Migration inhibitory factor related protein-8 (MRP-8) is an autocrine chemotactic factor for periodontal ligament cells.

Previous studies have suggested that human periodontal ligament (PDL) cells secrete a chemotactic factor which stimulates motility in an autocrine manner. Here we report the partial amino acid sequence of a purified factor which shows 100% homology with human migration inhibitory factor related protein-8 (MRP-8). In addition, reverse-transcription polymerase chain-reaction (RT-PCR) analysis revealed that mRNA encoding MRP-8 was expressed in cultured human PDL cells. To confirm that MRP-8 is chemotactic for PDL cells, we synthesized 25 mer peptides overlapped by 5 amino acids covering the entire MRP-8 protein and tested them for their chemotactic activities. The data indicated that amino acid residues 21-45 showed chemotactic activity for cultured human PDL cells. The maximum chemotactic response was observed at the concentration of 10(-15) mol/mL for human PDL cells. The chemotactic activity was estimated to be approximately 1000-fold higher than that of platelet-derived growth factor (PDGF), insulin-like growth factors-I and -II (IGF-I, -II), and epidermal growth factor (EGF) when compared on a molar basis. Since MRP-8 is reported to be produced mainly by neutrophils and monocytes, the result of the current study may suggest another important role of MRP-8 in human PDL cells.

Expression of PrP mRNA is regulated by a fragment of MRP8 in human fibroblasts.

Prion protein (PrPc) is expressed in many tissues, both in human and animals. The scrapie isoform of PrPc has been shown to cause neurodegeneration. In other studies it has been demonstrated that overexpression of the PrP gene can result in nonneuronal tissue degradation. Little is known, however, about the normal function of PrPc and prion protein gene regulation. Using cultured periodontal ligament cells as an experimental model, we have demonstrated the stimulation of PrP mRNA expression by MRP8 (migration inhibitory factor-related protein). Additionally, we have shown that PDGF has an opposite effect acting as a suppresser. We propose that a correlation exists between PrPc mRNA expression and cell growth arrest and differentiation.

Comparison of in vitro proliferative capacity of human periodontal ligament cells in juvenile and aged donors.

OBJECTIVE: The aim of this study is to compare the in vitro proliferative capacity of periodontal ligament (PDL) cells from aged and juvenile donors. MATERIALS AND METHODS: Flow-cytometric analysis of the cell cycle was used to compare the length of each cell cycle, and the ratio of the cells progressing through the cycles between four PDL cells from juvenile donors and four cells from aged donors. Then, replicative capacity of the PDL cells from three juvenile and three aged donors was compared by serial cultures. Finally, expression of c-fos was compared between cells proliferating and cells which had reached senescent. RESULTS: Flow-cytometric analysis of the cell cycle had revealed that although there were no differences in the length of each phase of the cell cycle, significant differences were found in the ratio of the cells entering from Gap I to DNA synthesis phase of the cell cycle (P < 0.025). Replicative capacity was much longer in two cells from juvenile donors (about 20 population doublings), while all cells from aged donors showed short dividing abilities (less than eight population doublings), hence entered senescent phases shortly. Additionally, no c-fos was detected in cells which had reached senescence upon stimulation with serum. CONCLUSIONS: It is generally believed that aged humans have an impaired wound healing ability. We believe that more fibrotic PDL tissues seen in aged humans might be the reason for this, and suggest that this phenomena might be due to the progressive accumulation of senescent cell populations.

Comparative study of the chemotactic responses of periodontal ligament cells and gingival fibroblasts to polypeptide growth factors.

