Bladder Extracellular Matrix Research Articles

Bladder acellular matrix seeding with autologous cultured vaginal smooth cells repaired rabbit vaginal defect

Objective To evaluate the feasibility of bladder extracellular matrix (BACM) seeding with autologous cultured vaginal smooth muscle cells (VSMC) repaired rabbit vaginal defect.Methods This study included 24 female rabbits.BACM and vaginal acellular matrix (VACM) were obtained from 8 rabbits by decellularization process.The other 16 rabbits were randomly divided into experimental and control groups.Vaginal tissue biopsies ( ～ 1 cm2) were harvested from female New Zealand rabbits.VSMC were cultured and stained with a-smooth muscle actin antibodies. Cultured VSMC cells were seeded on BACM ( experimental group) or VACM ( control group) at a cell density of 1 × 107 cells/cm2.The cell-seeded matrixes were cultured for 5days.In experimental group of 8 rabbits,a 2 cm segment of vagina was resected and replaced with BACM seeding with VSMC.Then the regenerative segment was studied with histological technique by hematoxylin-eosin staining after 3,6 and 12 weeks postoperative aud vaginography was performed at 12 weeks postoperative.The 8 rabbits in control group underwent the exact same procedure as above but the vaginal defect was repaired with VACM seeding with VSMC,Results The prepared BACM and VACM were transparent,HE staining and scanning electron microscopy showed the two acellular matrix was both consisted of abundant network of fibers with the regular arrangement,without cellular debris.Primary culture of VSMC was successfully established and passaged,and was uniformly spindle-shaped in the confluent state and showed a characteristic hill and valley'formation.Immunohistochemical staining showed VSMC in culture stained positively with a-smooth muscle actin antibodies.VSMC began to adhere to the BACM 5 hour after implanting and the number of the adhered cells increased with time.Cells gradually expanded and showed the typical morphology of smooth muscle cells.Three weeks after implantation in vivo,the luminal surface of matrix was completely covered by vaginal epithelial tissue,a layer of smooth muscle cells was formed in the outer surface of the matrix.Multilayered vaginal epithelial and improved development of organized muscle bundles was observed after 6 weeks.The regenerative tissue was equivalent to the normal vaginal tissue at 12 weeks postoperatively.Vaginography demonstrated the maintenance of full patency and a wide vaginal caliber without fibrosis and graft rejection.There was no significant difference in all evaluated items between experimental and control groups.Conclusions BACM has the same regenerative process as VACM in the replacement of vaginal defect.Moreover,BACM has wider source and appears to be an suitable material for vaginal replacement. Key words: Urinary bladder; Extracellular matrix; Vagina/cytology; Myocytes, smooth muscle; Vagina/abnormalities; Rabbits

Use of Urinary Bladder Matrix, a Bioactive, Acellular Scaffold, in Transplant Donor Scars and Androgenetic Alopecia: Initial Clinical Experience

Introduction: Emerging regenerative medicine technologies have yet to be utilized in hair restoration surgery. Urinary bladder extracellular matrix (UBM) has shown promise in a wide variety of applications, inducing site-specific remodeling of injured tissue. This case series describes one clinician's experience in the first use of this regenerative material in hair restoration surgery. Materials and Methods: Twenty subjects underwent one of several protocols: (1) Treatment of 3-mm biopsies within donor site scars with UBM versus untreated controls, (2) Removal of a strip of donor site scar tissue and treatment of the site with UBM, (3) Soaking of occipital hair follicle grafts in a solution of UBM, or (4) Soaking of beard or temple hair follicle grafts. Results: At 6 months, hair was observed in biopsy sites that contained UBM, including donor scar areas that would not normally regrow hair, in contrast to the control biopsy sites. Similar results were observed for the strip excisions. UBM-soaked occipital, beard, and temple hair grafts consistently yielded higher than expected numbers of hair follicles. Conclusions: These cases provide anecdotal support for the hypothesized benefits of UBM regenerative technology in restoration therapy for men and women with androgenetic hair loss.

