Induction of transforming growth factor-beta 1 on dentine pulp cells in different culture patterns

Recent studies have documented that TGF-beta1 takes part in dental pulp tissue repair. Moreover, dental pulp cells have the potential to differentiate into odontoblast-like cells and produce reparative dentine in this process. However, the molecular mechanisms and potential interactions between TGF-beta1 and dental pulp cells are not clear due to the complexity of the pulp/dentine microenvironment. In this study, we investigated the induction of TGF-beta1 on the dental pulp cells in cell culture, tissue culture and three-dimensional culture patterns. These results demonstrated that TGF-beta1 significantly increased the proliferation of cells and activity of ALPase. Dental pulp cells cultured in the presence of TGF-beta1 formed mineralization nodules. In the organ culture, dental pulp cells treated with TGF-beta1 differentiated into odontoblast-like cells and formed a pulp-dentinal complex; and TGF-beta1 significantly induced synthesis of dentine relative proteins DSPP, DMP-1. The dental pulp cells share some characteristics of the odontoblast, such as a parallel arrangement with columnar form and a unilateral cell process. Together, these data indicate that TGF-beta1 can make dental pulp cells differentiated into odontoblast-like cells and form the pulp-dentinal complex. Moreover, these results suggest that TGF-beta1 is an important regulatory factor in odontoblast differentiation during tooth development and pulp repair.     

Recruitment of dental pulp cells by dentine and pulp extracellular matrix components

The present study aimed to determine whether dentine tissue and preparations of extracellular matrix (ECM) from pulp (pECM) and dentine (dECM), and breakdown products, influenced pulp cell migration. Chemotaxis transwell and agarose spot assays demonstrated that both dentine and pulp ECM molecules acted as chemoattractants for primary pulp cells. Chemoattractant activities of dECM and pECM were enhanced when subjected to acid and enzymatic breakdown, respectively. This enhanced activity following physiologically relevant breakdown may be pertinent to the disease environment. Pulp cell migration in response to dental ECMs was dependent on an active rho pathway. Recruited cells exhibited increased stem cell marker expression indicating that dental ECMs and their breakdown products selectively attract progenitor cells that contribute to repair processes. In conclusion, combined these results indicate that ECM molecules contribute to cell recruitment necessary for regeneration of the dentine-pulp complex after injury.     

Survival of rat functional dental pulp cells in vascularized tissue engineering chambers

Regenerative endodontics aims to preserve, repair or regenerate the dental pulp tissue. Dental pulp stem cells, have a potential use in dental tissue generation. However, specific requirements to drive the dental tissue generation are still obscured. We established an in vivo model for studying the survival of dental pulp cells (DPC) and their potential to generate dental pulp tissue. DPC were mixed with collagen scaffold with or without slow release bone morphogenic protein 4 (BMP-4) and fibroblast growth factor 2 (FGF2). The cell suspension was transplanted into a vascularized tissue engineering chamber in the rat groin. Tissue constructs were harvested after 2, 4, 6, and 8 weeks and processed for histomorphological and immunohistochemical analysis. After 2 weeks newly formed tissue with new blood vessel formation were observed inside the chamber. DPC were found around dentin, particularly around the vascular pedicle and also close to the gelatin microspheres. Cell survival, was confirmed up to 8 weeks after transplantation. Dentin Sialophosphoprotein (DSPP) positive matrix production was detected in the chamber, indicating functionality of dental pulp progenitor cells. This study demonstrates the potential of our tissue engineering model to study rat dental pulp cells and their behavior in dental pulp regeneration, for future development of an alternative treatment using these techniques.     

