Basal Lamina Deposition Research Articles

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 SignificanceIt 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.

Improved Corneal Wound Healing through Modulation of Gap Junction Communication Using Connexin43-Specific Antisense Oligodeoxynucleotides

Gap junctions play a major role in corneal wound healing. This study used reproducible models of corneal wound healing to evaluate the effect of a gap junction channel modulator, connexin43 (Cx43) antisense oligodeoxynucleotides (AsODN), on corneal healing dynamics. A mechanical scrape wound model was used to evaluate Cx43 AsODN penetration and initial wound reepithelialization 12 hours postsurgery. Thereafter, detailed analyses of corneal edema, inflammation, and healing were performed in an excimer laser surface ablation model. In vivo confocal microscopy determined clinical parameters (edema, haze) and cellular changes (stromal hypercellularity, reepithelialization), whereas histology and immunohistochemistry were used to quantify stromal edema, inflammation, and reepithelialization. Cx43 AsODN penetrated through the hydrophilic stroma where the epithelium had been removed and accumulated in the basal epithelium close to the wound edge. Twelve hours after scrape wounding, Cx43 AsODN-treated eyes showed a significant reduction in wound area compared with the vehicle alone (1.59±0.37 and 2.29±0.58 mm2, respectively, P<0.01). After excimer laser ablation, stromal edema and inflammation were reduced, with endothelial structures being clearly visible, and reepithelialization rates were again increased in Cx43 AsODN-treated eyes. Histologic analysis confirmed reduced edema in the central wound site and at the periphery of treated corneas (P<0.05), whereas immunohistochemistry showed lower Cx43 levels (P<0.05), reduced myofibroblast activation, and improved epithelial basal lamina deposition in antisense-treated wounds (P<0.01). Application of Cx43 AsODN to the cornea reduces stromal edema and inflammation, promoting faster wound closure and a more uniform repair of the epithelial basal lamina after laser ablation.

Aging and the Hepatic Sinusoidal Endothelium

Old age in many species including humans is associated with substantial thickening and defenestration of the liver sinusoidal endothelial cell, sporadic deposition of collagen and basal lamina in the extracellular space of Disse and increased numbers of fat engorged, non‐activated stellate cells. Associated immunohistochemical age‐related changes include peri‐sinusoidal upregulation of von Willebrands factor, VEGFR‐2, collagen I and IV and ICAM‐1. There is also a small reduction in the fractal dimension of the microvascular architecture but without any change in vascular dispersion. On the other hand, unlike most liver disease, there is no change in expression of alpha‐SMA, desmin or VEGF, reduced expression of caveolin‐1 and stellate cells are not activated. These age‐related changes have been termed pseudocapillarization in order to differentiate them from capillarization seen in cirrhosis. Pseudocapillarization is reversed by caloric restriction indicating it is likely to be a primary aging change. There is an associated impairment of endocytosis. In aged rats the loss of fenestrations leads to impaired transfer of lipoproteins from the sinusoid into the space of Disse. This provides a novel mechanism and therapeutic target for impaired chylomicron remnant clearance and post‐prandial hyperlipidemia associated with old age.

D024 Lysyl oxidase like 2 regulates vascular cells migration and basal lamina organisation