Selective recruitment of periodontal ligament cells to a previously exposed root surface is believed to enhance periodontal regeneration. It has been hypothesized that competition from gingival fibroblasts may reduce the potential of periodontal regeneration. We compared the migratory responses of PDL cells and gingival fibroblasts to a variety of biologicals. Parallel experiments designed to examine the directed migration responses of both periodontal ligament cells (PDL cells) and gingival fibroblasts (GF) isolated from the same donors were conducted using Platelet Derived Growth Factor (PDGF), Insulin Like Growth Factor-I, -II (IGF-I, -II), Epidermal Growth Factor (EGF), Transforming Growth Factor-beta (TGF-beta), and the chemotactic factor derived from the conditioned culture media of PDL cells (termed PDL-CTX) as attractants. Both PDL cells and GF exhibited dose-dependent migratory responses when challenged with PDGF, IGF-I, IGF-II, EGF, and TGF-beta. However, when these cells were challenged with PDL-CTX, only PDL cells migrated in a specific dose-dependent manner, while GF were refractive to PDL-CTX stimulation. Additionally, concentrated conditioned culture media from cultures of gingival fibroblasts did not stimulate PDL cell migratory responses. In other experiments, antibody directed against PDGF, FGF, TGF-beta, IGF-I, IGF-II, NGF, and EGF did not inhibit the PDL-CTX-elicited response in PDL cells. Previous studies have suggested that success of periodontal therapy depends on the specific attachment, migration, and proliferation of selected periodontal ligament cells. The data presented in this manuscript suggest that both PDL cells and gingival fibroblasts respond to a multitude of growth factors. PDL-CTX was found to be PDL-cell-specific for directed migration. Thus, we conclude that any biological therapeutic regime for periodontal regeneration should include PDL-cell-specific agents.

Periodontal ligament cells are chemotactic to fibroblast collagenase.

Periodontal ligament (PDL) cell motility and the passage of PDL cells along a root surface are important components of tissue remodeling during periodontal regeneration. Proteolytic enzymes, including fibroblast collagenase, have been demonstrated to play an important role in tissue remodeling. Previous studies have shown that PDL cells chemotactically respond to a variety of matrix and growth factors. We therefore studied the effects of type I collagen fragments and fibroblast collagenase on PDL cell migration, since PDL cells have been shown to adhere preferentially to partially demineralized root surfaces with exposed type I collagen. Gingival epithelial cells were used as a control cell population. We report that PDL cells but not gingival epithelial cells preferentially migrate in a dose-dependent manner to both fibroblast collagenase and to type I collagen degradation products. Epithelial cell migration to fibroblast collagenase and type I collagen fragments was observed. Antibody to type I collagen inhibited the type I collagen fragment-mediated migration. Collagenase pre-treatment of PDL cells enhanced PDL cell migration to type I collagen fragments. In other assays, enzyme inhibitors were shown to decrease the collagenase-mediated PDL cell motility. Epithelial cells were shown to migrate preferentially to 92-kDa type IV collagenase and type IV collagen degradation products. Antibody to type IV collagen inhibited type IV collagen-induced epithelial cell migration. Taken together, these data suggest a role for collagenase in the fine control of PDL cell migration in tissue remodeling during periodontal regeneration.

Isolation, purification, and partial characterization of an autocrine periodontal ligament cell chemotactic factor.

Periodontal ligament (PDL) cells are believed to play a critically important role in the regeneration of the periodontium. We have suggested that polypeptide growth factors can enhance periodontal regeneration by stimulating PDL cell chemotaxis and mitogenesis. This manuscript describes the identification of a novel chemotactic factor isolated from human PDL cells which we named PDL-CTX. PDL-CTX induces the directed migration of human PDL cells in vitro and was found to be a more potent chemotactic agent than other known growth factors. Additionally, PDL-CTX has no chemotactic effect on gingival fibroblasts or gingival epithelial cells. Both tryptic digestion and boiling abolished PDL-CTX's biological activity. The designed purification method included Mono-S cation exchange, heparin-sepharose affinity, and microbore reverse-phase HPLC. The purified factor has a relative molecular weight of approximately 7000 daltons based on sodium dodecyl sulfate (SDS) gel analysis. The amino acid composition and partial amino acid sequence were determined from HPLC-purified material. These were determined to be unique. Further investigation of the biological functions of PDL-CTX on PDL cells and other ligament cells should help improve our understanding of ligament repair.

Mitogenic and chemotactic responses of human periodontal ligament cells to the different isoforms of platelet-derived growth factor.

