., 2004). In this study, we developed a sustained-release carrier of VEGF by utilizing a type-I collagen vitrigel membrane and accomplished the induction of angiogenesis by subcutaneously transplanting it into a rat.

Section snippets

Materials

Materials and chemicals were obtained as follows: recombinant rat β-NGF from R&D Systems (Minneapolis, MN); human VEGF, Dulbecco's modified Eagle's medium (DMEM) and phosphate buffered saline (PBS) from Sigma–Aldrich (St. Louis, MO); anti-NGF polyclonal antibody and anti-neuro filament polyclonal antibody from Chemicon (Temecula, CA); Type-I collagen acidic solution from Koken (Tokyo, Japan). Hybond-N nylon membrane from GE healthcare Biosciences (Amersham, UK): Goat anti-rabbit IgG conjugated

Nano-structure of a type-I collagen vitrigel membrane

The surface nano-structure of a type-I collagen vitrigel membrane (Fig. 1 A and B) was compared with that of a traditional type-I collagen gel (Fig. 1C and 1D) by SEM observations to clarify the architectural differences. The reticular network architecture composed of many collagen fibrils (Fig. 1A and 1C) and the characteristic cross-striations every ca. 70 nm for an individual collagen fibril (Fig. 1B and 1D) were observed in both type-I collagen forms of the vitrigel membrane and the

Discussion

The three dimensional collagen gel scaffolds can mimic the structural and biological features of native ECM. For instance, the collagen gels were utilized for reproducing the crosstalk co-culture models between two different types of cells such as endothelial cells and fibroblasts (Velazquez et al., 2002). Also, the collagen gel-coated multi-porous polycarbonate membrane was used for the chemotactic migration assay of endothelial cells (Tsujii et al., 1998). On the contrary, we previously

Acknowledgements

We are grateful to Dr. Tomohiro Tanemoto for surgical assistance. We thank Miss Yukiko Nakazawa, Mrs. Yuri Ohsumi, and Mrs. Yasuko Shimada for technical assistance in the sample preparation of collagen vitrigel membranes. We also thank the Multidisciplinary research laboratory system organized in the Graduate School of Engineering Science, Osaka University. This study was supported by a Grant-in-aid for Animal Re-modeling Project (No. 207) from National Institute of Agrobiological Sciences, and

References (26)

  • E. Cukierman et al.

    Cell interactions with three-dimensional matrices

    Curr. Opin. Cell Biol.

    (2002)
  • J. Fassett et al.

    Type I collagen structure regulates cell morphology and EGF signaling in primary rat hepatocytes through cAMP-dependent protein kinase A

    Mol. Biol. Cell

    (2006)
  • N. Ferrara et al.

    The biology of VEGF and its receptors

    Nat. Med.

    (2003)
  • Cited by (64)

    • Bi-layered carboxymethyl cellulose-collagen vitrigel dual-surface adhesion-prevention membrane

      2022, Carbohydrate Polymers
      Citation Excerpt :

      The CVM was first used as a cell culture substratum. A nylon membrane-framed CVM was useful for fabricating three-dimensional culture models with paracrine (Takezawa et al., 2007). A plastic cylinder-framed CVM (CVM chamber, commercially available as ad-MED Vitrigel®) facilitated the exposure of chemicals to culture models fabricated on CVM (Oshikata-Miyazaki & Takezawa, 2016; Uzu & Takezawa, 2020; Yamaguchi et al., 2013).

    • PDMS microstencil plate-supported fabrication of ultra-thin, condensed ECM membranes for separated cell coculture on both surfaces

      2019, Sensors and Actuators, B: Chemical
      Citation Excerpt :

      These membrane-based culture systems enable us to separately but closely assemble multiple cell types on both surfaces, and have been used to create placenta [26,27], cornea [23], liver [28], and lung [29] tissue models, all of which involve precise cell polarization and hierarchical cell organization. Additionally, paracrine signaling between different cell types on both surfaces through the membranes was investigated [22,30]. Despite great progress with these membranes in cell culture applications, the thickness of collagen vitrigel is typically in the range of 15–20 μm [31], significantly greater than the thickness of the basement membranes in vivo (typically less than 1 μm) [10].

    • Requirements for designing organ-on-a-chip platforms to model the pathogenesis of liver disease

      2019, Organ-on-a-chip: Engineered Microenvironments for Safety and Efficacy Testing
    • Protein-based gels: preparation, characterizations, applications in drug delivery, and tissue engineering

      2018, Polymeric Gels: Characterization, Properties and Biomedical Applications
    View all citing articles on Scopus
    View full text