Biologic scaffolds composed of central nervous system extracellular matrix

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.

Extracellular Matrix as an Inductive Template for Temporomandibular Joint Meniscus Reconstruction: A Pilot Study

A device consisting of powdered porcine urinary bladder extracellular matrix (UBM-ECM) encapsulated within sheets of the same material was investigated as a scaffold for temporomandibular joint (TMJ) meniscus reconstruction. Five dogs underwent unilateral resection of the native meniscus and replacement with a UBM-ECM device. Necropsies were performed at 3, 4, 8, 12, and 24 weeks. Two additional dogs underwent bilateral resection of the meniscus with replacement with a UBM-ECM device on 1 side, leaving the contralateral side empty as a control. Necropsies were performed at 24 weeks for bilaterally treated animals. Macroscopically, the UBM-ECM implants were remodeled rapidly and were indistinguishable from newly deposited host tissue at all time points. Microscopically, remodeling was characterized by a dense infiltration of predominantly CD68(+) mononuclear cells and smooth muscle actin-positive fibroblast-like cells at early time points changing with time to a sparse population of smooth muscle actin-negative spindle-shaped cells resembling those of the native fibrocartilaginous TMJ meniscus. Furthermore, the remodeling process showed deposition of predominantly type I collagen, the density and organization of which resembled those of the native meniscus by the 24-week time point. Ingrowth of calsequestrin-positive skeletal muscle tissue was also observed at the periphery of the remodeled UBM-ECM device and was similar to that found at the attachment site of the native meniscus to the surrounding soft tissues. Histologic results were identical for samples excised from both unilaterally and bilaterally treated animals. No adverse changes in the articulating surfaces of the condyle or fossa were observed in UBM-ECM-implanted joints. In the bilaterally treated animals, the unimplanted control side was characterized by degeneration and pitting of the articulating surfaces of both the condyle and the fossa, with disorganized bands of fibrous connective tissue observed within the joint space. Results of this study suggest that the UBM-ECM device provides an effective interpositional material while serving as an inductive template for reconstruction of the TMJ meniscus.

The bladder extracellular matrix. Part I: architecture, development and disease

From the earliest studies with epithelial cells implanted into detrusor muscle to later experiments on smooth muscle in defined collagen gels, cell niche and extracellular matrix (ECM) have been clearly shown to orchestrate cellular behavior and fate whether quiescent, migratory, or proliferative. Normal matrix can revert transformed cells to quiescence, and damaged matrix can trigger malignancy or dedifferentiation. ECM influence in disease, development, healing and regeneration has been demonstrated in many other fields of study, but a thorough examination of the roles of ECM in bladder cell activity has not yet been undertaken. Structural ECM proteins, in concert with adhesive proteins, provide crucial structural support to the bladder. Both structural and nonstructural components of the bladder have major effects on smooth muscle function, through effects on matrix rigidity and signaling through ECM receptors. While many ECM components and receptors identified in the bladder have specific known functions in the vascular smooth musculature, their function in the bladder is often less well defined. In cancer and obstructive disease, the ECM has a critical role in pathogenesis. The challenge in these settings will be to find therapies that prevent hyperproliferation and encourage proper differentiation, through an understanding of matrix effects on cell biology and susceptibility to therapeutics.

Myogenic differentiation of human bone marrow mesenchymal stem cells on a 3D nano fibrous scaffold for bladder tissue engineering

