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Biomedical subjects

C A McCulloch

Publications and source records attributed to C A McCulloch.

At least 55 records · Page 3Linked to original sources
PubMed1999

Tony Melcher's contributions to the regeneration of the periodontium.

The impact of Tony Melcher's scientific work on periodontal regeneration is discussed in light of his interests in cellular domains and bone healing, the physiology of the periodontal ligament, and his development of organ and cell culture methods. Collectively, his scientific accomplishments have pointed us in new directions for approaching periodontal regeneration and have helped to create new research opportunities, many of which were previously unrecognized. Tony's scientific ideas are insightful, numerous, and compelling; his ability to make these ideas come alive and relevant to clinical problems has been a major influence in shaping our current thinking on the biology of the periodontium and on periodontal regeneration.

Alveolar Process↗
PubMed1999

Assessment of a novel screening test for neutrophil collagenase activity in the diagnosis of periodontal diseases.

BACKGROUND: Increased levels of active neutrophil collagenase (MMP-8) in the gingival crevicular fluid (GCF) are associated with progressive periodontitis. The measurement of this enzyme in GCF could facilitate diagnosis. However, assays with sufficient sensitivity to detect collagenase in whole-mouth GCF currently use radiolabeled substrates and require several days to complete. To provide more rapid analyses of collagenase activity that are better adapted to clinical studies, we developed and validated a novel assay (soluble biotinylated-collagen assay: SBA) based on chemiluminescent detection of biotinylated collagen digestion products. METHODS: The concordance of the novel SBA assay with a radioactive collagen substrate assay was assessed by parallel analyses of enzyme from 35 neutrophil preparations and from 41 samples of GCF from periodontitis patients, followed by Pearson correlation analysis. To test whether the assay appropriately measured MMP-8 activity, enzyme activity was assessed after incubation with specific collagenase blockers. We examined the diagnostic utility of the SBA in cross-sectional and longitudinal analyses of 125 patients with adult periodontitis, 5 patients with early-onset periodontitis, 1 edentulous patient, and in 32 control patients without periodontitis. RESULTS: The assay detected <56 pg collagen degraded/hour/microl sample, which is comparable to the most sensitive radioactive assay. The total assay time was 22 hours and reproducibility on replicate measurements was high (r = 0.96). In direct comparisons of MMP-8 activity in GCF with enzyme from peripheral blood neutrophils using the SBA and radioactive assays, there was a high correlation (r = 0.97). As expected, EDTA and TIMP-1 and -2, known inhibitors of MMP-8, completely blocked enzyme activity with this assay. Cross-sectional and longitudinal analyses of GCF showed that MMP-8 activity was >18-fold higher in severe periodontitis than in stable periodontitis and decreased to <25% of pretreatment levels following therapy. Based on measurements of collagenase activity in different disease groups, we estimated a value of 80 nano units as a threshold for severe periodontitis. CONCLUSIONS: These results indicate that active MMP-8 is detected in GCF by a novel assay that is specific, simple, rapid, and reproducible and which may facilitate diagnostic discrimination between stable and progressive lesions.

Adult↗
PubMed1999

Stromal mesenchymal progenitor cells.

The precursors of bone, cartilage, fat and muscle cells are likely to be derived from more primitive mesenchymal cells which exhibit some of the characteristics of stem cells. Despite extensive study of stromal cell differentiation, neither mesenchymal stem cells or the more committed, tissue-specific progenitors have been well characterized. Here we describe the use of flow cytometry to isolate from fetal rat periosteum a population of small, relatively agranular cells (S cells) that display stem cell characteristics. After plating, S cells demonstrated extensive self-renewal with osteogenic potential. Electron microscopy showed that S cells have high nuclear:cytoplasmic ratios with large condensed nuclei and a paucity of cytoplasmic organelles. Freshly sorted suspensions of immunocytochemically stained S cells did not express differentiation-associated markers such as type I, II, and III collagens, alkaline phosphatase or osteopontin. However following attachment, S cells became immunopositive for collagens I, II, III, osteopontin and also for the cell surface receptor CD44, which mediates cell attachment to hyaluronan and osteopontin. S-cells showed two discrete populations of surface-stained protein by sulforhodamine, wheat germ agglutinin and Thy-1. In contrast, large (L) cells that did not exhibit stem cell characteristics exhibited low staining levels for Thy-1 and for wheat germ agglutinin. These studies demonstrate that viable osteogenic precursor cells with the stem cell characteristics of self-renewal, high proliferative capacity and multipotentiality can be enriched from heterogeneous stromal cell populations with simple flow cytometric methods.

