Macrophages have been classified as having plastic phenotypes which exist within a spectrum between M1 (classically activated; pro-inflammatory) and M2 (alternatively activated; regulatory, homeostatic). To date, the effects of polarization towards a predominantly M1 or M2 phenotype have been studied largely in the context of response to pathogen or cancer. Recently, M1 and M2 macrophages have been shown to play distinct roles in tissue remodeling following injury. In the present study, the M1/M2 paradigm was utilized to examine the role of macrophages in the remodeling process following implantation of 14 biologically derived surgical mesh materials in the rat abdominal wall. In situ polarization of macrophages responding to the materials was examined and correlated to a quantitative measure of the observed tissue remodeling response to determine whether macrophage polarization is an accurate predictor of the ability of a biologic scaffold to promote constructive tissue remodeling. Additionally the ability of M1 and M2 macrophages to differentially recruit progenitor-like cells in vitro, which are commonly observed to participate in the remodeling of those ECM scaffolds which have a positive clinical outcome, was examined as a possible mechanism underlying the differences in the observed remodeling responses. The results of the present study show that there is a strong correlation between the early macrophage response to implanted materials and the outcome of tissue remodeling. Increased numbers of M2 macrophages and higher ratios of M2:M1 macrophages within the site of remodeling at 14 days were associated with more positive remodeling outcomes (r2=0.525–0.686, p<0.05). Further, the results of the present study suggest that the constructive remodeling outcome may be due to the recruitment and survival of different cell populations to the sites of remodeling associated with materials that elicit an M1 versus M2 response. Both M2 and M0 macrophage conditioned medias were shown to have higher chemotactic activities than media conditioned by M1 macrophages (p<0.05). A more thorough understanding of these issues will logically influence the design of next generation biomaterials and the development of regenerative medicine strategies for the formation of functional host tissues.
Most tumor cells are characterized by increased genomic instability and chromosome segregational defects, often associated with hyperamplification of the centrosome and the formation of multipolar spindles. However, extra centrosomes do not always lead to multipolarity. Here, we describe a process of centrosomal clustering that prevented the formation of multipolar spindles in noncancer cells. Noncancer cells needed to overcome this clustering mechanism to allow multipolar spindles to form at a high frequency. The microtubule motor cytoplasmic dynein was a critical part of this coalescing machinery, and in some tumor cells overexpression of the spindle protein NuMA interfered with dynein localization, promoting multipolarity.
The extracellular matrix (ECM) has been successfully used as a scaffold for constructive remodeling of multiple tissues in both preclinical studies and in human clinical applications. The basement membrane is a specialized form of the ECM that supports and facilitates the growth of epithelial cell populations. The morphology and the molecular composition of the ECM, including the basement membrane, vary depending upon the organ from which the ECM is harvested and the methods by which it is processed for use as a medical device. Processing steps, such as decellularization, lyophilization, disinfection, and terminal sterilization, may affect the morphology and composition of an ECM scaffold, including, but not limited to, the integrity of a basement membrane complex. The present study evaluated the presence and integrity of a basement membrane complex in processed ECM derived from three different tissues: the urinary bladder, small intestine, and liver. Immunohistochemical determination of the presence and localization of three basement membrane molecules, collagen IV, laminin, and collagen VII, was conducted for each ECM scaffold. Scanning electron microscopy (SEM) was used to further explore the surface ultrastructure of selected ECM scaffolds. The effect of a surface basement membrane presence upon the pattern of in vitro growth of two separate cell types, NIH 3T3 fibroblasts and human microvascular endothelial cells (HMEC), was also evaluated for each ECM scaffold. Results showed that the only intact basement membrane complex was found on the luminal surface of the ECM derived from the urinary bladder and that the basement membrane was an effective barrier to penetration of the scaffold by the seeded cells. We conclude that the urinary bladder ECM but not the small intestine- or liver-derived ECM contains a surface with composition and morphology consistent with that of an intact basement membrane complex, that the basement membrane complex can survive processing, and that the basement membrane structure can modulate in vitro cell growth patterns.