A major focus of studies that center on regeneration of the periodontium is to determine the efficacy of the use of polypeptide growth factors. Platelet-derived growth factor has been reported to be a possible agent for clinical use. PDGF has various isoforms. Therefore, we decided to study the mitogenic and chemotactic responses of human periodontal ligament (PDL) cells to recombinant human PDGF-AB, AA, and BB. Addition of each isoform of PDGF to in vitro mitogenesis assays induced PDL cell proliferation in a dose-dependent manner. The maximum mitogenic effect was evident at the concentration of 100 ng/mL. In these assays, PDGF-BB was found to be the most potent mitogen. PDGF-AB elicited an intermediate response, and PDGF-AA was the least effective. The results of chemotaxis assays closely parallel those of the mitogenesis assays. PDGF-BB exhibited the most potent chemotactic effect. The maximal effect was observed at 10 ng/mL. The findings of these experiments indicate that PDGF-BB is more effective than the other isoforms in promoting mitogenesis and chemotaxis of PDL cells in vitro, and may therefore be a suitable ethical pharmaceutical for use in periodontal regeneration procedures.

Biologically active factors in the treatment of periodontal disease.

Cells in normal tissue progress through programs of growth, differentiation, and death. Many of the signals that start and stop this process are initiated by growth factors produced by other cells or by the resident cells. This network of intercellular signaling represents the basis for establishing and maintaining normal tissue architecture. Growth factors are released by some cells and conveyed to target cells, where they are bound to specific receptors, triggering a complex signal transduction cascade. Human periodontal ligament cells have been shown by in vitro assays to respond both mitogenically and chemotactically to a variety of growth factors including, but not limited to, platelet-derived growth factor, basic fibroblast growth factor, and transforming growth factor-beta. Evidence is presented for confirmation of an autocrine chemotactic and mitogenic human periodontal ligament cell-specific factor. This type of knowledge suggests that future periodontal regeneration procedures will rely on the exogenous addition of specific growth factors.

Periodontal regeneration: myth or reality?

Recent scientific advances have enabled scientists to understand some of the basic biological events leading both to an accelerated and improved healing of many tissues. These advances are now being applied to periodontal wound healing. Previous studies have indicated that various oncogenes are sequences related to growth factors and, that when these sequences are altered, cellular phenotype and especially cellular proliferation is altered. Data are presented using various human cell lines, including a c-myc-transfected periodontal ligament (PDL) cell line, which delineate the relationship between oncogenes, mRNA oncogene transcripts and polypeptide growth factors as inducers of cell phenotypic alterations, including adhesion, migration and proliferation. The polypeptide growth factors are a unique class of molecules that regulate cell phenotype both in vitro and in vivo. These demonstrable phenotypic conversions have indicated that such factors will play an important role in hard-soft tissue repair. Additionally, new data suggest that older PDL cells are not responsive to polypeptide stimulation; both the migratory and proliferative responses are diminished. A unique growth factor has been isolated and sequenced, which when applied to older PDL cells will reverse this refractory phenotype. These recent studies are extensively reviewed with emphasis and conclusions based on growth factor-induced periodontal regeneration. These studies stress the undeniable role that these novel approaches will have in future chairside periodontal practice.

The NC1 domain of type IV collagen promotes axonal growth in sympathetic neurons through interaction with the alpha 1 beta 1 integrin.

We have examined the effects of collagen IV on the morphological development of embryonic rat sympathetic neurons in vitro. In short-term (less than or equal to 24 h) culture, collagen IV accelerated process outgrowth, causing increases in the number of neurites and total neuritic length. Analysis of proteolytic fragments of collagen IV indicated that the NC1 domain was nearly as active as the intact molecule in stimulating process outgrowth; in contrast, the 7S domain and triple helix-rich fragments of collagen IV were inactive. Moreover, anti-NC1 antiserum inhibited neuritic outgrowth on collagen IV by 79%. In long-term (up to 28 d) cultures, neurons chronically exposed to collagen IV maintained a single axon but failed to form dendrites. Thus, the NC1 domain of collagen IV can alter neuronal development by selectively stimulating axonal growth. Comparison of collagen IV's effects to those of laminin revealed that these molecules exert quantitatively different effects on the rate of initial axon growth and the number of axons extended by sympathetic neurons. Moreover, neuritic outgrowth on collagen IV, but not laminin, was blocked by cycloheximide. We also observed differences in the receptors mediating the neurite-promoting activity of these proteins. Two different antisera that recognize beta 1 integrins each blocked neuritic outgrowth on both collagen IV and laminin; however, an mAb (3A3) specific for the alpha 1 beta 1 integrin inhibited collagen IV but not laminin-induced process growth in cultures of both sympathetic and dorsal root neurons. These data suggest that immunologically distinct integrins mediate the response of peripheral neurons to collagen IV and laminin.