Animals↗
PubMed1998

Role of mitochondrial membrane potential in concanavalin A-induced apoptosis in human fibroblasts.

Lectins induce apoptosis in a wide variety of cell types but the mechanisms of apoptotic induction are unknown. We examined the role of mitochondrial membrane potential (Psi m) in concanavalin A-induced apoptosis in human diploid fibroblasts. Cells were treated with Con A for 0.5, 1, 3, 5, and 24 h. Con A induced a time-dependent increase of the proportion of TUNEL+ ve cells over 24 h. Psi m was examined by staining cells with the mitochondria-specific fluorescent cationic dye JC-1. Comparison of JC-1 fluorescence within mitochondria by flow cytometry showed that after 3 h, Con A reduced Psi m in a subpopulation of apoptotic cells with smaller cell volume and with apoptotic nuclear morphology. In contrast, Psi m was unchanged in a separate population of viable cells with normal volume and normal nuclear morphology. Cyclosporin A protected cells against reduction of Psi m and also against nuclear condensation and morphological apoptosis. Measurement of intracellular calcium ion concentration ([Ca2+]i) by ratio fluorimetry of fura 2-loaded cells showed that Con A did not affect [Ca2+]i in viable cells but induced a progressive depletion of [Ca2+]i with generation of calcium oscillations in apoptotic cells. Assessment of Bcl-2 in Con A-treated cells demonstrated an initially strong increase in Bcl-2 protein and mRNA but the appearance of degraded Bcl-2 protein at 3 and 5 h after treatment, indicating an inadequate protective response to the Con A stimulation. Collectively, these data indicate that lectin-induced apoptosis in fibroblasts is associated with breakdown of Psi m, loss of [Ca2+]i homeostasis, and induced Bcl-2 expression.

Apoptosis↗
PubMed1998

Requirements of focal adhesions and calcium fluxes for interleukin-1-induced ERK kinase activation and c-fos expression in fibroblasts.

Interleukin-1 (IL-1) is an important inflammatory mediator and plays a central role in the destruction of connective tissue matrices in diseases such as arthritis and periodontitis. It is well established that IL-1 activation of the mitogen-activated protein (MAP) kinase pathway and induction of c-fos expression is a required step in the induction of matrix metalloproteinase expression involved in tissue degradation. Previous studies in our laboratory showed that IL-1-induced calcium flux is dependent on focal adhesion formation, suggesting a matrix-dependent restriction system for IL-1 signaling. Therefore, in the present study, we examined the consequences of this restriction on IL-1-mediated activation of the MAP kinase family and on c-fos expression. Treatment of human gingival fibroblasts with IL-1 activated extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase activity and induced c-fos expression in a dose- and time-dependent fashion. Plating cells on poly-L-lysine prevented focal adhesion formation, eliminated IL-1-induced calcium influx, abolished ERK stimulation, and blocked c-fos expression. Cells in suspension and hence with no suitable substratum for focal adhesion formation also showed no ERK activation or enhanced c-fos expression in response to IL-1. In contrast, eliminating focal adhesion formation or calcium depletion in cells plated on fibronectin had no effect on IL-1 stimulation of JNK and p38 kinases, demonstrating that their activation was mediated through pathways independent of focal adhesions and calcium. Calcium depletion abolished IL-1-induced calcium uptake, ERK activation, and c-fos expression. The focal adhesion dependence of IL-1-induced ERK activation and c-fos expression could be circumvented in cells plated on poly-L-lysine by simultaneous incubation with IL-1 and the calcium ionophore ionomycin. In transfection studies, IL-1 stimulation of serum responsive element (SRE) transcriptional activity was dependent on the presence of extracellular calcium. This is consistent with a requirement for calcium in the activation of ERKs and their involvement in the induction of c-fos expression through the SRE site on the 5' promoter of the c-fos gene. Our results demonstrate that in cells attached to substrates by focal adhesions, IL-1-mediated calcium flux is required for ERK activation and c-fos expression but not for JNK or p38 activation. We conclude that cellular interactions with the extracellular matrix play an important role in restricting ERK and c-fos-dependent processes.