Biologic materials from various species and tissues are commonly used as surgical meshes or scaffolds for tissue reconstruction. Extracellular matrix (ECM) represents the secreted product of the cells comprising each tissue and organ, and therefore provides a unique biologic material for selected regenerative medicine applications. Minimal disruption of ECM ultrastructure and content during tissue processing is typically desirable. The objective of this study was to systematically evaluate effects of commonly used tissue processing steps upon porcine dermal ECM scaffold composition, mechanical properties, and cytocompatibility. Processing steps evaluated included liming and hot water sanitation, trypsin/SDS/TritonX-100 decellularization, and trypsin/TritonX-100 decellularization. Liming decreased the growth factor and glycosaminoglycan content, the mechanical strength, and the ability of the ECM to support in vitro cell growth (p ≤ 0.05 for all). Hot water sanitation treatment decreased only the growth factor content of the ECM (p ≤ 0.05). Trypsin/ SDS/TritonX-100 decellularization decreased the growth factor content and the ability of the ECM to support in vitro cell growth (p ≤ 0.05 for both). Trypsin/TritonX-100 decellularization also decreased the growth factor content of the ECM but increased the ability of the ECM to support in vitro cell growth (p ≤ 0.05 for both). We conclude that processing steps evaluated in the present study affect content, mechanical strength, and/or cytocompatibility of the resultant porcine dermal ECM, and therefore care must be taken in choosing appropriate processing steps to maintain the beneficial effects of ECM in biologic scaffolds.
Whole-organ liver decellularization is possible with maintenance of structure and composition suitable to support functional hepatocytes.
Biologic scaffolds composed of extracellular matrix (ECM) are utilized in numerous regenerative medicine applications to facilitate the constructive remodeling of tissues and organs. The mechanisms by which the host remodeling response occurs are not fully understood, but recent studies suggest that both constituent growth factors and biologically active degradation products derived from ECM play important roles. The objective of the present study was to determine if degradation of ECM scaffold materials in vitro by methods that are biochemically and physiologically relevant can yield products that possess chemotactic and/or mitogenic activities for fully differentiated mammalian endothelial cells and undifferentiated multipotential progenitor cells. ECM harvested from porcine urinary bladder was degraded enzymatically with pepsin/hydrochloric acid or papain. The ECM degradation products were tested for chemoattractant properties utilizing either 48-well chemotaxis filter migration microchambers or fluorescence-based filter migration assays, and were tested for mitogenic properties in cell proliferation assays. Results showed that ECM degradation products possessed chemotactic and mitogenic activities for multipotential progenitor cells and that the same degradation products inhibited both chemotaxis and proliferation of differentiated endothelial cells. These findings support the concept that degradation products of ECM bioscaffolds are important modulators of the recruitment and proliferation of appropriate cell types during the process of ECM scaffold remodeling.
Biologic scaffolds prepared from the extracellular matrix (ECM) of decellularized mammalian tissues have been shown to facilitate constructive remodeling in injured tissues such as skeletal muscle, the esophagus, and lower urinary tract, among others. The ECM of every tissue has a unique composition and structure that likely has direct effects on the host response and it is plausible that ECM harvested from a given tissue would provide distinct advantages over ECM harvested from nonhomologous tissues. For example, a tissue specific muscle ECM scaffold may be more suitable for constructive remodeling of skeletal muscle than non-homologous ECM tissue sources. The present study describes an enzymatic and chemical decellularization process for isolating skeletal muscle ECM scaffolds using established decellularization criteria and characterized the structure and chemical composition of the resulting ECM. The results were compared to those from a non-muscle ECM derived from small intestine (SIS). Muscle ECM was shown to contain growth factors, glycosaminoglycans, and basement membrane structural proteins which differed from those present in SIS. Myogenic cells survived and proliferated on muscle ECM scaffolds in vitro, and when implanted in a rat abdominal wall injury model in vivo was shown to induce a constructive remodeling response associated with scaffold degradation and myogenesis in the implant area; however, the remodeling outcome did not differ from that induced by SIS by 35 days post surgery. These results suggest that superior tissue remodeling outcomes are not universally dependent upon homologous tissue derived ECM scaffold materials.
Acellular biologic scaffolds are commonly used to facilitate the constructive remodeling of three of the four traditional tissue types: connective, epithelial, and muscle tissues. However, the application of extracellular matrix (ECM) scaffolds to neural tissue has been limited, particularly in the central nervous system (CNS) where intrinsic regenerative potential is low. The ability of decellularized liver, lung, muscle, and other tissues to support tissue-specific cell phenotype and function suggests that CNS-derived biologic scaffolds may help to overcome barriers to mammalian CNS repair. A method was developed to create CNS ECM scaffolds from porcine optic nerve, spinal cord, and brain, with decellularization verified against established criteria. CNS ECM scaffolds retained neurosupportive proteins and growth factors and, when tested with the PC12 cell line in vitro, were cytocompatible and stimulated proliferation, migration, and differentiation. Urinary bladder ECM (a non-CNS ECM scaffold) was also cytocompatible and stimulated PC12 proliferation but inhibited migration rather than acting as a chemoattractant over the same concentration range while inducing greater rates of PC12 differentiation compared to CNS ECM. These results suggest that CNS ECM may provide tissue-specific advantages in CNS regenerative medicine applications and that ECM scaffolds in general may aid functional recovery after CNS injury.
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