Current strategies for engineering bladder tissues include a bladder biopsy for in vitro cell expansion for use in reconstructive procedures. However, this approach cannot be used in patients with bladder cancer who need a complete bladder replacement. Bone marrow mesenchymal stem cells (BMSC) might be an alternative cell source to better meet this need. We investigated the effects of soluble growth factors, bladder extracellular matrix (ECM), and 3D dynamic culture on cell proliferation and differentiation of human BMSC into smooth muscle cells (SMC). Myogenic growth factors (PDGF-BB and TGF-β1) alone, or combined either with bladder ECM or dynamic cultures, induced BMSC to express smooth muscle-specific genes and proteins. Either ECM or the dynamic culture alone promoted cell proliferation but did not induce myogenic differentiation of BMSC. A highly porous poly- l-lactic acid (PLLA) scaffold provided a 3D structure for maximizing the cell-matrix penetration, maintained myogenic differentiation of the induced BMSC, and promoted tissue remolding with rich capillary formation in vivo. Our results demonstrate that myogenic-differentiated BMSC seeded on a nano fibrous PLLA scaffold can be potentially used for cell-based tissue engineering for bladder cancer patients requiring cystoplasty.

Macrophage phenotype and remodeling outcomes in response to biologic scaffolds with and without a cellular component

Recently, macrophages have been characterized as having an M1 or M2 phenotype based on receptor expression, cytokine and effector molecule production, and function. The effects of macrophage phenotype upon tissue remodeling following the implantation of a biomaterial are largely unknown. The objectives of this study were to determine the effects of a cellular component within an implanted extracellular matrix (ECM) scaffold upon macrophage phenotype, and to determine the relationship between macrophage phenotype and tissue remodeling. Partial-thickness defects in the abdominal wall musculature of Sprague–Dawley rats were repaired with autologous body wall tissue, acellular allogeneic rat body wall ECM, xenogeneic pig urinary bladder tissue, or acellular xenogeneic pig urinary bladder ECM. At 3, 7, 14, and 28 days the host tissue response was characterized using histologic, immunohistochemical, and RT-PCR methods. The acellular test articles were shown to elicit a predominantly M2 type response and resulted in constructive remodeling, while those containing a cellular component, even an autologous cellular component, elicited a predominantly M1 type response and resulted in deposition of dense connective tissue and/or scarring. We conclude that the presence of cellular material within an ECM scaffold modulates the phenotype of the macrophages participating in the host response following implantation, and that the phenotype of the macrophages participating in the host response appears to be related to tissue remodeling outcome.

Enhanced recovery of mechanical function in the canine heart by seeding an extracellular matrix patch with mesenchymal stem cells committed to a cardiac lineage

The need to regenerate tissue is paramount, especially for the heart that lacks the ability to regenerate after injury. The urinary bladder extracellular matrix (ECM), when used to repair a right ventricular defect, successfully regenerated some mechanical function. The objective of the current study was to determine whether the regenerative effect of ECM could be improved by seeding the patch with human mesenchymal stem cells (hMSCs) enhanced to differentiate down a cardiac linage. hMSCs were used to form three-dimensional spheroids. The expression of cardiac proteins was determined in cells exposed to the spheroid formation and compared with nonmanipulated hMSCs. To determine whether functional calcium channels were present, the cells were patch clamped. To evaluate the ability of these cells to regenerate mechanical function, the spheroids were seeded on ECM and then implanted into the canine heart to repair a full-thickness right ventricular defect. As a result, many of the cells spreading from the spheroids expressed cardiac-specific proteins, including sarcomeric alpha-actinin, cardiotin, and atrial natriuretic peptide, as well as the cell cycle markers cyclin D1 and proliferating cell nuclear antigen. A calcium current similar in amplitude to that of ventricular myocytes was present in 16% of the cells. The cardiogenic cell-seeded scaffolds increased the regional mechanical function in the canine heart compared with the unmanipulated hMSC-seeded scaffolds. In addition, the cells prelabeled with fluorescent markers demonstrated myocyte-specific actinin staining with sarcomere spacing similar to that of normal myocytes. In conclusion, the spheroid-derived cells express cardiac-specific proteins and demonstrate a calcium current similar to adult ventricular myocytes. When these cells are implanted into the canine heart, some of these cells appear striated and mechanical function is improved compared with the unmanipulated hMSCs. Further investigation will be required to determine whether the increased mechanical function is due to a differentiation of the cardiogenic cells to myocytes or to other effects.