Blood↗
PubMed1998

The role of actin-binding protein 280 in integrin-dependent mechanoprotection.

To survive in a mechanically active environment, cells must adapt to variations of applied membrane tension. A collagen-coated magnetic bead model was used to apply forces directly to the actin cytoskeleton through integrin receptors. We demonstrate here that by a calcium-dependent mechanism, human fibroblasts reinforce locally their connection with extracellular adhesion sites by inducing actin assembly and by recruiting actin-binding protein 280 (ABP-280) into cortical adhesion complexes. ABP-280 was phosphorylated on serine residues as a result of force application. This phosphorylation and the force-induced actin reorganization were largely abrogated by inhibitors of protein kinase C. In a human melanoma cell line that does not express ABP-280, actin accumulation could not be induced by force, whereas in stable transfectants expressing ABP-280, force-induced actin accumulation was similar to human fibroblasts. Cortical actin assembly played a role in regulating the activity of stretch-activated, calcium-permeable channels (SAC) since sustained force application desensitized SAC to subsequent force applications, and the decrease in stretch sensitivity was reversed after treatment with cytochalasin D. ABP-280-deficient cells showed a > 90% increase in cell death compared with ABP-280 +ve cells after force application. We conclude that ABP-280 plays an important role in mechanoprotection by reinforcing the membrane cortex and desensitizing SACs.

Actins↗
PubMed1998

Osteogenic inhibition by rat periodontal ligament cells: modulation of bone morphogenic protein-7 activity in vivo.

Periodontal ligament width is precisely maintained throughout the lifetime of adult mammals but the biological mechanisms that inhibit ingrowth of bone into this soft connective tissue are unknown. As bone morphogenic proteins strongly stimulate osteogenesis and can induce ectopic bone formation in vivo, we tested the hypothesis that topical application of this powerful osteogenic agent will overwhelm the osteogenic inhibitory mechanisms of periodontal ligament cells and induce ankylosis. Wounds through the alveolar bone and periodontal ligament were created in 45 male Wistar rats. Defects were filled with either a collagen implant or collagen plus bone morphogenic protein (BMP-7), or were left unfilled (controls). Three animals per time period were killed on days 2, 5, 10, 21 and 60 after surgery for each wound type. Cellular proliferation and clonal growth in periodontal tissues were assessed by 3H-thymidine labeling 1 h before death, followed by radioautography. Cellular differentiation of soft and mineralizing connective tissue cell populations was determined by immunohistochemical staining of alpha-smooth muscle actin, osteopontin and bone sialoprotein. In regenerating periodontium, BMP-7 induced abundant bone formation by 21 days (2.5-fold greater than controls or collagen implant only; P<0.001), but by day 60 the volume of the newly formed bone had returned to baseline levels and was similar for all groups. Independent of the type of treatment, periodontal ligament width was unchanged throughout the experimental period (P>0.05). Animals treated with BMP-7 implants showed greatly increased cellular proliferation in the periodontal ligament adjacent to the wound site and in the regenerating alveolar bone at days 5 and 10 after wounding compared to the other treatment groups (P<0.005). Animals in the BMP-7 group exhibited similar spatial and temporal staining patterns for alpha-smooth muscle actin, osteopontin and bone sialoprotein as controls. Collectively, these data show that BMP-7 promoted the proliferation of precursor cells in the periodontal ligament but did not induce osteogenic differentiation in this compartment. Consequently a powerful osteogenic stimulus like BMP-7 cannot significantly perturb the mechanisms that regulate periodontal ligament width and maintain periodontal homeostasis.

