Disorganized collagen scaffold interferes with fibroblast mediated deposition of organized extracellular matrix in vitro

Center for Engineering in Medicine, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, USA.
Biotechnology and Bioengineering (Impact Factor: 4.13). 10/2012; 109(10):2683-98. DOI: 10.1002/bit.24533
Source: PubMed


Many tissue engineering applications require the remodeling of a degradable scaffold either in vitro or in situ. Although inefficient remodeling or failure to fully remodel the temporary matrix can result in a poor clinical outcome, very few investigations have examined in detail, the interaction of regenerative cells with temporary scaffoldings. In a recent series of investigations, randomly oriented collagen gels were directly implanted into human corneal pockets and followed for 24 months. The resulting remodeling response exhibited a high degree of variability which likely reflects differing regenerative/synthetic capacity across patients. Given this variability, we hypothesize that a disorganized, degradable provisional scaffold could be disruptive to a uniform, organized reconstruction of stromal matrix. In this investigation, two established corneal stroma tissue engineering culture systems (collagen scaffold-based and scaffold-free) were compared to determine if the presence of the disorganized collagen gel influenced matrix production and organizational control exerted by primary human corneal fibroblast cells (PHCFCs). PHCFCs were cultured on thin disorganized reconstituted collagen substrate (RCS--five donors: average age 34.4) or on a bare polycarbonate membrane (five donors: average age 32.4 controls). The organization and morphology of the two culture systems were compared over the long-term at 4, 8, and 11/12 weeks. Construct thickness and extracellular matrix organization/alignment was tracked optically with bright field and differential interference contrast (DIC) microscopy. The details of cell/matrix morphology and cell/matrix interaction were examined with standard transmission, cuprolinic blue and quick-freeze/deep-etch electron microscopy. Both the scaffold-free and the collagen-based scaffold cultures produced organized arrays of collagen fibrils. However, at all time points, the amount of organized cell-derived matrix in the scaffold-based constructs was significantly lower than that produced by scaffold-free constructs (controls). We also observed significant variability in the remodeling of RCS scaffold by PHCFCs. PHCFCs which penetrated the RCS scaffold did exert robust local control over secreted collagen but did not appear to globally reorganize the scaffold effectively in the time period of the study. Consistent with our hypothesis, the results demonstrate that the presence of the scaffold appears to interfere with the global organization of the cell-derived matrix. The production of highly organized local matrix by fibroblasts which penetrated the scaffold suggests that there is a mechanism which operates close to the cell membrane capable of controlling fibril organization. Nonetheless, the local control of the collagen alignment produced by cells within the scaffold was not continuous and did not result in overall global organization of the construct. Using a disorganized scaffold as a guide to produce highly organized tissue has the potential to delay the production of useful matrix or prevent uniform remodeling. The results of this study may shed light on the recent attempts to use disorganized collagenous matrix as a temporary corneal replacement in vivo which led to a variable remodeling response.