Development of a Model Bladder Extracellular Matrix Combining Disulfide Cross‐Linked Hyaluronan with Decellularized Bladder Tissue

[Image: see text] In this work we investigate the feasibility of modifying porcine-derived BAM to include HA with a view to developing a model, artificial extracellular matrix for the study of bladder cell-matrix interactions. HA-DPTH was incorporated into BAM disks and then cross-linked oxidatively to a disulfide containing hydrogel. Disks were seeded with bladder smooth muscle cells (BSMC) and UEC under three culture configurations and incubated for 3, 7, and 14 d. At each time point, matrix contraction was measured, and media supernatants assayed for cell-secreted gelatinase activity. To evaluate cell adherence and organization, triple immunofluorescent labeling of cell nuclei, actin cytoskeleton, and focal contacts was performed. HA-modified BAM exhibited a significant increase in matrix contraction and induced a higher level of cell-secreted gelatinase activity compared to unmodified BAM. Immunofluorescent labeling demonstrated that BSMCs remained adherent to both scaffold types over time. The distribution and organization of the cytoskeleton and focal contacts did not appear to be altered by the presence of HA. Interestingly, cellular infiltration into modified BAM was evident by 7 d and continued beyond 14 d, while BSMCs seeded onto unmodified BAM remained localized to the surface out to 14 d, with minimal infiltration evident only at day 28. These differences in cell infiltration support the gelatinase activity results. Increases in cell migration and matrix proteolysis in the presence of HA may be contributing factors toward BAM remodeling leading to increased matrix contraction with time. The model ECM developed in this work will be utilized for future studies aimed at elucidating the mechanisms controlling key remodeling events associated with bladder repair. Matrix contraction of cell-seeded BAM scaffolds.

The Basement Membrane Component of Biologic Scaffolds Derived from Extracellular Matrix

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.

Fiber architecture and biaxial mechanical behavior of porcine urinary bladder extracellular matrix

Fiber architecture and biaxial mechanical behavior of porcine urinary bladder extracellular matrix

Integrins expressed with bladder extracellular matrix after stretch injury in vivo mediate bladder smooth muscle cell growth in vitro.

It is unknown how bladder smooth muscle cells sense extrinsic mechanical stimuli. The integrins are a large versatile family of transmembrane mechanoreceptors that transduce extracellular matrix (ECM) alterations into the cell, thereby, regulating proliferation, differentiation and ECM synthesis. To our knowledge we provide the first evidence that the integrins may be involved in responses to whole bladder distention and bladder smooth muscle cell stretch. Bladders from 100 to 120 gm. rats were stretched to 40 cm. H2O for 5 minutes. Five to 96 hours after distention whole bladder mRNAs were isolated for analysis of temporal expression of collagen and integrin genes. Separately quiescent primary culture bladder smooth muscle cells from 1-day-old Sprague-Dawley rats were stretched cyclically for 4 hours. Relative expression of select integrin subunit mRNAs was assessed by semiquantitative reverse transcriptase-polymerase chain reaction. Integrin blockade with asparagine-glycine-arginine peptides was used to determine the role of integrins in stretch induced proliferation and the cell cycle in bladder smooth muscle cells. Within 24 hours bladder distention stimulated collagen expression 2-fold (type I) and 5-fold (type III). Collagen levels beyond 24 hours were 8-fold (type I) and 2-fold (type III) greater than in controls, revealing an inverse temporal type I-to-III ratio beyond 24 hours. Coordinate alterations were observed in integrin and collagen expression. In vitro bladder smooth muscle cell integrin beta1, beta3 and alphav subunit expression was increased by mechanical stretch 2.5, 3.8 and 5-fold, respectively, while alpha1 expression decreased. Asparagine-glycine-arginine peptide inhibition of integrin function significantly inhibited stretch induced bladder smooth muscle cell proliferation and exit from the G2/M phase of the cell cycle. To our knowledge these results demonstrate for the first time that that bladder distention initiates dynamic alterations in ECM expression. The ability of integrin blockade to suppress stretch induced bladder smooth muscle cell proliferation and the coordinate changes in bladder ECM and integrin expression suggest that integrins mediate key responses to mechanical stimuli in the bladder. Furthermore, cell cycle analysis of resting and stretched bladder smooth muscle cells revealed novel avenues for the examination of integrin and stretch regulation of bladder smooth muscle cell growth.

