Defining invasion in breast cancer: the role of basement membrane.

Basement membrane (BM) is an amorphous, sheet-like structure separating the epithelium from the stroma. BM is characterised by a complex structure comprising collagenous and non-collagenous proteoglycans and glycoproteins. In the breast, the thickness, density and composition of the BM around the ductal lobular system vary during differing development stages. In pathological conditions, the BM provides a physical barrier that separates proliferating intraductal epithelial cells from the surrounding stroma, and its absence or breach in malignant lesions is a hallmark of invasion and metastases. Currently, diagnostic services often use special stains and immunohistochemistry (IHC) to identify the BM in order to distinguish in situ from invasive lesions. However, distinguishing BM on stained sections, and differentiating the native BM from the reactive capsule or BM-like material surrounding some invasive malignant breast tumours is challenging. Although diagnostic use of the BM is being replaced by myoepithelial cell IHC markers, BM is considered by many to be a useful marker to distinguish in situ from invasive lesions in ambiguous cases. In this review, the structure, function and biological and clinical significance of the BM are discussed in relation to the various breast lesions with emphasis on how to distinguish the native BM from alternative pathological tissue mimicking its histology.

Mimicking the Natural Basement Membrane for Advanced Tissue Engineering.

Advancements in the field of tissue engineering have led to the elucidation of physical and chemical characteristics of physiological basement membranes (BM) as specialized forms of the extracellular matrix. Efforts to recapitulate the intricate structure and biological composition of the BM have encountered various advancements due to its impact on cell fate, function, and regulation. More attention has been paid to synthesizing biocompatible and biofunctional fibrillar scaffolds that closely mimic the natural BM. Specific modifications in biomimetic BM have paved the way for the development of in vitro models like alveolar-capillary barrier, airway models, skin, blood-brain barrier, kidney barrier, and metastatic models, which can be used for personalized drug screening, understanding physiological and pathological pathways, and tissue implants. In this Review, we focus on the structure, composition, and functions of in vivo BM and the ongoing efforts to mimic it synthetically. Light has been shed on the advantages and limitations of various forms of biomimetic BM scaffolds including porous polymeric membranes, hydrogels, and electrospun membranes This Review further elaborates and justifies the significance of BM mimics in tissue engineering, in particular in the development of in vitro organ model systems.

Designing Cardiovascular Implants Taking in View the Endothelial Basement Membrane.

Insufficient endothelialization of cardiovascular grafts is a major hurdle in vascular surgery and regenerative medicine, bearing a risk for early graft thrombosis. Neither of the numerous strategies pursued to solve these problems were conclusive. Endothelialization is regulated by the endothelial basement membrane (EBM), a highly specialized part of the vascular extracellular matrix. Thus, a detailed understanding of the structure-function interrelations of the EBM components is fundamental for designing biomimetic materials aiming to mimic EBM functions. In this review, a detailed description of the structure and functions of the EBM are provided, including the luminal and abluminal interactions with adjacent cell types, such as vascular smooth muscle cells. Moreover, in vivo as well as in vitro strategies to build or renew EBM are summarized and critically discussed. The spectrum of methods includes vessel decellularization and implant biofunctionalization strategies as well as tissue engineering-based approaches and bioprinting. Finally, the limitations of these methods are highlighted, and future directions are suggested to help improve future design strategies for EBM-inspired materials in the cardiovascular field.

Basement membrane proteins in various arterial beds from individuals with and without type 2 diabetes mellitus: a proteome study.