Administration, Topical↗
PubMed1998

Flow cytometric analysis of recombinant human osteogenic protein-1 (BMP-7) responsive subpopulations from fetal rat calvaria based on intracellular osteopontin content.

The bone morphogenetic proteins (BMPs) are characterized by their ability to induce both chondrogenic and osteogenic differentiation of mesenchymal cells in vivo and in vitro. Primary cultures of fetal rat calvarial cells contain a broad spectrum of osteogenic cells at various stages of differentiation, but the responsive subpopulations are incompletely characterized. To identify responsive cells in osteogenic cell differentiation, we have treated fetal rat calvarial cells with recombinant osteogenic protein-1 and used flow cytometric analyses of intracellular osteopontin, and of cartilage and bone nodule formation, to evaluate the effects. When administered as a single dose at confluence, osteogenic protein-1 stimulated bone nodule formation in fetal rat calvarial cultures in dose-dependently way. To determine the response of osteogenic subpopulations at two discrete stages of differentiation, fetal rat calvaria cells were cultured for 2 days (proliferative stage) or 12 days (early mineralization stage) and treated with 100 ng/ml recombinant osteogenic protein-1 for 12 h before analysis by flow cytometry. Flow cytometry analyses of cell suspensions revealed that osteogenic protein-1 increased the total protein content of cells, and selectively increased the mean expression of osteopontin and the size and granularity of osteopontin expressing cells, particularly at day 12, consistent with a stimulation of osteogenic differentiation and matrix formation. Pulse administration of 100 ng/ml osteogenic protein-1 to sorted, osteopontin-negative subpopulations enriched for stem cells reduced by more than four-fold the number and size of bone nodules while promoting chondrogenesis and adipogenesis. In contrast, a pulse administration of osteogenic protein-1 to more differentiated, large osteopontin-positive cells increased bone nodule formation two-fold. Continuous administration of 100 ng/ml osteogenic protein-1 to the large osteopontin-positive and small osteopontin-negative cell populations obliterated bone nodule formation and promoted chondrogenesis. We conclude that pulse administration of osteogenic protein-1 promotes osteogenic differentiation of cells committed to the osteogenic lineage, whereas undifferentiated periosteal cells are induced to differentiate along the chondrogenic pathway. In contrast, continuous exposure to osteogenic protein-1 promotes chondrogenesis in populations of committed osteogenic cells and in undifferentiated periosteal cells.

Adipocytes↗
PubMed1998

Analysis of intracellular osteopontin as a marker of osteoblastic cell differentiation and mesenchymal cell migration.

Formation and repair of the hard and soft connective tissues of teeth and their supporting structures require stem cells to divide, differentiate and migrate to generate specific tissues in a defined temporo-spatial sequence. We have used antibodies to osteopontin (OPN) and fluorescence-activated cell sorting (FACS) to determine the relationship between OPN expression and cell differentiation in cultures of fetal rat calvarial cells. At different stages of osteogenic differentiation, OPN was expressed by 60-98% of cells. Populations of small OPN-negative cells with low cellular granularity (S cells) were isolated and shown to be enriched in stem cells, characterised by a lack of differentiation markers, high proliferative potential, capacity for self renewal and multipotentiality. Within 24 h of plating, S cells attached, spread and started expressing OPN, CD44, and collagens types I, II and III. Confocal microscopy of OPN in differentiating cells revealed two distinct phenotypes; a perinuclear distribution, characteristic of secreted OPN, and an intracellular perimembranous distribution co-localising with the CD44 receptor, characteristic of migrating cells in which OPN was increased > 10-fold as measured by immunoblotting. These studies show that OPN is expressed early in mesenchymal cell differentiation and is related to cell migration as well as osteogenesis.

