Gli1+ Periodontal Mesenchymal Stem Cells in Periodontitis.

Periodontal mesenchymal stem cells (MSCs) play a crucial role in maintaining periodontium homeostasis and in tissue repair. However, little is known about how periodontal MSCs in vivo respond under periodontal disease conditions, posing a challenge for periodontium tissue regeneration. In this study, Gli1 was used as a periodontal MSC marker and combined with a Gli1-cre ERT2 mouse model for lineage tracing to investigate periodontal MSC fate in an induced periodontitis model. Our findings show significant changes in the number and contribution of Gli1+ MSCs within the inflamed periodontium. The number of Gli1+ MSCs that contributed to periodontal ligament homeostasis decreased in the periodontitis-induced teeth. While the proliferation of Gli1+ MSCs had no significant difference between the periodontitis and the control groups, more Gli1+ MSCs underwent apoptosis in diseased teeth. In addition, the number of Gli1+ MSCs for osteogenic differentiation decreased during the progression of periodontitis. Following tooth extraction, the contribution of Gli1+ MSCs to the tooth socket repair was significantly reduced in the periodontitis-induced teeth. Collectively, these findings indicate that the function of Gli1+ MSCs in periodontitis was compromised, including reduced contribution to periodontium homeostasis and impaired injury response.

Dental Pulp Stem Cell Polarization: Effects of Biophysical Factors.

Dental pulp stem cells (DPSCs) have the potential to polarize, differentiate, and form tubular dentin under certain conditions. However, the factors that initiate and regulate DPSC polarization and its underlying mechanism remain unclear. Identification of the factors that control DPSC polarization is a prerequisite for tubular dentin regeneration. We recently developed a unique bioinspired 3-dimensional platform that is capable of deciphering the factors that initiate and modulate cell polarization. The bioinspired platform has a simple background and confines a single cell on each microisland of the platform; therefore, it is an effective tool to study DPSC polarization at the single-cell level. In this work, we explored the effects of biophysical factors (surface topography, microisland area, geometry, tubular size, and gravity) on single DPSC polarization. Our results demonstrated that nanofibrous architecture, microisland area, tubular size, and gravity participated in regulating DPSC polarization by influencing the formation of the DPSC process and relocation of the Golgi apparatus. Among these factors, nanofibrous architecture, tubular size, and appropriate microisland area were indispensable for initiating DPSC polarization, whereas gravity served as an auxiliary factor to the process of DPSC polarization. Meanwhile, microisland geometry had a limited effect on DPSC polarization. Collectively, this work provides information on DPSC polarization and paves the way for the development of new biomaterials for tubular dentin regeneration.

Oral Sciences PhD Program Enrollment, Graduates, and Placement: 1994 to 2016.

For decades, dental schools in the United States have endured a significant faculty shortage. Studies have determined that the top 2 sources of dental faculty are advanced education programs and private practice. Those who have completed both DDS and PhD training are considered prime candidates for dental faculty positions. However, there is no national database to track those trainees and no evidence to indicate that they entered academia upon graduation. The objective of this study was to assess outcomes of dental school-affiliated oral sciences PhD program enrollment, graduates, and placement between 1994 and 2016. Using the American Dental Association annual survey of advanced dental education programs not accredited by the Commission on Dental Accreditation and data obtained from 22 oral sciences PhD programs, we assessed student demographics, enrollment, graduation, and placement. Based on the data provided by program directors, the average new enrollment was 33, and graduation was 26 per year. A total of 605 graduated; 39 did not complete; and 168 were still in training. Among those 605 graduates, 211 were faculty in U.S. academic institutions, and 77 were faculty in foreign institutions. Given that vacant budgeted full-time faculty positions averaged 257 per year during this period, graduates from those oral sciences PhD programs who entered academia in the United States would have filled 9 (3.6%) vacant faculty positions per year. Therefore, PhD programs have consistently generated only a small pipeline of dental school faculty. Better mentoring to retain talent in academia is necessary. Stronger support and creative funding plans are essential to sustain the PhD program. Furthermore, the oral sciences PhD program database should be established and maintained by dental professional organizations to allow assessments of training models, trends of enrollment, graduation, and placement outcomes.

Biglycan and Decorin Expression and Distribution in Palatal Adhesion.