BACKGROUND: Basement membrane (BM) accumulation is a hallmark of micro-vessel disease in diabetes mellitus (DM). We previously reported marked upregulation of BM components in internal thoracic arteries (ITAs) from type 2 DM (T2DM) patients by mass spectrometry. Here, we first sought to determine if BM accumulation is a common feature of different arteries in T2DM, and second, to identify other effects of T2DM on the arterial proteome. METHODS: Human arterial samples collected during heart and vascular surgery from well-characterized patients and stored in the Odense Artery Biobank were analysed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We included ascending thoracic aortas (ATA) (n = 10 (type 2 DM, T2DM) and n = 10 (non-DM)); laser capture micro-dissected plaque- and media compartments from carotid plaques (n = 10 (T2DM) and n = 9 (non-DM)); and media- and adventitia compartments from ITAs (n = 9 (T2DM) and n = 7 (non-DM)). RESULTS: We first extended our previous finding of BM accumulation in arteries from T2DM patients, as 7 of 12 pre-defined BM proteins were significantly upregulated in bulk ATAs consisting of > 90% media. Although less pronounced, BM components tended to be upregulated in the media of ITAs from T2DM patients, but not in the neighbouring adventitia. Overall, we did not detect effects on BM proteins in carotid plaques or in the plaque-associated media. Instead, complement factors, an RNA-binding protein and fibrinogens appeared to be regulated in these tissues from T2DM patients. CONCLUSION: Our results suggest that accumulation of BM proteins is a general phenomenon in the medial layer of non-atherosclerotic arteries in patients with T2DM. Moreover, we identify additional T2DM-associated effects on the arterial proteome, which requires validation in future studies.

Investigation of the effects of laminin present in the basal lamina of the peripheral nervous system on axon regeneration and remyelination using the nerve acellular scaffold.

This study aimed to investigate the effects of laminin (LN) located in the basal lamina, which are important components of the peripheral nervous system-extracellular matrix, on axon regeneration and remyelination. Nerve acellular scaffolds (NASs) (S-untreated) were prepared using the acellular technique. The active component LN in the NASs was blocked (S-LN- ) or upregulated (S-LN+ ); S-LN+ contained seven times more LN than did the S-untreated group. The adhesion capacity of Schwann cells (SCs) to the three types of NAS (S-untreated, S-LN- , and S-LN+ ) was assessed in vitro. Our results showed that the adhesion of SCs to the NASs was significantly reduced in the S-LN- group, whereas no difference was observed between the S-LN+ and S-untreated groups. The pretreated NASs were used to repair nerves in a nerve injury mouse model with the animals divided into four groups (S-LN- group, S-untreated group, S-LN+ group, and autograft group). Two weeks after surgery, although there was no difference in the S-LN- group, S-untreated group and S-LN+ group, the newly formed basal lamina in the S-LN- group were significantly lower than those in the other two groups. Four weeks after surgery, the S-LN+ group had higher numbers of newly generated axons and their calibers, more myelinated fibers, thicker myelin sheaths, increased myelin basic protein expression, and improved recovery of neural function compared to those of the S-LN- and S-untreated groups, but all of these parameters were significantly worse than those of the autograft group. Downregulation of the LN level in the NAS leads to a reduction in all of the above parameters.

Autoantibody Profile of a Cohort of 54 Italian Patients with Linear IgA Bullous Dermatosis: LAD-1 Denoted as a Major Auto-antigen of the Lamina Lucida Subtype.

Linear IgA bullous dermatosis (LABD) is characterized by presence of multiple IgA autoantibodies, and a comparatively lesser number of IgG antibodies, directed against different hemidesmosomal antigens. The main autoantigens are LAD-1, LABD-97, BP180 and BP230, type VII collagen and laminin 332. We retrospectively studied the serology of 54 Italian patients with LABD using enzyme-linked immunosorbent assay (ELISA), immunoblotting assay, and indirect immunofluorescence on monkey oesophagus and salt-split skin. Among these, indirect immunofluorescence of salt-split skin elicits the greatest sensitivity. Sixty-three percent of the sera were observed to be positive, with a lamina lucida pattern observed in 48%, a sub-lamina densa pattern in 2% and a mixed pattern in 13% of the cases. IgA reactivity to LAD-1 on immunoblotting was found in 52% of sera, to BP180-NC16A by ELISA in 32% and to BP230 in 26%. Only 17% of patients possessed circulating IgG autoantibodies. LAD-1 was determined to be a major autoantigen of the lamina lucida subtype. Combined serological assays demonstrated a high sensitivity (82%), suggesting that this approach could support diagnosis when a biopsy is not feasible or direct immunofluorescence results are negative.