Animals↗
PubMed1998

Treponema denticola outer membrane inhibits calcium flux in gingival fibroblasts.

Treponema denticola is a cultivable oral spirochete which perturbs the cytoskeleton in cultured cells of oral origin, but intracellular signalling pathways by which it affects actin assembly are largely unknown. As the outer membrane (OM) of Treponema denticola disrupts actin-dependent processes that normally require precise control of intracellular calcium, we studied the effects of an OM extract on internal calcium release, ligand-gated and calcium release-activated calcium channels, and related mechanosensitive cation fluxes in human gingival fibroblasts (HGF). Single-cell ratio fluorimetry demonstrated that in resting cells loaded with Fura-2, baseline intracellular Ca2+ concentration ([Ca2+]i) was not affected by treatment with OM extract, but normal spontaneous [Ca2+]i oscillations were dramatically increased in frequency for 20 to 30 min followed by complete blockade. OM extract inhibited ATP-induced and thapsigargin-induced release of calcium from intracellular stores by 40 and 30%, respectively. Addition of Ca2+ to the extracellular pool following depletion of intracellular Ca2+ by thapsigargin and extracellular Ca2+ by EGTA yielded 59% less replenishment of [Ca2+]i in OM extract-treated than in control HGF. In cells loaded with collagen-coated ferric oxide beads to stimulate integrin-dependent calcium release, baseline [Ca2+]i was nearly doubled but was not significantly different in control and OM extract-treated cells. Magnetically generated tensile forces on the beads induced >300% increases of [Ca2+]i above baseline. Cells preincubated with OM extract exhibited dose-dependent and time-dependent reductions in stretch-induced [Ca2+]i transients, which were due to neither loss of beads from the cells nor cell death. The T. denticola OM inhibitory activity was eliminated by heating the OM extract to 60 degrees C and by boiling but not by phenylmethylsulfonyl fluoride treatment. Thus nonlipopolysaccharide, nonchymotrypsin, heat-sensitive protein(s) in T. denticola OM can evidently inhibit both release of calcium from internal stores and uptake of calcium through the plasma membrane, possibly by interference with calcium release-activated channels.

Calcium↗
PubMed1997

Deregulation of collagen phagocytosis in aging human fibroblasts: effects of integrin expression and cell cycle.

Intracellular degradation of collagen by phagocytosis in fibroblasts is essential for physiological remodeling of the extracellular matrix in a wide variety of connective tissues but imbalances between degradation and synthesis can lead to loss of tissue collagen. As aging is associated with loss of dermal and periodontal collagen and with increased lysomomal enzyme content in fibroblasts, we examined the regulation of collagen phagocytosis by integrin expression and the cell cycle in an in vitro fibroblast aging model. Two different fibroblast lines (CL1; CL2) at the fourth subculture were passaged up to replicative senescence to model aging processes in vitro. Cells were incubated with collagen-coated or BSA-coated green fluorescent beads for 3 h to assess alpha 2 beta 1-integrin-mediated or nonspecific phagocytosis, respectively. Single-cell suspensions were stained with DAPI and sulforhodamine 101 to separate cycling G1 and noncycling G0 cells. Staining for alpha 2-integrin, bead internalization, and bivariate analyses of DNA/protein content were measured by three-color flow cytometry. Serum deprivation was used to induce increases in the proportion of G0 cells. For G1 cells, the proportion of collagen phagocytic cells was > 50% for all passages and collagen beads were internalized > 5-fold more frequently than BSA beads. In contrast, G0 cells with diploid DNA content but low protein content exhibited greatly reduced phagocytic capacity (< 10% of cells internalized collagen or BSA beads), the number of beads per cells was 4-fold less, and alpha 2 integrin expression was very low compared to G1 cells. The proportion of collagen phagocytic cells and the proportion of alpha 2-integrin-positive cells increased with transit through the cell cycle. At higher passage numbers mean cell volume and cytoplasmic granularity were reduced approximately 30% but at replicative senescence cells with large surface area and subdiploid DNA predominated. The proportion of collagen and BSA phagocytic G1 cells increased 1.5- and 5-fold, respectively, and the number of beads per cell increased < 3-fold. However, surface alpha 2-integrin staining remained unchanged. These data indicate that the collagen and nonspecific internalization pathways were greatly unregulated, independent of cell cycle phase, and that cellular aging in vitro strongly influences the specificity and rate of phagocytic processes in fibroblasts. We suggest that age-related loss of collagen in connective tissues undergoing turnover may be a manifestation of a deregulated increase of collagen phagocytosis in which the net loss of degraded collagen exceeds new synthesis.