Basic fibroblast growth factor stimulates hepatocyte growth factor/scatter factor secretion by human mesenchymal cells

Basic fibroblast growth factor (bFGF) together with other pleiotropic factors plays an important role in many complex physiological processes such as embryonic development, angiogenesis, and wound repair. Among these factors, hepatocyte growth factor/scatter factor (HGF/SF) which is secreted by cells of mesodermal origin exerts its mito- and motogenic activities on cells of epithelial and endothelial origin. Knowledge of the regulatory mechanisms of HGF/SF may contribute to the understanding of its role in physio-pathological processes. We observed that the secretion of HGF/SF by MRC-5 cells and by other fibroblast-derived cell cultures in conditioned media was enhanced by exposure to bFGF. HGF/SF was measured by the scatter assay, a bioassay for cell motility, and was further characterized by Western blot analysis with anti-HGF/SF antibodies. Exposure of MRC-5 cultures to 10 ng/ml of bFGF resulted already 6 h posttreatment in a threefold higher amount of scatter factor secreted into the medium as compared to untreated cultures. HGF/SF secretion was sustained after bFGF treatment for the following 72 h when increased amounts of HGF/SF were detected both in conditioned media as well as associated to the extracellular matrix. The secretion of HGF/SF in cell supernatants increased dose dependently upon treatment with bFGF starting from basal levels of 6 U/ml and reaching 27 U/ml at 30 ng/ml bFGF, plateauing thereafter. Upregulation of HGF/SF by IL-1, already described by others, was confirmed in this study. Based on our findings an articulated interaction can be speculated for bFGF, HGF/SF, and IL-1, e.g., in tissue regeneration during inflammatory processes or in wound healing. © 1996 Wiley-Liss, Inc.     

Gene transfer of hepatocyte growth factor by electroporation reduces bleomycin-induced lung fibrosis

Abnormal alveolar wound repair contributes to the development of pulmonary fibrosis after lung injury. Hepatocyte growth factor (HGF) is a potent mitogenic factor for alveolar epithelial cells and may therefore improve alveolar epithelial repair in vitro and in vivo. We hypothesized that HGF could increase alveolar epithelial repair in vitro and improve pulmonary fibrosis in vivo. Alveolar wound repair in vitro was determined using an epithelial wound repair model with HGF-transfected A549 alveolar epithelial cells. Electroporation-mediated, nonviral gene transfer of HGF in vivo was performed 7 days after bleomycin-induced lung injury in the rat. Alveolar epithelial repair in vitro was increased after transfection of wounded epithelial monolayers with a plasmid encoding human HGF, pCikhHGF [human HGF (hHGF) gene expressed from the cytomegalovirus (CMV) immediate-early promoter and enhancer] compared with medium control. Electroporation-mediated in vivo HGF gene transfer using pCikhHGF 7 days after intratracheal bleomycin reduced pulmonary fibrosis as assessed by histology and hydroxyproline determination 14 days after bleomycin compared with controls treated with the same vector not containing the HGF sequence (pCik). Lung epithelial cell proliferation was increased and apoptosis reduced in hHGF-treated lungs compared with controls, suggesting increased alveolar epithelial repair in vivo. In addition, profibrotic transforming growth factor-beta1 (TGF-beta1) was decreased in hHGF-treated lungs, indicating an involvement of TGF-beta1 in hHGF-induced reduction of lung fibrosis. In conclusion, electroporation-mediated gene transfer of hHGF decreases bleomycin-induced pulmonary fibrosis, possibly by increasing alveolar epithelial cell proliferation and reducing apoptosis, resulting in improved alveolar wound repair.     

Localization and synthesis of type III collagen and fibronectin in human reparative dentine

Summary The injury of dental pulp tissue, following caries, is accompanied by the deposit of a typical hard scar tissue known as reparative dentine which should be regarded as the mineralization of a new organic matrix. Highly purified antibodies were used in combination with immunoperoxidase or immunogold technique at the ultrastructural level to reveal the distribution and synthesis of types I and III collagen and fibronectin elaborated by typical matrix-forming cells in the new tissue.Specific immunoperoxidase labelling, on demineralized teeth, clearly demonstrated that type I collagen represents the main type of collagen (88%). It is associated with bundles of fine striated fibrils of type III collagen and in close vicinity with fibronectin and constituted, at least, the new organic matrix of reparative dentine.Immunogold staining gave precise localization mainly over Golgi apparatus for the 3 components, thus suggesting that the cells concerned should not be considered as new odontoblasts but rather as pulpal cells in the process of differentiation participating in the formation of new dentine. Moreover, these events are very similar to those observed during wound healing in other tissues.     