Letter Regarding the Article by Xue et al, “Functional Integration of Electrically Active Cardiac Derivatives From Genetically Engineered Human Embryonic Stem Cells With Quiescent Recipient Ventricular Cardiomyocytes”

To the Editor: The article by Xue et al1 on human embryonic stem cell-derived pacemakers illustrates that embryonic stem cells differentiated into spontaneously beating cardiocytes may function as biological pacemakers and mentions a potential limitation: The intrinsic pacemaker rate was slower than desirable. They suggest that incorporating HCN pacemaker channel genes might achieve more desirable rates, an idea consistent with our published results using HCN2 in gene- and adult human mesenchymal stem cell (hMSC)-based therapies.2–4 However, certain of the comments by Xue et al misinterpret our own work on HCN-loaded hMSCs. They state that “…these modified, undifferentiated, human mesenchymal stem cells are incapable of pacing quiescent cells because the former are neither electrically active nor genuine cardiac cells” (p 19). This statement suggests a misunderstanding of the rationale and underlying biophysics of the hMSC experiments. In fact, generation of pacemaker activity does not require delivery of an “excitable differentiated cardiac cell,” but only that the delivered cell (1) carry sufficient pacemaker current and (2) make gap junctions; thus, the hMSC-myocyte pair should behave as a pacemaker unit entirely equivalent to a single heart cell with substantial if. We clearly demonstrated both …

Myocardial tissue replacement with extracellular matrix scaffolds

Backaround Heart failure from Ml is a major health problem. Recent evidence suggests myocardiaf repair and recovery of LV function might be possible through tissue engineering such as autologous skeletal myoblast transplant. An alternative strategy is repair of infarcted regions with acellular biological matrix scaffolds.. -We implanted porcine urinary bladder extracellular matrix scaffolds (UBM) as full-thickness RV outflow tract replacement patches in 4 pigs and 1 dog; 1 pig received glutaraldehyde-treated pericardium as control. At 6 wk gross and microscopic structure was studied; immunohistochemistry was performed to assess cellular recruitment. m No postoperative signs of cardiac insufficiency were seen. Implant sates were pearly white at the epicardial aspect. Endocardial surfaces of UBM showed pearly white tissue interdigitated with abundant brown-colored tissue whereas GP showed mostly pearly white with sparse brown tissue. GP was a discrete foreign body in a fibrotic capsule with extensive inflammation including many giant cells. UBM was undiscernrble and the site was mixed tissue including myocardium, fibrosis, and extensive neovascularization. UBM immunohistochemistry showed myeloid cells at the endocardial aspect. Cells expressing muscle actin, with two morphologies were seen in UBM sites: stellateispindle-shaped resembling myofibrobfasts, and striated cells resembling cardiomyocytes. Neovascularization with vWfand smooth muscle actin-positive cells was abundant. Extensive replication was evident by PCNA immunostaining. The endocardium was vWf-positive endothelial cells. Conchsions UBM implants were incorporated into RV free wall by a tissue replacement process involving recruitment of circulating cells as well as appearance of muscle cells that may derive from adjacent myocardium. In contrast, GP was incorporated into a fibrotic capsule without a replacement process. Further research into potential utility of UBM for myocardial tissue repair and mechanisms of the replacement process is warranted.