Previous studies demonstrated that chondroitin sulfate proteoglycans (CSPGs) on apical surfaces of palatal medial edge epithelial (MEE) cells were necessary for palatal adhesion. In this study, we identified 2 proteoglycans, biglycan and decorin, that were expressed in the palatal shelves prior to adhesion. In addition, we established that these proteoglycans were dependent on transforming growth factor ß (TGFß) signaling. Laser capture microdissection was used to collect selected palatal epithelial cells from embryonic mouse embryos at various palate development stages. The expression of specific messenger RNA (mRNA) for biglycan and decorin was determined with quantitative real-time polymerase chain reaction. The TGFßrI kinase inhibitor (SB431542) was used in palatal organ cultures to determine if blocking TFGß signaling changed biglycan and decorin distribution. Immunohistochemistry of both biglycan and decorin revealed expression on the apical and lateral surfaces of MEE cells. Biglycan protein and mRNA levels peaked as the palatal shelves adhered. Decorin was less abundant on the apical epithelial surface and also had reduced mRNA levels compared to biglycan. Their proteins were not expressed on MEE cells of palates treated with SB431542, an inhibitor of TGFß signaling. The temporal expression of biglycan and decorin on the apical surface of MEE, combined with the evidence that these proteins were regulated through the TGFß pathway, indicated that they may be important for adhesion.

Epithelial-mesenchymal transformation during craniofacial development.

Epithelial to mesenchymal phenotype transition is a common phenomenon during embryonic development, wound healing, and tumor metastasis. This transition involves cellular changes in cytoskeleton architecture and protein expression. Specifically, this highly regulated biological event plays several important roles during craniofacial development. This review focuses on the regulation of epithelial-mesenchymal transformation (EMT) during neural crest cell migration, and fusion of the secondary palate and the upper lip.

Nicotine inhibits palatal fusion and modulates nicotinic receptors and the PI-3 kinase pathway in medial edge epithelia.

OBJECTIVES: To analyze the effects of nicotine on palatal fusion inhibition in vitro and determine if nicotine modulated transforming growth factor beta3 or phosphatidylinositol-3 kinase signaling. A second objective was to determine the localization and regulation of nicotinic receptors in the medial edge epithelia (MEE) during palatal fusion. DESIGN: Palatal shelves from embryonic day (E) 13.5 mice were cultured in serum free media and treated with 0, 0.06, 0.6, or 6 mM nicotine, nicotinic receptor antagonist alpha-bungarotoxin, or the combination of nicotine and alpha-bungarotoxin. Tissues harvested at 72 h were analyzed for epithelial-mesenchymal transformation (EMT) and fusion. MEE samples collected at 20 h were analyzed for phosphorylated Akt-Ser473, phosphorylated Smad2, and nicotinic receptors. RESULTS: Nicotine inhibited palatal fusion in vitro in a dose dependent manner. Activated Akt-Ser473 was greater in control MEE than in nicotine treated tissues; while there was no difference in activated Smad2 between groups. The alpha7 subunit of nicotinic receptor was expressed in MEE during palate fusion and increased in nicotine treated tissues. Alpha-bungarotoxin did not rescue the nicotine treated palates. CONCLUSION: Nicotine treatment had no effect on Smad2, but caused a down regulation of the PI-3 kinase pathway that may have contributed to inhibiting palatal fusion in vitro.

Erk and PI-3 kinase are necessary for collagen binding and actin reorganization in corneal epithelia.

PURPOSE: It was recently shown that phosphatidylinositol-(PI)3 kinase is upregulated in wounded rabbit corneal epithelia. Extracellular signal-regulated kinase (erk)-1 and -2 proteins and PI-3 kinase were activated in embryonic corneal epithelia after 1-hour stimulation by type I collagen. In the current investigation specific inhibitors of PI-3 kinase and mitogen-activated kinase-kinase (MEK-1 kinase) were used to determine the role of these signaling molecules in actin reorganization and collagen binding to isolated sheets of corneal epithelial tissue. METHODS: Effects of specific PI-3 kinase and MEK-1 inhibitors (LY294002, PD98059, respectively) were investigated in embryonic corneal epithelial tissues. Avian embryonic corneal epithelia were isolated as tissue sheets, organ cultured in the presence of these specific inhibitors, and stimulated with type I collagen. The tissues were evaluated for collagen-stimulated actin reorganization, erk-1 and -2 and PI-3 kinase activity, total filamentous actin accumulation, and collagen binding. RESULTS: The MEK-1 inhibitor PD98059 decreased erk-1 and -2 phosphorylation and blocked actin reorganization in a dose-dependent manner. The PI-3 kinase 85-kDa subunit was decreased 25% in LY294002-treated tissue, and collagen binding also decreased significantly in tissues treated with MEK-1 and PI-3 kinase inhibitors compared with control tissues. In addition, both inhibitors blocked actin cortical mat reorganization. CONCLUSIONS; PI-3 kinase and erk-1 and -2 signaling pathways are activated and necessary for collagen binding and integrin-mediated actin reorganization in embryonic avian corneal epithelium.

