183 research outputs found
Scattering of Light from Histologic Sections: A New Method for the Analysis of Connective Tissue
We have developed a light scattering technique that can be used to analyze the orientation and diameter of collagen fibers in histologic sections of connective tissue. Scattering patterns obtained by transmitting laser light through sections of tissue contain information both on the orientation, degree of alignment, and size of the constituent collagen fibers. Analysis of the azimuthal intensity distribution of scattered light yields numerical values of the degree of alignment by use of an orientation index, S, which is chosen to vary between 0 for randomly oriented fibers and 1 for a perfectly aligned arrangement. The average diameter of the collagen fibers is calculated from the scattering angle at which the intensity reaches its first minimum. These measurements are independent of the nature of histologic stain. The procedure is illustrated by measurements obtained with sections of the guinea pig dermis and of control scar. We conclude from our experiments that light scattering can complement the analysis of tissue architecture typically performed with the light microscope
Regeneration of injured skin and peripheral nerves requires control of wound contraction, not scar formation
We review the mounting evidence that regeneration is induced in wounds in skin and peripheral nerves by a simple modification of the wound healing process. Here, the process of induced regeneration is compared to the other two well-known processes by which wounds close, i.e., contraction and scar formation. Direct evidence supports the hypothesis that the mechanical force of contraction (planar in skin wounds, circumferential in nerve wounds) is the driver guiding the orientation of assemblies of myofibroblasts (MFB) and collagen fibers during scar formation in untreated wounds. We conclude that scar formation depends critically on wound contraction and is, therefore, a healing process secondary to contraction. Wound contraction and regeneration did not coincide during healing in a number of experimental models of spontaneous (untreated) regeneration described in the literature. Furthermore, in other studies in which an efficient contraction-blocker, a collagen scaffold named dermis regeneration template (DRT), and variants of it, were grafted on skin wounds or peripheral nerve wounds, regeneration was systematically observed in the absence of contraction. We conclude that contraction and regeneration are mutually antagonistic processes. A dramatic change in the phenotype of MFB was observed when the contraction-blocking scaffold DRT was used to treat wounds in skin and peripheral nerves. The phenotype change was directly observed as drastic reduction in MFB density, dispersion of MFB assemblies and loss of alignment of the long MFB axes. These observations were explained by the evidence of a surface-biological interaction of MFB with the scaffold, specifically involving binding of MFB integrins Îą[subscript 1]β[subscript 1] and Îą[subscript 2]β[subscript 1] to ligands GFOGER and GLOGER naturally present on the surface of the collagen scaffold. In summary, we show that regeneration of wounded skin and peripheral nerves in the adult mammal can be induced simply by appropriate control of wound contraction, rather than of scar formation.National Institutes of Health (U.S.) (Grant RO1 NS051320)National Institutes of Health (U.S.) (Grant 5âP41âEB015871â28)Horizon 2020 Framework Programme (European Commission) (Grant DLVâ658850)Singapore-MIT Alliance for Research and Technology (SMART) (Grant 5âP41âEB015871â28)Hamamatsu Corporatio
Quantifying the surface chemistry of 3D matrices in situ
Despite the major role of the matrix (the insoluble environment around cells) in physiology and pathology, there are very few and limited methods that can quantify the surface chemistry of a 3D matrix such as a biomaterial or tissue ECM. This study describes a novel optical-based methodology that can quantify the surface chemistry (density of adhesion ligands for particular cell adhesion receptors) of a matrix in situ. The methodology utilizes fluorescent analogs (markers) of the receptor of interest and a series of binding assays, where the amount of bound markers on the matrix is quantified via spectral multi-photon imaging. The study provides preliminary results for the quantification of the ligands for the two major collagen-binding integrins (ι[subscript 1]β[subscript 1], ι[subscript 2]β[subscript 1]) in porous collagen scaffolds that have been shown to be able to induce maximum regeneration in transected peripheral nerves. The developed methodology opens the way for quantitative descriptions of the insoluble microenvironment of cells in physiology and pathology, and for integrating the matrix in quantitative models of cell signaling.National Institutes of Health (U.S.) (RO1 NS051320)Singapore-MIT Alliance for Research and Technolog
Healing of Tendon Defects Implanted with a Porous Collagen-GAG Matrix: Histological Evaluation
There is currently no method to restore normal function in tendon injuries that result in a gap. The objective of this study was to evaluate the early healing of tendon defects implanted with a porous collagenâglycosaminoglycan (CG) matrix, previously shown to facilitate the regeneration of dermis and peripheral nerve. A novel animal model that isolates the tendon defect site from surrounding tissue during healing was employed. This model used a silicone tube to entubulate the surgically produced tendon gap of 10 mm, allowing for the evaluation of the effects of the analog of extracellular matrix on healing of tendon, absent the influences of the external environment. The results showed that tendon stumps induced synthesis of a tissue cable inside the silicone tube in both the presence and absence of CG matrix. The presence of the CG matrix, however, altered the process of tendon healing. Tubes filled with CG matrix contained a significantly greater volume of tissue at the time periods of evaluation: 3, 6, and 12 weeks. Granulation tissue persisted for a longer period of time in the lesion site of CG-filled defects, and the amount of dense fibrous tissue increased continuously during the period of study in defects filled with CG matrix. In contrast, the amount of dense fibrous tissue decreased after 6 weeks in originally empty tubes. In tubes that did not contain the CG matrix, the new tissue consisted of dense aggregates of crimped fibers with a wavelength and fiber bundle thickness that were significantly shorter than those in normal tendon, and consistent with the type of scar that is the end result of repair of many connective tissues. Although, CG-filled tubes contained dense fibrous tissue by 12 weeks, the tissue had no crimp. The CG matrix may have prolonged the synthesis of granulation tissue and delayed or prevented the formation of scar.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63265/1/ten.1997.3.187.pd
The effect of pore size on cell adhesion in collagen-GAG scaffolds.