Integrins Expressed With Bladder Extracellular Matrix After Stretch Injury In Vivo Mediate Bladder Smooth Muscle Cell Growth In Vitro

Integrins Expressed With Bladder Extracellular Matrix After Stretch Injury In Vivo Mediate Bladder Smooth Muscle Cell Growth In Vitro

Integrins Expressed With Bladder Extracellular Matrix After Stretch Injury In Vivo Mediate Bladder Smooth Muscle Cell Growth In Vitro

Integrins Expressed With Bladder Extracellular Matrix After Stretch Injury In Vivo Mediate Bladder Smooth Muscle Cell Growth In Vitro

Extracellular matrix and nuclear localization of beta ig-h3 in human bladder smooth muscle and fibroblast cells.

The extracellular matrix (ECM) plays an essential role in bladder structure and function. In this study, expression of beta ig-h3, a recently identified extracellular matrix protein, was investigated in human bladder tissue, and human bladder smooth-muscle (SMC) and fibroblast cells in vitro. SMCs secreted greater than three times the level of this protein compared with fibroblasts. The relative levels of beta ig-h3 mRNA in the two cell types reflected the protein expression. Immunohistochemical analysis demonstrated protein deposition in the ECM as well as cytoplasmic localization and, unexpectedly, nuclei. Anti-beta ig-h3 antibodies also stained the matrix surrounding the detrusor SMCs and nuclei of bladder fibroblasts, SMCs, and urothelium in intact bladder tissue. Western blot analyses of medium and matrix fractions obtained from cells in vitro revealed protein of approximately 70-74 kDa, whereas nuclear extracts contained a 65-kDa reactive protein band. We propose that although this protein is a structural component of bladder ECM, its nuclear localization suggests that it has other regulatory and/or structural functions.

Extracellular matrix and nuclear localization of βig-h3 in human bladder smooth muscle and fibroblast cells

The extracellular matrix (ECM) plays an essential role in bladder structure and function. In this study, expression of beta ig-h3, a recently identified extracellular matrix protein, was investigated in human bladder tissue, and human bladder smooth-muscle (SMC) and fibroblast cells in vitro. SMCs secreted greater than three times the level of this protein compared with fibroblasts. The relative levels of beta ig-h3 mRNA in the two cell types reflected the protein expression. Immunohistochemical analysis demonstrated protein deposition in the ECM as well as cytoplasmic localization and, unexpectedly, nuclei. Anti-beta ig-h3 antibodies also stained the matrix surrounding the detrusor SMCs and nuclei of bladder fibroblasts, SMCs, and urothelium in intact bladder tissue. Western blot analyses of medium and matrix fractions obtained from cells in vitro revealed protein of approximately 70-74 kDa, whereas nuclear extracts contained a 65-kDa reactive protein band. We propose that although this protein is a structural component of bladder ECM, its nuclear localization suggests that it has other regulatory and/or structural functions.

Changes in bladder extracellular matrix--overview.

For starting this session I have a couple of slides that will help me in giving a brief overview of this topic. This is one of those conceptual overviews, and not one that is rich with data. What we really ought to think about is the fact that the bladder, the way we normally think about it, tends to be a visual simplification. We think of it in terms from our textbooks of histology as a layer of glycosaminoglycans, mucosa, lamina propia, and muscle. So we always think about the cells we associate with bladder structure. However as we have seen over these past two days, there is a lot more to it than just these cell populations. So if you will just think about this analogy; think about this room here. This room has a nice aesthetically pleasing ceiling, nice wall paper, pictures, and all of those things are equivalent to those cells within the bladder wall that we normally look at. But what is behind these walls. There is steel within these walls that we don’t see that extends on down to the foundation to provide the ultimate structural support and that is indeed what the extracellular matrix is. It is the supporting matrix for the cells, and I just borrowed this slide from Paul Ehrlich because it shows so nicely what we are talking about. You have the matrix, in this case collagen, that is the scaffolding or the steel structure of the bladder. Then you have the cells that are intimately associated with it in such as way that you can’t always tell where the cell ends and the collagen begins.