Hypoxia stimulates angiogenesis during development, as well as in the course of ischemic cardiovascular diseases and tumor growth. In a hypoxic environment, endothelial cells (EC) are key players of the angiogenic response. Their activation leads to remodeling of the extracellular matrix (ECM): a degradation step generates a provisional ECM supporting EC proliferation and migration; assembly of a new basal lamina leads to pericyte recruitment and neovessel maturation. In order to identify endothelial ECM proteins regulated by hypoxia, 2D gel electrophoresis was performed on ECM samples prepared from cultured EC of micro or large vessels (HDMEC or HUVEC respectively). Mass spectrometry identified lysyl oxidase-like protein 2 (LOXL2) as a highly hypoxia-induced protein. Lysyl oxidases are secreted enzymes involved in ECM maturation through covalent cross-linking of its major components, collagens and elastin. The induction of LOXL2 expression was detected both at the mRNA and protein levels. Using siRNA, we demonstrated that LOXL2 is responsible for 65 % of lysyl oxidase total activity in hypoxic EC. In addition, LOXL2 protein was colocalised with type IV collagen in endothelial ECM. We further investigated the expression of LOXL2 in vivo. The enzyme was expressed in rat EC from retina during postnatal vascular development, and from adult skeletal muscle. In a murine model of hindlimb ischemia, LOXL2 was upregulated at the protein and mRNA levels. In situ hybridization revealed its expression in both EC and macrophages. Involvement of LOXL2 at different steps of the angiogenic process was further studied in vitro. We demonstrated that LOXL2 increases EC migration on fibronectin, as well as migration and tube formation in fibrin 3D gels. In addition, LOXL2 knock-down inhibited type IV collagen deposition in the ECM, suggesting a major role for LOXL2 in the organisation of endothelial basal lamina. Finally, whereas the efficiency of endothelial ECM for recruiting VSMC was increased by hypoxia, this effect was reduced upon knocking down endothelial LOXL2 expression. Altogether, these data suggest that LOXL2 plays a major role in EC migration, vascular basal lamina deposition and neovessel stabilisation during hypoxia-induced angiogenesis.

Old Age and the Hepatic Sinusoid

Morphological changes in the hepatic sinusoid with old age are increasingly recognized. These include thickening and defenestration of the liver sinusoidal endothelial cell, sporadic deposition of collagen and basal lamina in the extracellular space of Disse, and increased numbers of fat engorged, nonactivated stellate cells. In addition, there is endothelial up-regulation of von Willebrand factor and ICAM-1 with reduced expression of caveolin-1. These changes have been termed age-related pseudocapillarization. The effects of old age on Kupffer cells are inconsistent, but impaired responsiveness is likely. There are functional implications of these aging changes in the hepatic sinusoid. There is reduced sinusoidal perfusion, which will impair the hepatic clearance of highly extracted substrates. Blood clearance of a variety of waste macromolecules takes place in liver sinusoidal endothelial cells (LSECs). Previous studies indicated either that aging had no effect, or reduced the endocytic capacity of LSECs. However, a recent study in mice showed reduced endocytosis in pericentral regions of the liver lobules. Reduced endocytosis may increase systemic exposure to potential harmful waste macromolecules such as advanced glycation end products Loss of fenestrations leads to impaired transfer of lipoproteins from blood to hepatocytes. This provides a mechanism for impaired chylomicron remnant clearance and postprandial hyperlipidemia associated with old age. Given the extensive range of substrates metabolized by the liver, age-related changes in the hepatic sinusoid and microcirculation have important systemic implications for aging and age-related diseases.

Marked changes of the hepatic sinusoid in a transgenic mouse model of acute immune-mediated hepatitis

The liver sinusoidal endothelial cell (LSEC) is increasingly recognized as having an important role in hepatic immunity. However, the responses of LSECs and the hepatic sinusoid in immune-mediated hepatitis are poorly described. We studied a transgenic mouse model of acute immune-mediated hepatitis: Met-Kb mice injected with T cells from Des-TCR mice. Hepatitis was characterized by lymphocyte infiltrates causing severe but transient liver damage. There were marked changes in the ultrastructure of the LSEC five days after injection of the T cells that coincided with the peak of the hepatitis. The porosity of fenestrations in the LSEC decreased and the endothelium became thickened. LSECs appeared to be markedly activated. These changes were associated with narrowing of the space of Disse, loss of hepatocellular microvilli and deposition of basal lamina. Lymphocytes were seen passing through fenestrations. Loss of fenestration in the LSEC prevented hepatitis induced by a second injection of lymphocytes on day 5. Structural changes in the LSEC occur during the peak of a mouse model of immune-mediated hepatitis. These changes were associated with attenuation of subsequent liver damage, suggesting that they may influence immunological responses mediated by LSECs or the passage of lymphocytes through LSEC fenestrations.