Adult↗
PubMed1997

Characterization of stromal progenitor cells enriched by flow cytometry.

The progenitors for cells of bone, cartilage, fat, and muscle are thought to be derived from mesenchymal stem cells but despite extensive study of stromal cell differentiation, neither mesenchymal stem cells or the more committed, tissue-specific progenitors have been well-characterized. In this study we used flow cytometry to isolate from fetal rat periosteum a population of small, slowly cycling cells with low cytoplasmic granularity (S cells) that display stem cell characteristics. On plating, S cells exhibited a 90% higher labeling index with [3H]-thymidine compared to unsorted cells and when grown in culture generated cartilage, adipocyte, and smooth muscle phenotypes, in addition to bone. Only the S-cell population showed extensive self-renewal of cells with osteogenic potential. Electron microscopy showed that S cells have high nuclear:cytoplasmic ratios with large condensed nuclei and a paucity of cytoplasmic organelles. Freshly sorted suspensions of immunocytochemically stained S cells did not express differentiation-associated markers such as type I, II, and III collagens, alkaline phosphatase, or osteopontin. However, after attachment, S cells became immunopositive for collagens I, II, III, osteopontin, and also for the cell surface receptor CD44, which mediates cell attachment to hyaluronan and osteopontin. These studies show that viable osteogenic precursor cells with the stem cell characteristics of self-renewal, high proliferative capacity, and multipotentiality can be enriched from heterogeneous stromal cell populations with simple flow cytometric methods. These cells may be useful for regeneration of stromal tissues.

Animals↗
PubMed1997

Bisphosphonate modulates proliferation and differentiation of rat periodontal ligament cells during wound healing.

BACKGROUND: Periodontal ligament (PL) width is precisely maintained throughout the lifetime of adult mammals, but the biological mechanisms that regulate the spatial locations of the cell populations for bone, cementum, and PL are unknown. METHODS: As bisphosphonates induce ankylosis in mouse molar teeth, we used ethane-1-hydroxy-1, 1-bisphosphonate-(HEBP, Etidronate; Didronel) in combination with a periodontal window wound model to identify those cell populations involved in the regulation of PL width during the reformation of cellular domains after wounding. Four groups of Wistar rats were wounded by drilling through the alveolar bone and extirpation of the PL. Rats were administered HEBP for 1 week and then sacrificed or allowed to recover for an additional week prior to sacrifice. Control rats were sacrificed after 1 or 2 weeks. One hour prior to sacrifice, rats were injected with 3H-thymidine to label proliferating cells. Tissue sections were immunohistochemically stained for osteopontin (OPN) or bone sialoprotein (BSP) or were prepared for in situ hybridization (BSP) to identify extra- and intracellular expression of these non-collagenous bone proteins associated with periodontal healing. RESULTS: HEBP treatment for 1 week induced a twofold increase in the thickness of the alveolar bone matrix in which weak immuno-staining for OPN and BSP mRNA signal was seen. During the recovery phase the increased bone width was reduced but was still considerably thicker than in control (P < 0.001). OPN staining as well as the BSP mRNA signal were much more intense than at 1 week. HEBP induced a > 40% reduction of PL width which returned to normal dimensions following the recovery phase. HEBP also modulated PL cell proliferation and differentiation: PL cell counts and labelling indices were reduced fivefold after 1 week of HEBP but returned to control values after the recovery phase. In controls, PL cells did not express OPN and BSP, but after HEBP treatment, and particularly after the recovery phase, PL cells expressed both of these markers intensely. In contrast, gingival and pulp connective tissues that were contiguous with the PL were not stained for OPN and did not express BSP mRNA after HEBP treatment. CONCLUSIONS: While wounding induced transient increases of proliferation which were followed by repopulation of the extirpated tissue, the effects of HEBP on cell differentiation were independent of wounding. HEBP modulates the differentiation of PL cells and recruits cells that contribute to alveolar bone formation and loss of PL width homeostasis. Conceivably, bisphosphonates could be used therapeutically to selectively alter the differentiation of PL cells and promote the formation of alveolar bone and cementum.