Hyperbaric oxygen downregulates ICAM-1 expression induced by hypoxia and hypoglycemia: the role of NOS.

Hyperbaric oxygen (HBO) is being studied as a therapeutic intervention for ischemia/reperfusion (I/R) injury. We have developed an in vitro endothelial cell model of I/R injury to study the impact of HBO on the expression of intercellular adhesion molecule-1 (ICAM-1) and polymorphonuclear leukocyte (PMN) adhesion. Human umbilical vein endothelial cell (HUVEC) and bovine aortic endothelial cell (BAEC) induction of ICAM-1 required simultaneous exposure to both hypoxia and hypoglycemia as determined by confocal laser scanning microscopy, ELISA, and Western blot. HBO treatment reduced the expression of ICAM-1 to control levels. Adhesion of PMNs to BAECs was increased following hypoxia/hypoglycemia exposure (3. 4-fold, P < 0.01) and was reduced to control levels with exposure to HBO (P = 0.67). Exposure of HUVECs and BAECs to HBO induced the synthesis of endothelial cell nitric oxide synthase (eNOS). The NOS inhibitor nitro-L-arginine methyl ester attenuated HBO-mediated inhibition of ICAM-1 expression. Our findings suggest that the beneficial effects of HBO in treating I/R injury may be mediated in part by inhibition of ICAM-1 expression through the induction of eNOS.

Zyxin and vinculin distribution at the cell-extracellular matrix attachment complex (CMAX) in corneal epithelial tissue are actin dependent.

Avian embryonic corneal epithelia are two cell layers thick. If isolated without (-) basal lamina, the basal cells have unorganized actin and project cytoplasmic protrusions termed blebs. The actin-based cytoskeleton at the cell-extracellular matrix junction (termed the actin cortical mat) is disrupted. These epithelia respond to soluble extracellular matrix molecules by reorganizing the actin cortical mat. Sheets of epithelia were isolated + or -basal lamina. Epithelia isolated -basal lamina were cultured +/- laminin-1 and/or +/- cytochalasin D (CD). The intracellular localization of zyxin, vinculin, paxillin, focal adhesion kinase, and tensin was determined using indirect immunohistochemistry. Protein levels were determined by Western blot analysis. Zyxin and vinculin were concentrated in two areas of the tissue. The interface between the upper cell layer (periderm) and the basal cells. The second area of concentration was at the inferior 1-4 microns of the basal cells in an area with multiple actin bundles termed the actin cortical mat. The actin bundles align toward zyxin and vinculin that were located near basal lateral membranes. Zyxin was displaced from the basal compartment of blebbing basal cells. In contrast tensin, vinculin and focal adhesion kinase were found diffusely throughout the blebs. Zyxin and vinculin redistributed to the basal-lateral membranes as actin bundles reorganized in laminin-stimulated epithelia. In contrast to the altered protein distribution, extractable protein levels were similar in blebbing and laminin-stimulated epithelia. Zyxin, vinculin, and other associated proteins were disrupted in the CD-treated tissues and do not colocalize with each other or CD-induced actin aggregates. The intracellular localization of zyxin and vinculin were concentrated in distinct areas along the inferior basolateral membranes of basal cells termed the cell-extracellular matrix attachment complex (CMAX). The distribution of CMAX proteins was dependent upon actin bundle organization.

ECM-stimulated actin bundle formation in embryonic corneal epithelia is tyrosine phosphorylation dependent.