Preliminary analysis of the sinusoidal endothelium and space of Disse in ageing Papio hamadrayas

In the past it has been reported that the liver is free of any major age-related morphological changes apart from binucleate hepatocytes and lipofuscin deposition. Diminished hepatic blood flow and mass have been considered to be responsible for the decreased metabolic capacity seen in old age [1]. Recently we studied the ageing rat liver [2] and the ageing human liver [3] and found that there are a number of age-related changes to the perisinusoidal region at the ultrastructural level. These changes are called pseudocapillarization and are characterized by defenestration and thickening of the endothelium, basal lamina deposition and extracellular matrix deposition in the space of Disse (Figure ​(Figure1).1). Expression of von Willebrands factor in the sinusoidal cells in old rats was found together with increased perisinusoidal expression of collagen. In this preliminary study we have examined the livers of young and old baboons to confirm that pseudocapillarization is a generalized ageing change across species. Figure 1 Scanning electron micrographs taken from rats aged 4 months (a) and 24 months (b). The aged endothelium is defenestrated. Bar = 1 –m. Methods The animals used in this study were from a captive breeding colony of Papio hamadrayas. The experimental protocol was approved by the Central Sydney Area Health Service animal welfare committee. Liver tissue was collected by needle biopsy or from fresh post mortem animals. Half of each specimen was fixed for light microscopy and immunohistochemistry in 4% buffered paraformaldehyde and the other half was fixed for electron microscopy with 2% gluteraldehyde/ 3% paraformaldehyde in 0.1 M sodium-cacodylate buffer (0.1 M Sucrose, 2 mM CaCl2). Specimens fixed for light microscopy and immunohistochemistry were embedded in paraffin blocks. Sections from each animal were stained with H & E, Masson's trichrome and Wilder's reticulin. Immunohistochemistry was also used to detect the expression of Collagen IV, Laminin, Synaptophysin, Fibronectin, von Willebrands factor (vWF) and CD68. Tissue for transmission and scanning electron microscopy was prepared by standard methods [4] and randomly sampled for examination. Transmission electron micrographs were taken at 17 000– magnification (Philips CM120) and scanning electron micrographs were taken at 40 000– magnification (Philips XL30).

Age‐related pseudocapillarization of the human liver

Age-related changes in liver function are important because they may promote susceptibility to adverse drug reactions, neurotoxicity, atherosclerosis, and other important diseases in older people. Age-related changes in the rat hepatic sinusoidal endothelium, termed pseudocapillarization, have been described recently and these may contribute to hepatic impairment. The present study has examined surgical and post-mortem specimens with immunohistochemistry and transmission electron microscopy to determine whether pseudocapillarization also occurs in older humans. The age of the subject, independent of systemic disease or hepatic pathology in surgical and post-mortem samples of human liver, was associated with increased peri-sinusoidal expression of von Willebrand's factor, collagen I, collagen IV, and staining with Masson's trichrome. Electron microscopy revealed significant age-related thickening of the sinusoidal endothelium (young 165 +/- 17 nm, middle age 222 +/- 11 nm, older 289 +/- 9 nm, p < 0.001) with loss of fenestrations (young 7.7 +/- 0.7 per 10 micro m, middle age 3.6 +/- 0.5 per 10 micro m, older 1.5 +/- 0.4 per 10 micro m, p < 0.001), and age-related deposition of basal lamina and collagen. In conclusion, ageing in humans is associated with morphological changes in the sinusoidal endothelium and space of Disse which are presumptively related to the ageing process and potentially represent an important link between the ageing process and disease susceptibility.

Temporal progression of angiogenesis and basal lamina deposition after contusive spinal cord injury in the adult rat.