Electron Microscopic and Immunohistochemical Findings of the Epidermal Basement Membrane in Two Families with Nail-patella Syndrome.

Nail-patella syndrome is an autosomal dominant disorder characterized by nail dysplasia and skeletal anomaly. Some patients have been shown to have ultrastructural abnormalities of the glomerular basement membrane that result in nephrosis. However, little has been reported on the epidermal basement membrane in this condition. This paper reports 2 families with nail-patella syndrome. Direct sequencing analysis of LMX1B revealed that family 1 and family 2 were heterozygous for the mutations c.140-1G>C and c.326+1G>C, respectively. To evaluate the epidermal basement membrane zone, ultrastructural and immunohistochemical analyses were performed using skin specimens obtained from the dorsal thumb. Electron microscopy showed intact hemidesmosomes, lamina lucida, lamina densa, and anchoring fibrils. Immunofluorescence studies with antibodies against components of the epidermal basement membrane zone revealed a normal expression pattern among the components, including type IV collagen. These data suggest that nail dysplasia in patients with nail-patella syndrome is not caused by structural abnormalities of the epidermal basement membrane.

Urea-De-Epithelialized Human Amniotic Membrane for Ocular Surface Reconstruction.

The conjunctiva is a clear tissue covering the white part of the eye and lines the back of the eyelids. Conjunctival diseases, such as symblepharon, cause inflammation, discharges, and photophobia. The treatment often requires excision of large parts of conjunctiva. Tissue engineering of conjunctival cells using human amniotic membrane (HAM) denuded of its epithelium as a basement membrane scaffold has been shown to be effective for covering conjunctival defects. However, most epithelial denudation protocols are time-consuming and expensive or compromise HAM's basement membrane structure and matrix components. We have previously described a method to de-epithelialize HAM using ice-cold urea (uHAM). In this report, we used this method to provide tissue-engineered constructs with cultivated conjunctival epithelial cells on uHAM in two patients, one with a giant conjunctival nevus and the other with a large symblepharon. Autologous conjunctival epithelial cells harvested from incisional biopsies of these two patients were cultured on the uHAM scaffold. The transplantation of tissue-engineered constructs to patients' ocular surface immediately after the removal of lesions showed successful reconstruction of the ocular surface. Postoperatively, there were neither recurrence of lesions nor epithelial defects throughout the follow-up (up to 7 and 19 months, respectively). This report highlights the translational potential of an efficient and inexpensive method to prepare de-epithelialized HAM as a basement membrane scaffold for cell-based tissue-engineered treatments of ocular surface disorders. Stem Cells Translational Medicine 2019;8:620&626.

Development of an apoptosis-assisted decellularization method for maximal preservation of nerve tissue structure.

Preservation of tissue structure is often a primary goal when optimizing tissue and organ decellularization methods. Many current protocols nonetheless rely on detergents that aid extraction of cellular components but also damage tissue architecture. It may be more beneficial to leverage an innate cellular process such as apoptosis and promote cell removal without the use of damaging reagents. During apoptosis, a cell detaches from the extracellular matrix, degrades its internal components, and fragments its contents for easier clearance. We have developed a method that leverages this process to achieve tissue decellularization using only mild wash buffers. We have demonstrated that treating peripheral nerve tissue with camptothecin induced both an early marker of apoptosis, cleaved caspase-3 expression, as well as a late stage marker, TUNEL+ DNA fragmentation. Clearance of the cellular components was then achieved in an apoptosis-dependent manner using a gentle wash in hypertonic phosphate buffered saline followed by DNase treatment. This wash paradigm did not significantly affect collagen or glycosaminoglycan content, but it was sufficient to remove any trace of the cytotoxic compound based on conditioned media experiments. The resulting acellular tissue graft was immunogenically tolerated in vivo and exhibited an intact basal lamina microarchitecture mimicking that of native, unprocessed nerve. Hence, ex vivo induction of apoptosis is a promising method to decellularize tissue without the use of harsh reagents while better preserving the benefits of native tissue such as tissue-specific composition and microarchitecture. STATEMENT OF SIGNIFICANCE: Tissue decellularization has expanded the ability to generate non-immunogenic organ replacements for a broad range of health applications. Current technologies typically rely on the use of harsh agents for clearing cellular debris, altering the tissue structure and potentially diminishing the pro-regenerative effects. We have developed a method for effectively, yet gently, removing cellular components from peripheral nerve tissue while preserving the native tissue architecture. The novelty of this process is in the induction of programmed cell death - or apoptosis - via a general cytotoxin, thereby enabling antigen clearance using only hypertonic wash buffers. The resulting acellular nerve scaffolds are nearly identical to unprocessed tissue on a microscopic level and elicit low immune responses comparable to an isograft negative control in a model of subcutaneous implantation.