Previous studies demonstrated that corneal epithelial cells isolated without basal lamina respond to extracellular matrix (ECM) in an actin dependent manner; the basal cell surface flattens and the actin cortical mat reorganizes. We hypothesize that the actin reorganization is initiated by intracellular signaling mechanisms that includes tyrosine phoshporylation and activation of the Rho, MAP kinase, and PI3 kinase signal transduction pathways. Our goals were to develop a morphological assay to test this hypothesis by answering the following questions: 1) Do the actin bundle formations in the cortical mat have the same configuration in response to different ECM molecules? 2) What is the minimum time ECM molecules need to be in contact with the tissue for the actin to reorganize? 3) Will blocking tyrosine phosphorylation inhibit reorganization of the actin? 4) Are known signal transduction proteins phosphorylated in response to soluble matrix molecules? The actin cortical mat demonstrated distinct bundle configurations in the presence of different ECM molecules. Soluble fibronectin accumulated at the basal cell surfaces 75-fold over 30 min in a clustered pattern. The cells need contact with ECM for a minimum of 10 min to reform the actin bundles at 2 hr. In contrast, two substances that bind to heptahelical receptors to stimulate the Rho pathway, bombesin and lysophosphatidic acid, reorganized the actin bundles in 15-30 min. Focal adhesion kinase, p190 Rho-GAP, tensin, and paxillin were tyrosine phosphorylated in response to soluble fibronectin, type I collagen, or laminin 1. Erk-1, erk-2, and PI3 kinase were activated after 1 hr stimulation by type I collagen. Herbimycin A blocked actin reorganization induced by ECM molecules. In conclusion, we have developed two morphological assays to examine the response of corneal epithelial cells to ECM molecules. In addition, actin bundle reorganization involved tyrosine phosphorylation, MAP kinase, and PI3 kinase activation.

Chondrocyte-matrix attachment complexes mediate survival and differentiation.

Integrin mediated cell-extracellular matrix interactions are required for survival and differentiation of many cell types. In this review, the cell-matrix attachment complex (CMAX) is described for chondrocytes. The evidence that integrin-mediated signal transduction is necessary for normal chondrocyte differentiation and survival in various culture conditions and in vivo are reviewed. The possible signal transduction pathways stimulated by the extracellular matrix components are discussed with a review of current data from chondrocyte experiments. In addition, the influence of parathyroid hormone and transforming growth factor beta on chondrocyte survival has been included as they may function in concert with integrin mediated signal transduction. Finally, specific changes in gene expression preceding apoptosis are discussed. The current understanding of how integrin-mediated signals prevent apoptosis and implications of anchorage-dependent survival for development and differentiation of the chondrocyte phenotype are discussed.

Characterization of Bsk mice: I. The Bsk mutation does not involve a recombination of cornea-specific keratin 12 and skin-specific hair keratin genes.

PURPOSE: Bsk (bare skin) is an autosomal dominant mutation linked to the Krt 1 (type 1 keratin) locus of mouse chromosome 11. The adult Bsk mouse manifests hair loss and corneal opacity. To identify and characterize the keratin genes involved in this mutation, we examined the hypothesis proposing that the Bsk mutation might involve a recombination event between cornea-specific (K12) and hair-specific (mHa 1, 2, 3 and 4) type I keratin genes. METHODS: The Bsk phenotype was examined by histochemical analysis, using light and electron microscopy. RFLP was used for their genotyping, and possible keratin gene expression was examined by immunohistochemical staining, Western analysis, RT-PCR and Northern hybridization. RESULTS: Northern hybridization, RT-PCR and Western blot analysis revealed that mHa 1, 2, 3 and 4 keratins are expressed in the skin, but not in cornea, whereas the expression of K12 is limited to the corneas of the Bsk mice. These data ruled out the hypothesis that Bsk phenotype results from a recombination event between K12 and mHa 1, 2, 3 and 4. Ultrastructural and biochemical analyses also indicated that Bsk does not involve negative dominant mutations of keratin 12, mHa 1, 2, 3 and 4, epidermal-specific keratin 10, or basal cell-specific keratin 14. Expression of an acidic 50 kD keratin, recognized by monoclonal antibody AK 2, was up-regulated in the injured corneas of normal mice as well as Bsk corneas. CONCLUSION: The gene linked to the Bsk mutation remains unknown. The pathological changes in the skin and corneas may be secondary to the loss of protecting hairs and lashes by an unknown mechanism.

Chondrocyte survival and differentiation in situ are integrin mediated.