After spinal cord injury (SCI), the absence of an adequate blood supply to injured tissues has been hypothesized to contribute to the lack of regeneration. In this study, blood vessel changes were examined in 28 adult female Fischer 344 rats at 1, 3, 7, 14, 28, and 60 days after a 12.5 g x cm NYU impactor injury at the T9 vertebral level. Laminin, collagen IV, endothelial barrier antigen (SMI71), and rat endothelial cell antigen (RECA-1) immunoreactivities were used to quantify blood vessel per area densities and diameters in ventral gray matter (VGM), ventral white matter (VWM), and dorsal columns (DC) at levels ranging 15 mm rostral and caudal to the epicenter. This study demonstrates an angiogenic response, defined as SMI71/RECA-1-immunopositive endothelial cells that colocalize with a robust deposition of basal lamina and basal lamina streamers, 7 days after injury within epicenter VGM. This angiogenesis diminishes concurrent with cystic cavity formation. GAP43- and neurofilament- (68 kDa and 210 kDa) immunopositive fiber outgrowth was associated with these new blood vessels by day 14. Between 28 and 60 days after injury, increases in SMI71-immunopositive blood vessel densities were observed in the remaining VWM and DC with a corresponding increase in vessel diameters up to 15 mm rostral and caudal to the epicenter. This second angiogenesis within VWM and DC, unlike the acute response observed in VGM, did not correspond to any previously described changes in locomotor behaviors in this model. We propose that therapies targeting angiogenic processes be directed at the interval between 3 and 7 days after SCI.

Localization of focal adhesion kinase in differentiating Schwann cell/neuron cultures.

Previous studies have shown that Schwann cells (SCs) differentiate into myelin-forming or ensheathing cells only under conditions which allow the deposition of basal lamina and extracellular collagen [Bunge (1993) Peripheral Neuropathy, pp. 299-316]. SC adhesion to basal lamina is mediated by beta1 integrins and function blocking antibodies to beta1 integrins inhibit myelination [Fernandez-Valle et al. (1993) Development 119:867-880]. Recently, focal adhesion kinase (FAK), a cytoplasmic non-receptor tyrosine kinase, was found to mediate beta1 integrin-dependent signalling in a variety of cultured cell types adhering to ECM components such as fibronectin [reviewed in Schwartz et al. (1995) Ann. Rev. Cell Biol. 11:549-599; Ilic et al. (1997) J. Cell Sci. 110:401-407]. In the present study, we have determined more precisely the respective time courses of ECM deposition and myelination. In addition, we have studied by immunocytochemistry, immuno-gold labelling, and electron microscopy the expression and subcellular localization of FAK in nondifferentiating SCs and in SCs differentiating into myelinating cells. We show that the development of basal lamina and extracellular collagen fibrils precedes by 3 days the appearance of the first myelin sheaths. FAK was detected by immunocytochemistry or immuno-gold labelling only in SCs differentiating in the presence of ascorbic acid. Localization of FAK to the abaxonal plasma membrane was dependent upon ECM deposition. Cytochalasin D did not prevent or disrupt localization of FAK to the plasma membrane. These data support the possibility that FAK acts as an intermediate in the pathway by which basal lamina regulates SC differentiation.

Addition of Purified Basal Lamina Molecules Enables Schwann Cell Ensheathment of Sympathetic Neurites in Culture

Our aim was to determine the effect of purified basal lamina components on the differentiation of non-myelin-forming Schwann cells accompanying rat sympathetic neurons in culture. Previous work has demonstrated that Schwann cells contacting superior cervical ganglion neurons cultured in the presence of ascorbate and serum fall to effectively deposit matrix and ensheathe neurites unless fibroblasts are also present. We questioned if an increase in the amount of available basal lamina components could mimic fibroblast-induced Schwann cell differentiation. Superior cervical ganglion neuron plus Schwann cell cultures were supplemented with purified basal lamina molecules for up to 7 weeks. The addition of laminin, type IV collagen, heparan sulfate proteoglycan, or a combination of all three components, led to increased basal lamina deposition and increased neurite ensheathment compared with cultures with no additions. In long-term cultures receiving component additions, however, ensheathment was exaggerated, and the Schwann cells were hypertrophic. The ensheathing conformations adopted by these Schwann cells surpassed normal-appearing ensheathment and resembled more unusual unmyelinated nerve morphology found in vivo by others in normal senescence and in several peripheral neuropathies. These experiments show that increased availability of exogenous basal lamina components leads to increased basal lamina deposition and that basal lamina elaboration can have dramatic effects on the morpholgy of nonmyelinating Schwann cells. These findings suggest that influences of extracellular matrix should be considered when unusual Schwann cell/axon conformations are seen in vivo.