The biological activity of scaffolds used in tissue engineering applications hypothetically depends on the density of available ligands, scaffold sites at which specific cell binding occurs. Ligand density is characterized by the composition of the scaffold, which defines the surface density of ligands, and by the specific surface area of the scaffold, which defines the total surface of the structure exposed to the cells. It has been previously shown that collagen-glycosaminoglycan (CG) scaffolds used for studies of skin regeneration were inactive when the mean pore size was either lower than 20 microm or higher than 120 microm (Proc. Natl. Acad. Sci., USA 86(3) (1989) 933). To study the relationship between cell attachment and viability in scaffolds and the scaffold structure, CG scaffolds with a constant composition and solid volume fraction (0.005), but with four different pore sizes corresponding to four levels of specific surface area were manufactured using a lyophilization technique. MC3T3-E1 mouse clonal osteogenic cells were seeded onto the four scaffold types and maintained in culture. At the experimental end point (24 or 48 h), the remaining viable cells were counted to determine the percent cell attachment. A significant difference in viable cell attachment was observed in scaffolds with different mean pore sizes after 24 and 48 h; however, there was no significant change in cell attachment between 24 and 48 h for any group. The fraction of viable cells attached to the CG scaffold decreased with increasing mean pore size, increasing linearly (R2 = 0.95, 0.91 at 24 and 48 h, respectively) with the specific surface area of the scaffold. The strong correlation between the scaffold specific surface area and cell attachment indicates that cell attachment and viability are primarily influenced by scaffold specific surface area over this range (95.9-150.5 microm) of pore sizes for MC3T3 cells
Influence of freezing rate on pore structure in freeze-dried collagen-GAG scaffolds.
The cellular structure of collagen-glycosaminoglycan (CG) scaffolds used in tissue engineering must be designed to meet a number of constraints with respect to biocompatibility, degradability, pore size, pore structure, and specific surface area. The conventional freeze-drying process for fabricating CG scaffolds creates variable cooling rates throughout the scaffold during freezing, producing a heterogeneous matrix pore structure with a large variation in average pore diameter at different locations throughout the scaffold. In this study, the scaffold synthesis process was modified to produce more homogeneous freezing by controlling of the rate of freezing during fabrication and obtaining more uniform contact between the pan containing the CG suspension and the freezing shelf through the use of smaller, less warped pans. The modified fabrication technique has allowed production of CG scaffolds with a more homogeneous structure characterized by less variation in mean pore size throughout the scaffold (mean: 95.9 microm, CV: 0.128) compared to the original scaffold (mean: 132.4 microm, CV: 0.185). The pores produced using the new technique appear to be more equiaxed, compared with those in scaffolds produced using the original technique
In Situ Quantification of Surface Chemistry in Porous Collagen Biomaterials
Cells inside a 3D matrix (such as tissue extracellular matrix or biomaterials) sense their insoluble environment through specific binding interactions between their adhesion receptors and ligands present on the matrix surface. Despite the critical role of the insoluble matrix in cell regulation, there exist no widely-applicable methods for quantifying the chemical stimuli provided by a matrix to cells. Here, we describe a general-purpose technique for quantifying in situ the density of ligands for specific cell adhesion receptors of interest on the surface of a 3D matrix. This paper improves significantly the accuracy of the procedure introduced in a previous publication by detailed marker characterization, optimized staining, and improved data interpretation. The optimized methodology is utilized to quantify the ligands of integrins ι[subscript 1]β[subscript 1], ι[subscript 2]β[subscript 1] on two kinds of matched porous collagen scaffolds, which are shown to possess significantly different ligand density, and significantly different ability to induce peripheral nerve regeneration in vivo. Data support the hypothesis that cell adhesion regulates contractile cell phenotypes, recently shown to be inversely related to organ regeneration. The technique provides a standardized way to quantify the surface chemistry of 3D matrices, and a means for introducing matrix effects in quantitative biological models.National Institutes of Health (U.S.) (Grant RO1 NS051320)National Institutes of Health. National Institute for Biomedical Imaging and Bioengineering (Biomechanics Training Grant T32EB006348)National Science Foundation (U.S.). Graduate Research Fellowship Program (Grant DGE-1122374)Singapore-MIT Alliance for Research and Technology (SMART). BioSym IRGComputation and Systems Biology Programme of Singapore--Massachusetts Institute of Technology AllianceNational Institutes of Health (U.S.) (Grant 9P41EB015871-26A1