Modulation of extracellular matrix proteins in rat liver during development.

The expression and localization of extracellular matrix proteins in rat liver was investigated as a function of liver development. Levels of extracellular matrix proteins were measured by dot-blot or immunoblot protocols using monospecific antibodies prepared against collagen types I, III and IV; laminin; fibronectin; and fibronectin receptor. Proline and hydroxyproline levels from extracted liver collagen were quantitated by Pico Tag analysis. It was observed that the content of type IV collagen and fibronectin in the rat liver increased two to four times during the perinatal period. In contrast, levels of laminin and collagen types I and III decreased up to twofold in developing rat livers. The content of fibronectin receptor during ontogeny was decreased four times in an inverse relationship to fibronectin molecules. Fibronectin receptor and extracellular matrix proteins displayed no difference in apparent molecular weight as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots. Indirect immunofluorescence staining of frozen thin liver sections revealed that the pattern of localization of extracellular matrix proteins in the nonvascular regions of fetal liver was punctate rather than restricted to a specific region such as the perisinusoidal area of adult livers. Similarly, fibronectin receptor was also present, mainly in the sinusoidal area of adult livers, whereas fetal sections were diffusely stained. Our findings suggest that the differential modulation of extracellular matrix proteins and their localization in the developing rat livers undergo a dramatic alteration in the composition and structural organization of matrix material, which may act to modulate proliferation and to promote the differentiation of liver cells during development.

Directed migration of murine and human tumor cells to collagenases and other proteases.

Tumor cell motility and the passage of tumor cells through various tissue matrices, including basement membrane, are important components of the metastatic process. Proteolytic enzymes, including a type IV collagen-specific collagenase, have been demonstrated to play a significant role in extracellular matrix and basement membrane degradation. In addition, exogenous collagenase has been shown to enhance the motility of some tumor cells independent of its effect on collagen-containing material. Previous studies have also indicated that collagen fragments are chemotactic for many tumor cells. We therefore studied the effect of type I and type IV collagen-specific collagenases, other enzymes involved in collagenase activation and connective tissue degradation, and subsequent collagen degradation products on the directed migration of tumor cells. We report that type I and type IV collagen-specific mammalian collagenases were potent chemoattractants as were native type I and type IV collagens and collagen fragments. Collagenase inhibitor SC44483 inhibited the type IV collagenase-stimulated migration. Collagenase pretreatment of the tumor cells potentiated the migratory response of the tumor cells to collagen and collagen fragments. The plasminogen activator, urokinase, as well as plasminogen itself also enhanced the directed migration of tumor cells in concentrations that suggest involvement of the appropriate cell surface receptor. The chemotactic response of tumor cells to the proteases studied extends the prior report of a role for collagenases and other matrix-active enzymes in tumor cell behavior in addition to matrix degradation.

Neovascularization of surface demineralized dentin.

Uneventful healing of the wound site created by periodontal reconstructive surgery is crucial for the long term survival of the dentition. Wound healing has been shown to be initiated and mediated by matrix components and polypeptide growth factors. Neovascularization (or angiogenesis) is one of the most important events in the healing process of a wound site. Any increase in the degree and/or rate of neovascularization could result in more rapid or complete healing. Previously, we have shown that basic fibroblast growth factor (bFGF) selectively enhances periodontal ligament cell migration and proliferation. In addition, we have shown that FGF stimulates human umbilical vein endothelial cell migration and proliferation. In this study we examined whether human umbilical vein endothelial cells could be influenced to form capillary-like structures in a type I collagen stroma and on dentin surfaces in response to fibroblast growth factor (FGF). We observed tubule-like structures formed from a monolayer of endothelial cells within a type I collagen sponge in response to a gradient of FGF. Furthermore, we observed tubule-like structures formed from self-association of individual endothelial cells on partially demineralized dentin surfaces in response to FGF. Proliferation of human endothelial cells on dentin was dose dependent and maximally stimulated at a concentration of 10 ng/ml FGF. These data indicate that FGF can induce endothelial cell migration, proliferation and tubule formation on dentin.

Repopulation of dentin surfaces by periodontal ligament cells and endothelial cells. Effect of basic fibroblast growth factor.