Accelerated Structural Maturation Induced by Synapsin I at Developing Neuromuscular Synapses of Xenopus laevis

The role of synapsin I, a synaptic vesicle-associated phosphoprotein, in the maturation of nerve-muscle synapses was investigated in nerve-muscle co-cultures prepared from Xenopus embryos loaded with the protein by the early blastomere injection method. The stage of maturation of the synapses was analysed by electron microscopy as well as by whole-cell patch-clamp recording. The acceleration in the functional maturation of neuromuscular synapses induced by synapsin I was accompanied by a profound rearrangement in the ultrastructure of the nerve terminal. Nerve terminals formed by synapsin I-loaded neurons were characterized by a higher number of small synaptic vesicles organized in clusters and predominantly localized close to the nerve terminal plasma membrane, a smaller number of large dense-core vesicles and no significant change in the number of coated vesicles. Precocious development of active zone-like structures as well as deposition of basal lamina into the synaptic cleft were also observed at these synapses. These results support a role for synapsin I in the architectural changes which occur during synaptogenesis and lead to the maturation of quantal neurotransmitter release mechanisms.

The matrix form of collagen and basal microporosity influence basal lamina deposition and laminin synthesis/secretion by stratified human keratinocytes in vitro.

The ability of the collagen matrix form to support the formation of a basal lamina by cultured normal human epidermal keratinocytes (NHEK) was determined using transmission electron microscopy. The collagen matrix forms tested in this study were a) a dry type I collagen film and b) a type I collagen gel. NHEK were grown for 14 days on the following five different substrates: plain plastic culture dishes without the addition of collagen (PP); plain plastic culture dishes overlaid with a dry, aldehyde-crosslinked type I collagen film (DCF-P); plain plastic culture dishes overlaid with an aldehyde-crosslinked type I collagen gel (GEL-P); Millipore Millicell CM microporous membranes overlaid with a dry, aldehyde-crosslinked type I collagen film (DCF-CM); and Millipore Millicell CM microporous membranes overlaid with an aldehyde-crosslinked type I collagen gel (GEL-CM). NHEK maintained for 2 wk on PP and DCF-P were unable to secrete a basal lamina. NHEK grown for 2 wk on the GEL-P and GEL-CM substrates, however, secreted a contiguous basal lamina at the GEL-NHEK interface. To determine if the appearance of this basal lamina correlated with laminin synthesis, laminin was immunoprecipitated from cellular extracts, as well as media from the apical and basal chambers. NHEK grown on the GEL-P substrate synthesized more laminin than did NHEK grown on the other four alternative substrates. In addition, NHEK grown on GEL-CM were able to direct more laminin to the basal compartment than NHEK grown on DCF-CM substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Germinal matrix microvascular maturation correlates inversely with the risk period for neonatal intraventricular hemorrhage

The risk period for intraventricular hemorrhage (IVH) of the preterm neonate is the first 3–4 postnatal days. For infants of <34 weeks' gestation, this risk period is independent of gestational age. We hypothesized that this risk period is attributable to the perinatal induction of maturation of the germinal matrix microvasculature and tested this hypothesis by examining changes in the classical ultrastructural features of the blood-brain barrier over the first ten postnatal days in the newborn beagle model for neonatal IVH. Newborn beagle pups ( n = 6) were anesthetized and systemically perfused and the brains were removed and prepared for electron microscopic examination. Examination of electron micrographs from the germinal matrix of animals on the first, fourth and tenth postnatal days demonstrated no difference in perimeter lengths and capillary and endothelial cell areas; in contrast, luminal areas significantly decreased across postnatal age ( P = 0.04). Significant increases were found in basement membrane area between days 1 and 4 ( P = 0.01) and tight junction length (day 1 vs. day 10, P = 0.02). In addition, on day 1, 19% of germinal matrix capillary perimeter was determined not to be covered by supporting cell processes, while by day 10, only 5% was bare. In contrast, the microvessels of the white matter exhibited no changes in these parameters during these three time points. These studies are consistent with the concept that basal lamina deposition and organization precede increases in endothelial cell tight junction formation and coverage by supporting cells.