Image informatics for studying signal transduction in cells interacting with 3D matrices
Cells sense and respond to chemical stimuli on their environment via signal transduction pathways, complex networks of proteins whose interactions transmit chemical information. This work describes an implementation of image informatics, imaging-based methodologies for studying signal transduction networks. The methodology developed focuses on studying signal transduction networks in cells that interact with 3D matrices. It utilizes shRNA-based knock down of network components, 3D high-content imaging of cells inside the matrix by spectral multi-photon microscopy, and single-cell quantification using features that describe both cell morphology and cell-matrix adhesion pattern. The methodology is applied in a pilot study of TGFβ signaling via the SMAD pathway in fibroblasts cultured inside porous collagen-GAG scaffolds, biomaterials similar to the ones used clinically to induce skin regeneration. Preliminary results suggest that knocking down all rSMAD components affects fibroblast response to TGFβ1 and TGFβ3 isoforms in different ways, and suggest a potential role for SMAD1 and SMAD5 in regulating TGFβ isoform response. These preliminary results need to be verified with proteomic results that can provide solid evidence about the particular role of individual components of the SMAD pathway.National Institutes of Health (U.S.) (RO1 NS051320)Singapore-MIT Alliance for Research and Technolog
The effect of pore size on permeability and cell attachment in collagen scaffolds for tissue engineering.
The permeability of scaffolds and other three-dimensional constructs used for tissue engineering applications is important as it controls the diffusion of nutrients in and waste out of the scaffold as well as influencing the pressure fields within the construct. The objective of this study was to characterize the permeability/fluid mobility of collagen-GAG scaffolds as a function of pore size and compressive strain using both experimental and mathematical modeling techniques. Scaffolds containing four distinct mean pore sizes (151, 121, 110, 96 microns) were fabricated using a freeze-drying process. An experimental device was constructed to measure the permeability of the scaffold variants at different levels of compressive strain (0, 14, 29 and 40% while a low-density open-cell foam cellular solids model utilizing a tetrakaidecahedral unit cell was used to accurately model the permeability of each scaffold variant at all level of applied strain. The results of both the experimental and the mathematical analysis revealed that scaffold permeability increases with increasing pore size and decreases with increasing compressive strain. The excellent comparison between experimentally measured and predicted scaffold permeability suggests that cellular solids modelling techniques can be utilized to predict scaffold permeability under a variety of physiological loading conditions as well as to predict the permeability of future scaffolds with a wide variety of pore microstructures
Use of the parabiotic model in studies of cutaneous wound healing to define the participation of circulating cells
Previous experimental studies to assess the contribution of blood-borne circulating (BBC) cells to cutaneous wound healing have relied on discontinuous pulsing of labeled BBC elements or bone marrow transplant protocols. Such approaches do not allow the examination of stable BBC cells that have matured in a physiologically normal host. We have used a parabiotic murine model for cutaneous wound healing to evaluate the relative contribution of stable populations of peripheral blood cells expressing the green fluorescent protein (GFP) transgene in otherwise normal animals. Circulating cells (mature and immature) expressing the GFP transgene were easily detected and quantified in wounds of GFPâ parabiotic twins during all evaluated stages of the healing response. Using multiple antibody probes, the relative contribution of various subsets of BBC cells could be comparatively assessed. In early wounds, some cells expressing mesenchymal epitopes were documented to be of hematopoietic origin, indicating the utility of this model in assessing cell plasticity in the context of tissue regeneration and repair. Application of this approach enables further investigation into the contribution of peripheral blood in normal and abnormal healing responses.National Institutes of Health (U.S.) (NIH 5 T32 HL007627- 22 Physician-Scientist Training Grant)National Institutes of Health (U.S.) (NIH/NIDDK (5 P30 DK36836-20))Brigham and Womenâs Hospital (Program in Dermatopathology core grant (SDRC))National Institutes of Health. (U.S.). Department of Health and Human Services (Brigham and Womenâs Hospitalâs Program in Dermatopathology core grant (SPORE)
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