Comparative adsorption profiles of basal lamina proteome and gingival cells onto dental and titanium surfaces.

Titanium (Ti) dental implants are susceptible to bacterial infections and failure due to lack of proper epithelial seal. Epithelial cells establish a strong epithelial seal around natural teeth by the deposition of basal lamina (BL) proteins that adsorb on the tooth surface. This seal can even be re-established onto cementum or dentin following injury or periodontal therapy. However, it is unclear how tooth surfaces promote this cell attachment and protein adsorption. Understanding the interactions between BL proteins and epithelial cells with dentin and Ti will facilitate the development of implant surfaces that promote the formation of an epithelial seal and improve the success of periodontal therapy and wound healing on natural teeth. To study these interactions, we used a surface proteomic approach to decipher the adsorption profile of BL proteins onto Ti and dentin, and correlated these adsorption profiles with in vitro interactions of human gingival fibroblasts and epithelial cells. Results showed that dentin adsorbed higher amounts of key BL proteins, particularly laminin and nidogen-1, and promoted more favorable interactions with epithelial cells than Ti. Next, dentin specimens were deproteinized or partially demineralized to determine if its mineral or protein component was responsible for BL adsorption and cell attachment. Deproteinized (mineral-rich) and partially demineralized (protein-rich) dentin specimens revealed BL proteins (i.e. laminin and nidogen-1) and epithelial cells interact preferentially with dentinal proteins rather than dentin mineral. These findings suggest that, unlike Ti, dentin and, in particular, dentinal proteins have a selective affinity to BL proteins that enhance epithelial cell attachment. STATEMENT OF SIGNIFICANCE: It is remains unclear why natural teeth, unlike titanium dental implants, promote the formation of an epithelial seal that protects them against the external environment. This study used a surface screening approach to analyze the adsorption of proteins produced by epithelial tissues onto tooth-dentin and titanium surfaces, and correlate it with the behaviour of cells. This study shows that tooth-dentin, in particular its proteins, has a higher selective affinity to certain adhesion proteins, and subsequently allows more favourable interactions with epithelial cells than titanium. This knowledge could help in developing new approaches for re-establishing and maintaining the epithelial seal around teeth, and could pave the way for developing implants with surfaces that allow the formation of a true epithelial seal.