Hepatocyte growth factor inhibits the formation of the basement membrane of alveolar epithelial cells in vitro

Hepatocyte growth factor (HGF) is a pulmotrophic factor for the regeneration of injured pulmonary tissue. We investigated the role of HGF in basement membrane formation during wound healing by immortalized alveolar type II epithelial cells that could form a continuous basement membrane when they were cultured on collagen fibrils in the presence of entactin-contaminated laminin-1. Cells cultured with 5.0 ng/ml HGF neither formed a continuous basement membrane on collagen fibrils nor maintained a continuous basement membrane architecture on a basement membrane substratum. The cells showed increased secretion of matrix metalloproteinase-9 and urokinase-type plasminogen activator, and the HGF-induced inhibition of basement membrane formation was attenuated by addition of 200 ng/ml tissue inhibitor of matrix metalloproteinase-1. Cells sequentially exposed to HGF and 1.0 ng/ml transforming growth factor-beta1 had enhanced basement membrane formation compared with those receiving these reagents in the reverse order or concurrently. HGF simultaneously stimulated proliferation and migration of the cells so that it advanced wound closure on the basement membrane substratum. The present results indicate that the role of HGF in wound healing is the stimulation of reepithelization, but this factor may also contribute to the degradation of the basement membrane.     

Motogenic effect of recombinant HGF on airway epithelial cells during the in vitro wound repair of the respiratory epithelium

Cell migration is the earliest mechanism involved in the wound repair process of the respiratory epithelium and could be potentially enhanced by growth factors. In the present work, we investigated the localisation of the hepatocyte growth factor (HGF) receptor (c-Met) during wound repair and evaluated the effect of recombinant HGF (rHGF) on cell migration by using an in vitro model of airway epithelial wound repair. By using immunohistochemical methods, we observed that the immunoreactivity of the c-Met proto-oncogene was increased in epithelial cells engaged in the process of tissue repair. The incubation of wounded cultures with increasing concentrations of rHGF (0.2, 2, 20, and 200 ng/ml) induced a significant (P < 0.02) dose-dependent effect on the wound repair index, with a maximum effect produced at 20 ng/ml (+31.3%). The cell migration speed reached 50.2 micrometer/h at this concentration, compared to 20.4 micrometer/h in the absence of rHGF. No significant effect on cell proliferation was observed in the repairing area in the presence of rHGF. These results suggest that rHGF is able to improve the wound repair process of the airway epithelium by increasing cell migration.     

Odontoblasts: the cells forming and maintaining dentine

Odontoblasts are tall columnar cells located at the periphery of the dental pulp. They derive from ectomesenchymal cells originated by migration of neural crest cells during the early craniofacial development. Odontoblasts form the dentine, a collagen-based mineralized tissue, through secretion of its collagenous and noncollagenous organic matrix components and by control the mineralization process. A conspicuous cell process arises from the cell body of odontoblasts and penetrates into the mineralized dentine. After dentinogenesis, odontoblasts deposit new layers of dentine throughout life and might also form a type of reactionary/reparative dentine in response to dental caries and other external factors may affect teeth.     

Localization and synthesis of type III collagen and fibronectin in human reparative dentine. Immunoperoxidase and immunogold staining

The injury of dental pulp tissue, following caries, is accompanied by the deposit of a typical hard scar tissue known as reparative dentine which should be regarded as the mineralization of a new organic matrix. Highly purified antibodies were used in combination with immunoperoxidase or immunogold technique at the ultrastructural level to reveal the distribution and synthesis of types I and III collagen and fibronectin elaborated by typical matrix-forming cells in the new tissue. Specific immunoperoxidase labelling, on demineralized teeth, clearly demonstrated that type I collagen represents the main type of collagen (88%). It is associated with bundles of fine striated fibrils of type III collagen and in close vicinity with fibronectin and constituted, at least, the new organic matrix of reparative dentine. Immunogold staining gave precise localization mainly over Golgi apparatus for the 3 components, thus suggesting that the cells concerned should not be considered as new odontoblasts but rather as pulpal cells in the process of differentiation participating in the formation of new dentine. Moreover, these events are very similar to those observed during wound healing in other tissues.     