Transforming growth factor-beta blocks myelination but not ensheathment of axons by Schwann cells in vitro

Mechanisms regulating Schwann cell differentiation into a myelinating or a mature nonmyelinating phenotype during development are poorly understood. Humoral factors such as members of the transforming growth factor-beta (TGF-beta) family, which are found in the developing and adult mammalian nervous system and are known to affect cell differentiation, could be involved. We tested the effects of TGF-beta isoforms on the ensheathment and myelination of dorsal root ganglion (DRG) neurons by Schwann cells in vitro. Rat embryonic DRG neurons and Schwann cells from the sciatic nerve were isolated, purified, and recombined. In serum-free conditions, TGF-beta blocked both Schwann cell myelination and the expression of the myelin-related molecules galactocerebroside, P0, myelin-associated glycoprotein, and myelin basic protein. In contrast, the expression of molecules characteristic of mature nonmyelinating Schwann cells, including neural-cell adhesion molecule, L1, and nerve growth factor receptor, was maintained when compared to Schwann cells in nondifferentiated cultures. Notably, the expression of glial fibrillary acidic protein, which is expressed only in mature nonmyelinating Schwann cells in vivo, was increased 10-fold in our cultures by TGF-beta. Electron microscopic analysis indicated that in the presence of TGF-beta, basal lamina deposition by Schwann cells was slightly increased. Most importantly, many axons in TGF-beta-treated cultures received ensheathment typical of mature nonmyelinated nerves. These effects of TGF-beta were partially reversed by specific neutralizing anti-TGF-beta antibodies. We interpret these results as evidence that TGF-beta regulates Schwann cell differentiation in vitro by blocking the expression of the myelinating phenotype and promoting the development of the nonmyelinating phenotype.

Cultured incisors display major modifications in basal lamina deposition without further effect on odontoblast differentiation.

Matrix-mediated epithelio-mesenchymal interactions play a crucial role in the control of dental cytodifferentiations. Ultrastructural observation of the epithelio-mesenchymal junction in cultured embryonic mouse molars showed discrete zones with duplicated or multilayered basal laminae. The use of synthetic peptides demonstrated that the process was RGD*-independent, did not involve the YIGSR* sequence present on laminin and could occur spontaneously. Cultured incisors showed a similar but much more dramatic multiplication of the basal laminae. Furthermore, the deposition of multilayered basal laminae was specific for the labial aspect of the tooth and could be detected after 6 h of culture. Despite these alterations, preodontoblasts differentiated and gradients of differentiation were maintained, suggesting that among basement membrane constituents, the basal lamina itself does not play a critical role. More important is the inner dental epithelium which may still control odontoblast differentiation by means of diffusible molecules able to reach surface receptors expressed by preodontoblasts or matrix receptors underlying the basal lamina. Gradients of odontoblast differentiation could result from a progressive acquisition of competence by preodontoblasts.

Cultured incisors display major modifications in basal lamina deposition without further effect on odontoblast differentiation

Cultured incisors display major modifications in basal lamina deposition without further effect on odontoblast differentiation

Fibroblasts Promote Schwann Cell Basal Lamina Deposition and Elongation in the Absence of Neurons in Culture