Circulating Vascular Basement Membrane Fragments are Associated with the Diameter of the Abdominal Aorta and Their Expression Pattern is Altered in AAA Tissue.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is characterised by enhanced proteolytic activity, and extracellular matrix (ECM) remodelling in the vascular wall. Type IV and XVIII collagen/endostatin are structural proteins in vascular basement membrane (VBM), a specialised ECM structure. Here the association between plasma levels of these collagens with the aortic diameter and expansion rate is studied, and their expression in aortic tissue characterised. METHODS: This was a retrospective population based cohort study. Type IV and XVIII collagen/endostatin were analysed in plasma by ELISA assay in 615 men, divided into three groups based on the aortic diameter: 1) normal aorta ≤ 25 mm, 2) sub-aneurysmal aorta (SAA) 26-29 mm, and 3) AAA ≥ 30 mm. Follow up data were available for 159 men. The association between collagen levels and aortic diameter at baseline, and with the expansion rate at follow up were analysed in ordinal logistic regression and linear regression models, controlling for common confounding factors. Tissue expression of the collagens was analysed in normal aorta (n = 6) and AAA (n = 6) by immunofluorescence. RESULTS: Plasma levels of type XVIII collagen/endostatin (136 ng/mL [SD 29] in individuals with a normal aorta diameter, 154 ng/ml [SD 45] in SAA, and 162 ng/ml [SD 46] in AAA; p = .001) and type IV collagen (105 ng/mL [SD 42] normal aorta, 124 ng/ml [SD 46] SAA, and 127 ng/ml [SD 47] AAA; p = .037) were associated with a larger aortic diameter. A significant association was found between the baseline levels of type XVIII/endostatin and the aortic expansion rate (p = .035), but in the multivariable model, only the initial aortic diameter remained significantly associated with expansion (p = .005). Altered expression patterns of both collagens were observed in AAA tissue. CONCLUSION: Plasma levels of circulating type IV and XVIII collagen/endostatin increase with AAA diameter. The expression pattern of VBM proteins is altered in the aneurysm wall.

[Basement membrane-derived matricryptins as a new target molecule for heart failure treatment].

Extracellular matrix (ECM) is a complex of non-cellular macromolecules which is indispensable not only for maintaining tissue structure but also for regulating the functions of surrounding cells via cell-ECM interaction. Recently, a number of ECM fragments termed "matricryptins" have been identified as novel endogenous bioactive substances, which usually have different bioactivity from that of original ECM. Because most matricryptins derived from basement membrane have anti-angiogenic and anti-tumor effects, they have been initially studied for developing anti-tumor agents. Meanwhile, there are several reports indicating that the expression of basement membrane-derived matricryptins changes in the heart tissue from experimental animal models as well as in the circulating blood from patients with cardiac diseases. Thus, it is logical to hypothesize that the basement membrane-derived matricryptins play roles in the development of cardiac diseases. In this article, we would like to introduce current findings on the functions of matricryptins including ours and discuss the possibility that they serve as a novel target for the treatment of heart failure.

Multiple and repeated sampling increases the sensitivity of direct immunofluorescence testing for the diagnosis of mucous membrane pemphigoid.

BACKGROUND: Mucous membrane pemphigoid (MMP) is an autoimmune disease characterized by the predominant blistering of mucosal surfaces and the linear deposition of complement, IgG, or IgA along the basement membrane detected by direct immunofluorescence (DIF) test. OBJECTIVE: To assess the impact of multiple and repeated DIF sampling on establishing the diagnosis of MMP. METHODS: We reviewed the results of DIF studies in 136 nonlesional biopsies from 78 patients who were immunologically confirmed to have MMP. RESULTS: Thirty-six of 52 patients (69%) who underwent only 1 biopsy at the first workup were positive. In 13 cases, the initial single biopsy was negative, and later biopsies were positive. Twenty-two of 26 patients (85%) who underwent multiple biopsies at the initial workup showed ≥1 positive DIF test result. Simultaneously obtained biopsies yielded discordant positive and negative findings in 11 patients. Overall, 74 of 78 patients (95%) had ≥1 positive result by DIF test. In the remaining 4 cases, the diagnosis was confirmed by the detection of circulating autoantibodies against BP180. LIMITATIONS: This is a retrospective, single-center study. CONCLUSION: Our data demonstrate that multiple and repeated biopsies increase the sensitivity of the DIF test for MMP diagnosis. Negative DIF test findings in cases clinically suggestive of MMP should prompt repeat biopsies.

A bioartificial environment for kidney epithelial cells based on a supramolecular polymer basement membrane mimic and an organotypical culture system.