Keratinocyte and hepatocyte growth factors in the lung: roles in lung development, inflammation, and repair

A growing body of evidence indicates that the epithelial-specific growth factors keratinocyte growth factor (KGF), fibroblast growth factor (FGF)-10, and hepatocyte growth factor (HGF) play important roles in lung development, lung inflammation, and repair. The therapeutic potential of these growth factors in lung disease has yet to be fully explored. KGF has been best studied and has impressive protective effects against a wide variety of injurious stimuli when given as a pretreatment in animal models. Whether this protective effect could translate to a treatment effect in humans with acute lung injury needs to be investigated. FGF-10 and HGF may also have therapeutic potential, but more extensive studies in animal models are needed. Because HGF lacks true epithelial specificity, it may have less potential than KGF and FGF-10 as a targeted therapy to facilitate lung epithelial repair. Regardless of their therapeutic potential, studies of the unique roles played by these growth factors in the pathogenesis and the resolution of acute lung injury and other lung diseases will continue to enhance our understanding of the complex pathophysiology of inflammation and repair in the lung.     

Bone morphogenetic proteins in dentin regeneration for potential use in endodontic therapy

The human dentition is indispensable for nutrition and physiology. The teeth have evolved for mastication of food. Caries is a common dental problem in which the dentin matrix is damaged. When the caries is deep and the dental pulp is exposed, the pulp has to be removed in many cases, resulting ultimately in loss of the tooth. Therefore, the regeneration of dentin-pulp complex is the long-term goal of operative dentistry and endodontics. The key elements of dentin regeneration are stem cells, morphogens such as bone morphogenetic proteins (BMPs) and a scaffold of extracellular matrix. The dental pulp has stem/progenitor cells that have the potential to differentiate into dentin-forming odontoblasts in response to BMPs. Pulpal wound healing consists of stem/progenitor cells release from dental pulp niche after noxious stimuli such as caries, migration to the injured site, proliferation and differentiation into odontoblasts. There are two main strategies for pulp therapy to regenerate dentin: (1) in vivo method of enhancing the natural healing potential of pulp tissue by application of BMP proteins or BMP genes, (2) ex vivo method of isolation of stem/progenitor cells, differentiation with BMP proteins or BMP genes and transplantation to the tooth. This review summarizes recent advances in application of BMPs for dentin regeneration and possible use in endodotic therapy.     

Origins of bone repair in the armour of fossil fish: response to a deep wound by cells depositing dentine instead of dermal bone

The outer armour of fossil jawless fishes (Heterostraci) is, predominantly, a bone with a superficial ornament of dentine tubercles surrounded by pores leading to flask-shaped crypts (ampullae). However, despite the extensive bone present in these early dermal skeletons, damage was repaired almost exclusively with dentine. Consolidation of bone, by dentine invading and filling the vascular spaces, was previously recognized in Psammolepis and other heterostracans but was associated with ageing and dermal shield wear (reparative). Here, we describe wound repair by deposition of dentine directly onto a bony scaffold of fragmented bone. An extensive wound response occurred from massive deposition of dentine (reactionary), traced from tubercle pulp cavities and surrounding ampullae. These structures may provide the cells to make reparative and reactionary dentine, as in mammalian teeth today in response to stimuli (functional wear or damage). We suggest in Psammolepis, repair involved mobilization of these cells in response to a local stimulatory mechanism, for example, predator damage. By comparison, almost no new bone is detected in repair of the Psammolepis shield. Dentine infilling bone vascular tissue spaces of both abraded dentine and wounded bone suggests that recruitment of this process has been evolutionarily conserved over 380 Myr and precedes osteogenic skeletal repair.     