Download full-text


Available from: Nima Saeidi, Feb 27, 2014
  • Source
    • "They also increase the alignment of cells and matrix within each layer of selfassembled sheets derived from corneal fibroblasts (Guillemette et al., 2009). On Transwell filters which have parallel, aligned grooves on the surface, human corneal fibroblasts stimulated with ascorbate analogs secrete and organize a more cornea-like ECM as compared to fibroblasts plated on planar substrates or disorganized collagen ECM (Guo et al., 2007; Karamichos et al., 2014, 2010; Ren et al., 2008; Saeidi et al., 2012). Thus substrate topography can modulate both the organization and differentiation of corneal fibroblasts. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The generation of cellular forces and the application of these physical forces to the ECM play a central role in mediating matrix patterning and remodeling during fundamental processes such as developmental morphogenesis and wound healing. In addition to growth factors and other biochemical factors that can modulate the keratocyte mechanical phenotype, another key player in the regulation of cell-induced ECM patterning is the mechanical state of the ECM itself. In this review we provide an overview of the biochemical and biophysical factors regulating the mechanical interactions between corneal keratocytes and the stromal ECM at the cellular level. We first provide an overview of how Rho GTPases regulate the sub-cellular pattern of force generation by corneal keratocytes, and the impact these forces have on the surrounding ECM. We next review how feedback from local matrix structural and mechanical properties can modulate keratocyte phenotype and mechanical activity. Throughout this review, we provide examples of how these biophysical interactions may contribute to clinical outcomes, with a focus on corneal wound healing. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Experimental Eye Research 04/2015; 133. DOI:10.1016/j.exer.2014.09.003 · 2.71 Impact Factor
  • Source
    • "Both the collagen implant and the PDS sheath had significant roles in aligning the newly regenerated tissue. Saeidi et al [47] showed that using a disorganized scaffold as a guide to produce highly organized tissue has the potential to delay the production of useful matrix or prevent uniform remodeling. Liu et al [48] showed that the alignment of the electrospun fibrous scaffold has a significant role in cellular alignment in vitro. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Healing of large tendon defects is challenging. We studied the role of collagen implant with or without polydioxanone (PDS) sheath on the healing of a large Achilles tendon defect model, in rabbits. Sixty rabbits were divided into three groups. A 2 cm gap was created in the left Achilles tendon of all rabbits. In the control lesions, no implant was used. The other two groups were reconstructed by collagen and collagen-PDS implants respectively. The animals were clinically examined at weekly intervals and their lesions were observed by ultrasonography. Blood samples were obtained from the animals and were assessed for hematological analysis and determination of serum PDGF level, at 60 days post injury (DPI). The animals were then euthanized and their lesions were assessed for gross and histopathology, scanning electron microscopy, biomechanical testing, dry matter and hydroxyproline content. Another 65 pilot animals were also studied grossly and histopathologically to define the host implant interaction and graft incorporation at serial time points. The treated animals gained significantly better clinical scoring compared to the controls. Treatment with collagen and collagen-PDS implants significantly increased the biomechanical properties of the lesions compared to the control tendons at 60DPI (P<0.05). The tissue engineered implants also reduced peritendinous adhesion, muscle fibrosis and atrophy, and increased ultrasonographical echogenicity and homogenicity, maturation and differentiation of the collagen fibrils and fibers, tissue alignment and volume of the regenerated tissue compared to those of the control lesions (P<0.05). The implants were gradually absorbed and substituted by the new tendon. Implantation of the bioimplants had a significant role in initiating tendon healing and the implants were biocompatible, biodegradable and safe for application in tendon reconstructive surgery. The results of the present study may be valuable in clinical practice.
    PLoS ONE 09/2013; 8(9):e73016. DOI:10.1371/journal.pone.0073016 · 3.23 Impact Factor
  • Source
    • "Within tissue engineering, many scaffold-based approaches have been reported, forming a large body of research concerning the synthesis of fibrocartilage and other tissues [10] [11] [12]. However, these methods often face issues, such as a lack of cellto-cell communication, the prevention of mechanotransduction, and the impairment or absence of ECM remodeling [13] [14]. For this reason, scaffoldless tissue engineering strategies, such as bio-printing and cell sheet engineering, have recently emerged [15] [16]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Knee meniscus fibrocartilage is frequently injured, resulting in approximately 1 million procedures annually in the U.S. and Europe. Its near-avascularity contributes heavily to its inability to heal, and places it as a prime candidate for replacement through regenerative medicine. Here, we describe a novel approach to increase extracellular matrix organization, rather than content, in order to augment the mechanical properties of engineered tissue. To synthesize fibrocartilage, we employ a self-assembling process, which is free of exogenous scaffolds and relies on cell-to-cell interactions to form all-biologic constructs. When treated with the signaling phospholipid lysophosphatidic acid (LPA), tissue constructs displayed increased tensile properties and collagen organization, while total collagen content remained unchanged. LPA-treated constructs exhibited greater DNA content, indicative that the molecule exerted a signaling effect. Furthermore, LPA-treated cells displayed significant cytoskeletal reorganization. We conclude that LPA induced cytoskeletal reorganization and cell-matrix traction, which resulted in matrix reorganization and increased tensile properties. This study emphasizes the potential of non-traditional stimuli, such as signaling phospholipids, for use in tissue development studies. The extension of these results to other collagen-rich tissues represents a promising avenue for future exploration.
    Biochemical and Biophysical Research Communications 02/2013; 433(1). DOI:10.1016/j.bbrc.2013.02.048 · 2.30 Impact Factor
Show more