Renal applications in healthcare, such as renal replacement therapies and nephrotoxicity tests, could potentially benefit from bioartificial kidney membranes with fully differentiated and functional human tubular epithelial cells. A replacement of the natural environment of these cells is required to maintain and study cell functionality cell differentiation in vitro. Our approach was based on synthetic supramolecular biomaterials to mimic the natural basement membrane (BM) on which these cells grow and a bioreactor to provide the desired organotypical culture parameters. The BM mimics were constructed from ureidopyrimidinone (UPy)-functionalized polymer and bioactive peptides by electrospinning. The resultant membranes were shown to have a hierarchical fibrous BM-like structure consisting of self-assembled nanofibres within the electrospun microfibres. Human kidney-2 (HK-2) epithelial cells were cultured on the BM mimics under organotypical conditions in a custom-built bioreactor. The bioreactor facilitated in situ monitoring and functionality testing of the cultures. Cell viability and the integrity of the epithelial cell barrier were demonstrated inside the bioreactor by microscopy and transmembrane leakage of fluorescently labelled inulin, respectively. Furthermore, HK-2 cells maintained a polarized cell layer and showed modulation of both gene expression of membrane transporter proteins and metabolic activity of brush border enzymes when subjected to a continuous flow of culture medium inside the new bioreactor for 21 days. These results demonstrated that both the culture and study of renal epithelial cells was facilitated by the bioartificial in vitro environment that is formed by synthetic supramolecular BM mimics in our custom-built bioreactor. Copyright © 2015 John Wiley & Sons, Ltd.

Biomimetic, ultrathin and elastic hydrogels regulate human neutrophil extravasation across endothelial-pericyte bilayers.

The vascular basement membrane-a thin, elastic layer of extracellular matrix separating and encasing vascular cells-provides biological and mechanical cues to endothelial cells, pericytes, and migrating leukocytes. In contrast, experimental scaffolds typically used to replicate basement membranes are stiff and bio-inert. Here, we present thin, porated polyethylene glycol hydrogels to replicate human vascular basement membranes. Like commercial transwells, our hydrogels are approximately 10µm thick, but like basement membranes, the hydrogels presented here are elastic (E: 50-80kPa) and contain a dense network of small pores. Moreover, the inclusion of bioactive domains introduces receptor-mediated biochemical signaling. We compare elastic hydrogels to common culture substrates (E: >2GPa) for human endothelial cell and pericyte monolayers and bilayers to replicate postcapillary venules in vitro. Our data demonstrate that substrate elasticity facilitates differences in vascular phenotype, supporting expression of vascular markers that are increasingly replicative of venules. Endothelial cells differentially express vascular markers, like EphB4, and leukocyte adhesion molecules, such as ICAM-1, with decreased mechanical stiffness. With porated PEG hydrogels we demonstrate the ability to evaluate and observe leukocyte recruitment across endothelial cell and pericyte monolayers and bilayers, reporting that basement membrane scaffolds can significantly alter the rate of vascular migration in experimental systems. Overall, this study demonstrates the creation and utility of a new and accessible method to recapture the mechanical and biological complexity of human basement membranes in vitro.

Basement Membrane Mimics of Biofunctionalized Nanofibers for a Bipolar-Cultured Human Primary Alveolar-Capillary Barrier Model.

In vitro reconstruction of an alveolar barrier for modeling normal lung functions and pathological events serve as reproducible, high-throughput pharmaceutical platforms for drug discovery, diagnosis, and regenerative medicine. Despite much effort, the reconstruction of organ-level alveolar barrier functions has failed due to the lack of structural similarity to the natural basement membrane, functionalization with specific ligands for alveolar cell function, the use of primary cells and biodegradability. Here we report a bipolar cultured alveolar-capillary barrier model of human primary cells supported by a basement membrane mimics of fully synthetic bifunctional nanofibers. One-step electrospinning process using a bioresorbable polyester and multifunctional star-shaped polyethylene glycols (sPEG) enables the fabrication of an ultrathin nanofiber mesh with interconnected pores. The nanofiber mesh possessed mechanical stability against cyclic expansion as seen in the lung in vivo. The sPEGs as an additive provide biofunctionality to fibers through the conjugation of peptide to the nanofibers and hydrophilization to prevent unspecific protein adsorption. Biofunctionalized nanofiber meshes facilitated bipolar cultivation of endothelial and epithelial cells with fundamental alveolar functionality and showed higher permeability for molecules compared to microporous films. This nanofiber mesh for a bipolar cultured barrier have the potential to promote growth of an organ-level barrier model for modeling pathological conditions and evaluating drug efficacy, environmental pollutants, and nanotoxicology.