Chondrocytes in specific areas of the chick sternum have different developmental fates. Cephalic chondrocytes become hypertrophic and secrete type X collagen into the extracellular matrix prior to bone deposition. Middle and caudal chondrocytes remain cartilaginous throughout development and continue to secrete collagen types II, IX, and XI. The interaction of integrin receptors with extracellular matrix molecules has been shown to affect cytoskeleton organization, proliferation, differentiation, and gene expression in other cell types. We hypothesized that chondrocyte survival and differentiation including the deposition into interstitial matrix of type X collagen may be integrin receptor mediated. To test this hypothesis, a serum-free organ culture sternal model that recapitulates normal development and maintains the three-dimensional relationships of the tissue was developed. We examined chondrocyte differentiation by five parameters: type X collagen deposition into interstitial matrix, sternal growth, actin distribution, cell shape, and cell diameter changes. Additional sterna were analyzed for apoptosis using a fragmented DNA assay. Sterna were organ cultured with blocking antibodies specific for integrin subunits (alpha2, alpha3, or beta1). In the presence of anti-beta1 integrin (25 microg/ml, clone W1B10), type X collagen deposition into interstitial matrix and sternal growth were significantly inhibited. In addition, all chondrocytes were significantly smaller, the actin was disrupted, and there was a significant increase in apoptosis throughout the specimens. Addition of anti-alpha2 (10 microg/ml, clone P1E6) or anti-alpha3 (10 microg/ml, clone P1B5) integrin partially inhibited type X collagen deposition into interstitial matrix; however, sternal growth and cell size were significantly decreased. These data are the first obtained from intact tissue and demonstrate that the interaction of chondrocytes with extracellular matrix is required for chondrocyte survival and differentiation.

Increased cell diameter precedes chondrocyte terminal differentiation, whereas cell-matrix attachment complex proteins appear constant.

BACKGROUND: Chondrocytes in specific areas of chick sterna have different developmental fates. Cephalic chondrocytes become hypertrophic and secrete type X collagen into the extracellular matrix, whereas middle and caudal chondrocytes remain cartilagenous throughout development, continuing to secrete collagen types II, IX, and XI. In this report, we ask if the cell size and cytoarchitecture of chondrocytes differ in cephalic, middle, and caudal portions of whole sterna prior to and during hypertrophy. In addition, what is the distribution of integrin subunits and actin associate proteins in differentiating chondrocytes? METHODS: Phalloidin was used to stain filamentous actin, and immunohistochemistry was used to localize the distribution of collagen molecules, integrin receptor subunits, and actin-associated proteins. RESULTS: Chondrocytes stained for filamentous actin demonstrated that on day 14 cephalic chondrocytes had a significantly larger diameter than middle and caudal chondrocytes. Day 17 chondrocytes in nonhypertrophic cephalic and middle regions of sterna were significantly smaller than hypertrophic chondrocytes and significantly larger than caudal chondrocytes. In contrast to day 14 chondrocytes, day 17 chondrocytes in the hypertrophic region demonstrated similar diameters at all cartilagenous depths. The beta 1 integrin subunit appeared punctate and associated with cell membranes, allowing nonpolarized interactions with extracellular matrix molecules. The distribution of alpha integrin subunits was similar to the beta 1 integrin subunit, although alpha integrin subunits also appeared cytoplasmic. Actin-associated proteins, vinculin, and alpha-actinin, were associated with F-actin, but vinculin was more specifically localized to the ends of the actin filaments. Focal adhesion kinase was diffusely distributed throughout the cytoplasm but also demonstrated areas of colocalization with vinculin. Zyxin and paxillin demonstrated a punctate distribution, although paxillin was slightly more diffuse. Using immunohistochemical detection, no difference in integrin subunit or actin associated protein distribution could be determined between chondrocytes and hypertrophic chondrocytes. CONCLUSIONS: The increased chondrocyte diameter observed in cephalic regions of sterna on day 14 suggests that intracellular changes may precede the specific hypertrophic marker, type X collagen, by several days. In addition, the presence of integrin subunits, which are known to interact with collagen and cytoskeletal proteins, suggests that communication may exist between chondrocytes and their extracellular matrix via these receptor molecules.

Intracellular distribution of type II collagen mRNA and prolyl 4-hydroxylase in embryonic avian corneal epithelia.