Animals↗
PubMed1997

Collagen phagocytosis and apoptosis are induced by high level alkaline phosphatase expression in rat fibroblasts.

Study of fibroblast origins and lineages is complicated by the lack of unambiguous markers that could be used to identify discrete subpopulations on the basis of functional attributes. We have studied the role of the membrane-anchored hydrolytic enzyme tissue-nonspecific alkaline phosphatase (TN-AP) and the placental alkaline phosphatase (PL-AP) in collagen phagocytosis and in the deletion of cells by apoptosis. Rat-2 cells, which do not constitutively express AP, were transfected with full-length rat TN-AP or PL-AP cDNAs to determine the impact of the TN-AP collagen-binding domain on cell function. Various levels of expression were driven by early (strong) or late (weak) SV40 promoters in the plasmid construct. Controls were transfected with plasmids that did not contain AP cDNA. AP expression in transfected cells was confirmed by Northern blotting, histochemical analysis, and SDS-PAGE analysis of membrane-anchored enzyme released by phosphatidyl inositol phospholipase C. Low levels of TN-AP expression increased cell spreading slightly, nearly doubled the percentage of collagen phagocytic cells (up to 80%), and increased the number of internalized collagen-coated fluorescence beads per cell. In cells transfected with PL-AP (i.e., no collagen-binding domain), collagen phagocytosis was not affected. Internalization of BSA beads was also not affected by either AP isozyme, indicating that AP was selective for integrin-mediated phagocytosis. In single cells, histochemically demonstrable TN-AP activity on cell membranes was colocalized with the binding of collagen beads, but this colocalization was not detected in cells transfected with PL-AP. Phagocytosis was inhibited by antibodies to the alpha 2 integrin and to AP but not by levamisole, an inhibitor of AP phosphohydrolytic activity. High-level TN-AP expression caused a fivefold reduction of cell proliferation and was associated with the development of cells with sub-G1 DNA content, nuclear condensation, and nuclear budding. In AP-positive cultures, there was a greatly increased number of floating cells; nick-labeling of DNA by terminal transferase and biotinylated dUTP showed a 15-fold increase of stained cells. These data indicate that low-level TN-AP expression enhances collagen phagocytosis, presumably through the TN-AP collagen-binding domain. High-level AP expression promotes cell deletion by apoptosis. We suggest that the expression of AP by fibroblasts indicates a novel role for this enzyme in collagen degradation by phagocytosis.