Synthetic hydrogels mimicking basement membrane matrices to promote cell-matrix interactions.

Naturally-derived materials have been extensively used as 3D cellular matrices as their inherent bioactivity makes them suitable for the study of many cellular processes. Nevertheless, lot-to-lot variability, inability to decouple biochemical and biophysical properties and, in some types, their tumor-derived nature limits their translational potential and reliability. One innovative approach to overcome these limitations has focused on incorporating bioactivity into cytocompatible, synthetic hydrogels that present tunable physicochemical properties. This review provides an overview of successful approaches to convey basement membrane-like bioactivity into 3D artificial hydrogel matrices in order to recapitulate cellular responses to native matrices. Recent advances involving biofunctionalization of synthetic hydrogels via incorporation of bioactive motifs that promote cell-matrix interactions and cell-directed matrix degradation will be discussed. This review highlights how the tunable physicochemical properties of biofunctionalized synthetic hydrogel matrices can be exploited to study the separate contributions of biochemical and biophysical matrix properties to different cellular processes.

Proteomic definitions of basement membrane composition in health and disease.

Basement membranes are formed from condensed networks of extracellular matrix (ECM) proteins. These structures underlie all epithelial, mesothelial and endothelial sheets and provide an essential structural scaffold. Candidate-based investigations have established that predominant components of basement membranes are laminins, collagen type IV, nidogens and heparan sulphate proteoglycans. More recently, global proteomic approaches have been applied to investigate ECM and these analyses confirm tissue-specific ECM proteomes with a high degree of complexity. The proteomes consist of structural as well as regulatory ECM proteins such as proteases and growth factors. This review is focused on the proteomic analysis of basement membranes and illustrates how this approach can be used to build our understanding of ECM regulation in health and disease.

The role of human fibronectin- or placenta basement membrane extract-based gels in favouring the formation of polarized salivary acinar-like structures.

Head and neck cancer patients treated with radiotherapy commonly experience hyposalivation and oral/tooth infections, leading to a reduced quality of life. Clinical management is currently unsatisfactory for dry mouth. Thus, there is a need for growing salivary fluid-secreting (acinar) cells for these patients. However, functionally-grown salivary acinar cells are cultured in Matrigel, a product that cannot be used clinically, owing to its source from a mouse sarcoma. Therefore, finding a gel suitable for clinical use and possessing properties similar to that of Matrigel would allow biopsied salivary cells to be expanded in vitro and transplanted into the mouths of xerostomic patients. This study tested gels made with human placenta basement membrane extract (BME) or fibronectin for the growth and differentiation of human salivary biopsies into acinar cells. We report here that, following expansion of primary human salivary gland epithelial cells (huSGs) in serum-free medium, using these gels (made from human proteins) allowed morphological and functional differentiation of salivary ductal cells into acinar-like cells. These (human) gels gave comparable results to Matrigel, such as differentiation into polarized acinar 3D units or monolayers with tight junction proteins (claudin-1, -2, -3) and exhibiting adequate transepithelial electrical resistance, acinar proteins (AQP5, α-amylase, mucin-1, NKCC1) and acinar adhesion-related cell markers (CD44, CD166). Ultrastructural, mRNA and protein analyses confirmed the formation of differentiated acinar polarized cells. The mitotic activity was highest with human placenta BME gel. This human culture model provided a reproducible approach to studying human salivary cell expansion and differentiation for tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.