BACKGROUND: The distribution of endoplasmic reticulum (ER) proteins and type II collagen mRNA in whole mount preparations of embryonic corneal epithelia was investigated. We asked, do transmission electron microscopy (TEM) and confocal microscopy of chick prolyl 4-hydroxylase (CPH) show similar ER distributions as CPH is necessary for collagen biosynthesis? METHODS: The overall distribution of ER was analyzed by TEM. Collagen secreting ER was localized with antibodies to chick prolyl 4-hydroxylase (CPH, CPH-alpha, or CPH-beta subunits). Type II collagen mRNA distribution was determined by in situ hybridization. RESULTS: The ER localized with TEM, and immunohistochemistry using anti-CPH antibodies, showed similar distribution patterns. Immunostaining for CPH and CPH-beta was prominent in both periderm and basal cells and also appeared to stain the periderm apical surface. CPH-alpha was less intense, only localizing to ER regions. The basal cell CPH distribution appeared perinuclear in optical sections that contained nuclei, but occupied nearly all the cytoplasm in a reticular pattern above and below nuclei, similar to that seen with TEM cross-sections. Epithelia double labeled with propidium iodide and CPH showed overlapping cytoplasmic staining. The distribution of type II collagen mRNA was similar to the ER staining pattern, appearing to represent a subset of total ER. CONCLUSIONS: This study demonstrates that ER markers have a similar distribution as type II collagen mRNA in embryonic avian corneal epithelia. In addition the CPH subunits had distinct, but overlapping distributions, suggesting that they may act independently.

Distribution of F-actin, vinculin and integrin subunits (alpha 6 and beta 4) in response to corneal substrata.

Our goal was to determine the early response of corneal epithelial cells to living modified stromal substrates. We examined the distribution of integrin subunits (alpha 6 and beta 4), vinculin and the organization of F-actin in epithelial cells after cell-matrix and cell-cell hypothesized that the distribution of proteins in the cell matrix attachment complex would be altered if the substrate was modified. Integrin subunits, alpha 6 and beta 4, were chosen as they play a role in cell matrix adhesion and adhesion site formation. Corneal epithelial cells were cultured from explants and seeded on three corneal substrates (a stroma containing an intact basal lamina, a cornea lacking a basal lamina and a cornea treated with alkali). After 3 h of incubation, the tissue was fixed and stained with monoclonal antibodies specific for vinculin and for the integrin subunits alpha 6 and beta 4. The organization of F-actin was assessed using rhodamine phalloidin. The localization of the proteins was recorded with confocal laser scanning microscopy. Filamentous actin delineated the lateral cell membranes of corneal epithelial cells. The organization of actin and distribution of vinculin and integrin subunits of epithelial cells cultured on stromal substrates containing a basal lamina mimicked a simple epithelial organization. In contrast, when cells are cultured on the substrate lacking a basal lamina and alkaline treated corneal substrates the distribution of the specific proteins examined was altered. Vinculin and alpha 6 were present along membranes of cells cultured on substrates lacking a basal lamina and were diffuse in cells cultured on the alkaline substrates. These studies demonstrate that changes in the distribution of adhesion and cytoskeletal proteins in response to different surfaces may contribute to the healing dynamics in different wounds.

Precursors of macrophages in embryonic rat lungs fail to exhibit granulocyte-forming potential.

BACKGROUND: Mesenchyme-like macrophage (M) precursors called angular cells are present in rat lungs on the thirteenth day of gestation and by then can differentiate into outright macrophages. Based on studies of bone marrow-derived cells, it is widely believed that the macrophage line necessarily proceeds from a colony-forming unit with dual granulocyte-macrophage potential (CFU-GM). In embryos this seems doubtful since macrophages are already scattered throughout the body before the first granulocytes appear. We examined the question in organ cultured 14 day prenatal rat lungs after having shown earlier that the macrophage population developed in explants is increased by exposure to M- and GM-colony-stimulating factors (CSFs) but is unaffected by multi (IL-3)- or granulocyte (G)-CSF. Reportedly retinoic acid (RA) shifts CFU-GM strongly towards granulocytic differentiation and inhibits mitosis of unipotential macrophage precursors but not differentiated cells. Transforming growth factor beta 1 (TGF) inhibits multipotential blood progenitors but allows proliferation of committed precursors, and TGF together with GM-CSF induces granulocytopoiesis from CFU-GM. METHODS: Lung pairs were grown on a serum-containing medium or one supplemented either by RA, TGF, or TGF/GM-CSF to form a control and three experimental groups. A fourth experiment compared responses to M-CSF exposure and M-CSF/TGF. Macrophage population growth was estimated by measuring the areas of coronas formed by macrophages emerged from the explants. F-actin was stained with fluorescein-labeled phalloidin. RESULTS: In all experiments macrophages were produced unmixed with granulocytes. By +8 days they had largely emerged to form coronas about the lungs. In cultures exposed to RA, macrophages were less intensely stained for actin and slower to emerge than controls. At +8 days, however, coronal areas were not significantly different from controls, as was also true for the TGF group. In contrast, coronal areas of cultures grown with TGF/GM-CSF were much larger. At +17 days, mean coronal area of TGF cultures was about half that of controls (P < 0.05), whereas mean coronal area of the TGF/GM-CSF group was 5.4 times greater (P < 0.001). Macrophages from control and TGF-exposed cultures responded to M-CSF by an increase in coronal area which was greater among cultures given M-CSF alone than those given TGF + M-CSF (both P < 0.005). CONCLUSIONS: Macrophage precursors in embryonic lungs are distinct from CFU-GM.