Alkaline Phosphatase↗
PubMed1997

Paclitaxel selectively induces mitotic arrest and apoptosis in proliferating bovine synoviocytes.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by chronic progressive destruction of joints involving several disease processes, such as villous hypertrophy, proliferation of synovial lining cells, and infiltration of inflammatory cells. Synovial cell activation and proliferation is thought to be a key step in the destruction of cartilaginous and bony tissues in RA joints. In view of the invasive properties of synoviocytes in RA, we conducted in vitro studies to determine the mechanism of action of paclitaxel (Taxol) on synoviocytes, which may account for the inhibition of joint destruction found when this agent is administered. METHODS: Cultured synovial cells were treated with various concentrations of paclitaxel and were evaluated by cell viability, fluorescence microscopy, flow cytometry of DAPI-stained cells, and electron microscopy. RESULTS: The data indicated that paclitaxel inhibited synoviocyte proliferation by a G2/M phase block and was toxic to synoviocytes by inducing apoptosis. Confluent cells such as chondroyctes and synoviocytes were not affected by paclitaxel. Synchronization of synovioyctes at the G1/S boundary effectively abolished paclitaxel-induced apoptosis. CONCLUSION: The data indicate that induction of apoptosis in synoviocytes might be dependent on transit through the cell cycle, specifically through G2 and mitosis. Further, paclitaxel was selectively toxic to proliferating synoviocytes but spared nonproliferating synoviocytes and chondrocytes. These results demonstrate that paclitaxel can inhibit synovial cell proliferation and pannus formation in RA joints in vivo. We suggest that paclitaxel be considered as a prototypical compound for a new class of potential chondroprotective agents.

Animals↗
PubMed1997

Collagen implants do not preserve periodontal ligament homeostasis in periodontal wounds.

An improved understanding of the differentiation of periodontal ligament cells could facilitate the development of new treatment approaches for overcoming the loss of specialized cell types caused by periodontitis. To study healing of wounded periodontal tissues and the differentiation of mineralizing connective tissue cells in periodontal ligament, we have examined the influence of wound size and collagen implantation on the regeneration of periodontium and on immunohistochemical staining for osteopontin and bone sialoprotein. Four groups of Wistar rats were wounded by drilling through the alveolar bone and by extirpation of the periodontal ligament. Wounds were 0.6 or 1.8 mm in diameter and defects were either implanted with collagen gels or were treated without implants. Rats were killed at 1 wk or 2 months after wounding and tissue sections were stained with monoclonal antibodies against rat osteopontin and bone sialoprotein. Collagen implants strongly increased staining for osteopontin and bone sialoprotein in defects at 1 wk. By 2 months alveolar bone healed completely regardless of the wound size but in large defects, periodontal ligament width was significantly reduced with or without implants. In large wounds at 2 months, collagen implants inhibited bone regeneration and there was stronger staining for osteopontin and bone sialoprotein in the bone replacing the implant, indicating that collagen prolonged bone remodelling. We conclude that implantation of exogenous collagen affects alveolar bone healing but does not preserve the width of the regenerated periodontal ligament. Therefore collagen does not appear to contribute to homeostasis in the periodontium following wounding.

Alveolar Process↗
PubMed1997

Is fibroblast heterogeneity relevant to the health, diseases, and treatments of periodontal tissues?

There are wide variations of gene expression and strikingly different responses to extracellular signals among different fibroblast populations. This has prompted a large number of in vitro studies which suggest that fibroblasts are not homogeneous but instead comprise multiple subpopulations with extensive site-to-site and intra-site variations. Conceivably, either fibroblasts are not all created equal, or, alternatively, discrete subpopulations may emerge in development, inflammatory lesions, or wound healing. While the heterogeneous nature of cultured fibroblasts has been known for some time, are these variations relevant to our understanding of the biology of oral tissues, their involvement in disease, and their response to therapy? Since fibroblasts are the predominant cell type in soft connective tissue matrices, the regulation of their proliferative, synthetic, and degradative behavior is likely to be important in tissue physiology and pathology. In this review, we use the current literature to assess whether fibroblast subpopulations really make a difference in the health and disease of periodontal tissues. We address the following questions: (1) Is fibroblast heterogeneity a real in vivo phenomenon? (2) How can we advance our knowledge of phenotypic variations and the regulation of fibroblast differentiation? (3) Could a knowledge of fibroblast heterogeneity have an impact on the development of new approaches to pathogenesis and the treatment of periodontal tissues?

Apoptosis↗