The regeneration of connective tissue attachment is a major goal of clinical periodontics. Recent investigations on biochemically mediated periodontal regeneration have attempted to define the various biological response modifiers which may provide a mechanism for periodontal regeneration. Fibronectin and endothelial cell growth factor have been shown to selectively enhance periodontal ligament (PDL) cell adhesion, migration, and proliferation. In addition, dentin preconditioned with tetracycline HCl (TTC) or citric acid (CA) supports PDL cell adhesion, presumably by exposing collagen fibers. We have now extended these studies to include basic fibroblast growth factor (b-FGF) as a potential meditor of periodontal regeneration. Using AFSCM (assays for specific cell migration), b-FGF in concentrations as low as 10 ng per dentin block significantly stimulated PDL cell chemotaxis, while the antibody against b-FGF inhibited both the chemotactic and proliferative characteristics of the mitogen. We also found that 5 ng and above of b-FGF per dentin block significantly stimulated human endothelial cell migration and proliferation. Using 125I-b-FGF, we demonstrated that the factor binds to native dentin. This binding was increased when the dentin blocks were preconditioned by TTC or CA and reduced when the dentin was subsequently treated with collagenase. 125I-b-FGF also bound with moderate affinity to a type I collagen affinity column whereas the binding to a hydroxylapatite affinity column was negligible. The combination of FN and b-FGF was a marginally more potent chemo-attractant than b-FGF alone for PDL cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Healing, regeneration, and repair: prospectus for new dental treatment.

Recent advances in our understanding of growth and development have led us to the realization that previously unattainable tissue regeneration and repair are now within the scope of patient care. Concurrent and complementary use of nonbiological substitutes, with complete biological integration and host acceptance, is becoming a leading recognized alternative to the loss of function of biological tissues. This manuscript will examine the implications of the new biotechnology in medical sciences for dental healing, regeneration, and repair. These concepts, when coupled with genetic engineering, could produce enormous changes in the quality of life.

Laminin: molecular organization and biological function.

Laminin, the most abundant glycoprotein molecule found in basement membrane, has multiple functions in eukaryotic tissues. It serves to attach epithelial cells to basement membrane, aids development and migration of specific cell types in growth and maturation, and has been implicated in tumor metastasis and some types of infection. Current concepts of the molecular organization and myriad functions of the laminin molecule are reviewed.

Repair of periodontal furcation defects in beagle dogs following reconstructive surgery including root surface demineralization with tetracycline hydrochloride and topical fibronectin application.

This study examines the effects of root surface demineralization and topical fibronectin as adjuncts to reconstructive periodontal surgery. In 14 beagle dogs, horizontal periodontal defects were surgically induced around the mandibular premolars followed by a 6-week period without plaque control. Reconstructive surgery of the defects was subsequently carried out. The root surfaces were debrided and superficially demineralized with citric acid or tetracycline hydrochloride, with or without subsequent application of fibronectin. Mucoperiosteal flaps were raised to cover most of the crowns and sutured. The animals were sacrificed 12 weeks after surgery and block sections of the teeth and surrounding tissues were processed for histology. Analysis included incidence of furcation defects presenting with an epithelial lining, quantification of connective tissue repair relative to the furcation circumference, and regeneration of alveolar bone relative to the furcation defect height. The incidence of root resorption and ankylosis was also analyzed. Within the limitations of this study it was concluded that: (1) citric acid conditioning of the root surface frequently resulted in complete connective tissue repair of the furcation defect; (2) root resorption and ankylosis were prevalent features of the healing response; (3) citric acid and tetracycline treatment had similar potential to induce connective tissue repair and resulted in corresponding incidences of root resorption and ankylosis; (4) application of fibronectin to demineralized root surfaces did not enhance the amount of connective tissue repair and did not alter the pattern of root resorption and ankylosis.

Extracellular matrices and polypeptide growth factors as mediators of functions of cells of the periodontium. A review.

This is a review of the interactions between cells and their extracellular matrices and polypeptide growth factors. The review not only attempts to provide a basic understanding of the functions of extracellular matrices and polypeptide growth factors but, in addition, suggests the role these biological molecules may play in periodontal regeneration. It is conceivable that future periodontal therapy will include the treatment of a scaled root with biological response modifiers to predictably attain a true new connective tissue attachment.