We and other investigators have repeatedly observed that, although purified populations of Schwann cells cultured in the absence of neurons immunostain for some basal lamina constituents, assembled basal lamina is not seen ultrastructurally. In this study we show that purified rat Schwann cells cultured with either fibroblasts or diffusible substances from fibroblasts acquire basal lamina; this basal lamina is most robust and continuous when Schwann cells and fibroblasts are cultured together. The diffusible fibroblast factor(s) is ascorbate-dependent and heat-sensitive and exhibits a molecular weight greater than 30-50,000. The addition of either a combination of basal lamina components or purified laminin also results in the deposition of lengths of basal lamina. Extracellular matrix elaboration promoted by fibroblast factors is linked to dramatic changes in the morphology of Schwann cells; the cells become elongated and aligned with respect to each other. The addition of purified laminin partly mimics this effect. These changes may represent an early response of the Schwann cell to the acquisition of basal lamina preceding further differentiation in vivo. Our observations resemble those reported for other epithelial cell-fibroblast interactions and thus reaffirm the identity of the Schwann cell as an epithelial cell sensitive to the influence of mesenchymally derived factors. We propose that fibroblast stimulation of Schwann cell extracellular matrix deposition probably occurs in normal development with axons and in abnormal situations without axons, such as peripheral nerve injury and tumor formation.

Fibroblasts are required for Schwann cell basal lamina deposition and ensheathment of unmyelinated sympathetic neurites in culture.

The ability to purify and recombine populations of peripheral neurons, Schwann cells and fibroblasts in tissue culture has enabled us to examine the contribution of fibroblasts to Schwann cell basal lamina assembly and ensheathment of unmyelinated rat superior cervical ganglion neurites in vitro. Purified perinatal superior cervical ganglion neurons were grown in culture either with Schwann cells or with Schwann cells plus fibroblasts derived from either superior cervical ganglion capsule or cranial periosteum. The cultures were maintained for 2-8 weeks on a collagen substratum in a medium known to promote Schwann cell differentiation (myelin, basal lamina formation) in the presence of dorsal root ganglion neurons. The extent of Schwann cell differentiation (ensheathment, basal lamina formation) in the presence of superior cervical ganglion neurons was evaluated in this study using electron microscopy. In superior cervical ganglion neuron plus Schwann cell cultures (without fibroblasts), Schwann cells achieved only a moderate degree of ensheathment; also, Schwann cell basal lamina was discontinuous and extracellular collagen fibrils were sparse. Although only discontinuous basal lamina was demonstrable by electron microscopy in these cultures, surprisingly, Schwann cell/neurite fascicles were uniformly immunostained for laminin, type IV collagen, and heparan sulfate proteoglycan. The addition of fibroblasts to superior cervical ganglion neuron plus Schwann cell cultures increased the deposition of basal lamina around the Schwann cell/neurite units, the number of collagen fibrils, and the extent of neurite ensheathment. We propose that the presence of basal lamina increases the Schwann cell's ability to ensheathe superior cervical ganglion neurites, possibly through an augmentation of specific extracellular matrix components or by increasing in some way the capacity of these components to become organized into basal lamina. We conclude that, unlike dorsal root ganglion neurons, superior cervical ganglion neurons are unable to stimulate full Schwann cell extracellular matrix expression with the result that these Schwann cells require the extraneuronal influence of fibroblasts to deposit basal lamina and attain their mature phenotype in culture.

Differentiation of epithelial cells on microporous membranes

Microporous membranes support the differentiation of a variety of cell types including the Madin-Darby canine kidney (MDCK) cell line and normal human epidermal keratinocytes (NHEK). Coincident with culturing these cells on microporous membranes is a change in the cytoskeletal architecture of the cells to a more in vivo-like morphology and the secretion of a basal lamina-like structure, the latter not being apparent when the cells are grown in conventional cell culture plates. The potential regulatory role of the cytoskeleton in basal lamina formation was investigated using both transmission electron microscopy and immunoprecipitation of the extracellular matrix component, laminin. Disruption of the MDCK microtubular network with nocodazole caused the early formation of an electron-dense structure resembling a basal lamina. Disruption of the microtubular network of both MDCK and NHEK cells caused an increase in synthesis or retention of beta laminin or both. Disruption of the MDCK microfilament network with cytochalasin D also increased the synthesis or retention of beta laminin or both, but in addition resulted in different laminin-immunoreactive proteins secreted from the cells. Thus both ultrastructural evidence and biochemical data suggest that altering the cytoskeleton of epithelial cells might affect basal lamina formation. Our data suggest that the cytoskeleton be considered one of the possible regulators of basal lamina deposition.