Cellular invasion and collagen type IX in the primary corneal stroma in vitro.

During different stages in the development of the avian cornea, various collagen types have been shown to participate in matrix formation and have been implicated in morphogenesis. One of these is the fibril-associated collagen type IX. This molecule is present when the primary corneal stroma is in a compact state, but rapidly disappears just prior to stromal swelling and its invasion by mesenchymal cells. The temporospatial pattern of the disappearance of type IX collagen in the developing cornea suggests that this molecule may be involved in stabilizing the primary corneal stromal matrix by interacting either with other type IX collagen molecules or with other matrix components. To explore further whether the removal of type IX collagen is involved in stromal swelling, we have employed an in vitro culture system in which swelling of the primary stroma and mesenchymal cell invasion can be experimentally manipulated by culturing chick corneal explants on a Nuclepore filter support in the presence or absence of an associated lens. We have also examined the effect of exogenously added human recombinant tissue inhibitor of metalloproteinases (TIMP-1) on the presence of type IX collagen and cellular invasion. When stage 25-26+ corneal explants were cultured with an associated lens, the primary stroma did not swell; immunohistochemically detectable type IX collagen was still present, and mesenchymal cell invasion failed to occur. Conversely, when the same stages of corneal explants were cultured without an associated lens, the primary stroma swelled; type IX collagen disappeared, and mesenchymal cell migration occurred. Under both conditions, however, the type II collagen of the stroma, which is known to be a component of the striated fibrils, remained clearly detectable and with time even seemed to increase in amount. This result is consistent with the proposition that type IX collagen is one factor involved in maintaining the primary stroma as a compact matrix, possibly by functioning as a bridging/stabilizing factor. When TIMP was added to cultures of corneal explants, type IX collagen remained detectable in focal regions, suggesting that one or more metalloproteinases are involved in the removal of the type IX collagen. In addition, some of these type IX-containing regions contained mesenchymal cells, suggesting that in addition to type IX collagen other factors are likely to be involved in regulating mesenchymal cell migration.

The intracellular distribution of vinculin and alpha 2 integrin in epithelial cells and chondrocytes.

The purpose of this study was to demonstrate the presence of vinculin and alpha 2 integrin in chondrocytes in situ and epithelial cells. We also determined that the appropriate fixation and extraction protocols for immunohistochemistry and laser scanning confocal microscopy for an integral membrane protein and an actin-associated protein in cultured cells and whole tissue was different. Cultured epithelial cells, whole mount human cornea and avian cartilage were fixed and prepared using a number of standard procedures used for indirect fluorescence immunohistochemistry. The distribution of vinculin was cell-type and fixation-specific. Chondrocytes and cultured epithelial cells demonstrated vinculin in areas that appear to be associated with filamentous actin. Vinculin was associated with cell membranes in human cornea. The expression of alpha 2 integrin observed in chondrocytes fixed with methanol, paraformaldehyde, or formaldehyde is consistent with its role in cell-substrate interaction, but may also suggest a role in dividing and differentiating cells. The localization of alpha 2 integrin in human corneal epithelia supports its role as a cell-cell adhesion molecule. The cytoplasmic distribution of vinculin and alpha 2 integrin in tissues fixed without detergent extraction suggests that the fixation step may be sufficient for antibody penetration and antigen extraction. These studies are the first report of vinculin and alpha 2 integrin in embryonic chondrocytes. In addition we have shown that confocal laser scanning microscopy combined with proper fixation and extraction protocols may optimize the localization of antigens in cultured and whole mount cells.