p38 and ERK1/2 coordinate cellular migration and proliferation in epithelial wound healing: evidence of cross-talk activation between MAP kinase cascades

One important action of growth factors is their participation in tissue repair; however, the signaling pathways involved are poorly understood. In a model of corneal wound healing, we found that two paracrine growth factors, hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF), induced rapid and marked activation and prompt nuclear accumulation of phospho-p38 (p-p38) and -ERK1/2 (p-ERK1/2), but not of JNK (p-JNK1/2), in corneal epithelial cells. Interruption of p38 and ERK1/2 signaling pathways by pretreatment with inhibitors SB203580 and PD98059 and subsequent stimulation with HGF or KGF abolished the activation and nuclear localization. Inhibition of either one of these mitogen-activated protein kinases, p38 or ERK1/2, induced a robust cross-activation of the other. In immunofluorescence studies of wounded cornea, p-p38, unlike p-ERK1/2, was immediately detectable in epithelium after injury. Inhibition of p38 by SB203580 blocked migration of epithelial cells almost completely. In contrast, PD98059 seemed to slightly increase the migration, through concomitant activation of p38. Unlike ERK1/2, p38 did not significantly contribute to proliferation of epithelial cells. Inhibition of either the ERK1/2 or p38 pathway resulted in delayed corneal epithelial wound healing. Interruption of both signaling cascades additively inhibited the wound-healing process. These findings demonstrate that both p38 and ERK1/2 coordinate the dynamics of wound healing: while growth factor-stimulated p38 induces epithelial migration, ERK1/2 activation induces proliferation. The cross-talk between these two signal cascades and the selective action of p38 in migration appear to be important to corneal wound healing, and possibly wound healing in general, and may offer novel drug targets for tissue repair.     

Human progenitor cells from bone marrow or adipose tissue produce VEGF, HGF, and IGF-I in response to TNF by a p38 MAPK-dependent mechanism

Accumulating evidence suggests that progenitor cells may decrease destructive inflammation and reduce tissue loss by antiapoptotic mechanisms. However, they remain poorly characterized, and many questions remain regarding the mechanisms by which they may positively affect wound healing, tissue remodeling, or tissue regeneration. It has been speculated that various growth factors are responsible, but what components of the wound milieu stimulate progenitor cell production of growth factors and by what mechanisms? We hypothesized that tumor necrosis factor-alpha (TNF-alpha) stimulated progenitor cell secretion of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin-like growth factor I (IGF-I) by a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism. Human mesenchymal stem cells (hMSCs) and human adipose progenitor cells (hAPCs) were divided into four groups: control, p38 MAPK inhibitor (p38MKI), TNF, and TNF + p38MKI. After 24 h of incubation, supernatants were harvested for ELISA of VEGF, HGF, and IGF-I. Cells were collected for Western blot analysis of p38 MAPK activation. Secretion of VEGF, HGF, and IGF-I in hMSCs and hAPCs was significantly increased by stimulation with TNF and was associated with increased activation of p38 MAPK. The p38 MAPK inhibitor decreased production of TNF-stimulated VEGF, HGF, and IGF-I in hMSCs and hAPCs. However, p38 MAPK inhibitor alone had no effect on production of growth factors. These data demonstrate that progenitor cells are potent sources of VEGF, HGF, and IGF-I. TNF, a prominent tissue cytokine, strongly stimulated production of growth factors by hMSCs and hAPCs via a p38 MAPK-dependent mechanism.     

Biomimetic Remineralization of Demineralized Dentine Using Scaffold of CMC/ACP Nanocomplexes in an In Vitro Tooth Model of Deep Caries

Currently, it is still a tough task for dentists to remineralize dentine in deep caries. The aim of this study was to remineralize demineralized dentine in a tooth model of deep caries using nanocomplexes of carboxymethyl chitosan/amorphous calcium phosphate (CMC/ACP) based on mimicking the stabilizing effect of dentine matrix protein 1 (DMP1) on ACP in the biomineralization of dentine. The experimental results indicate that CMC can stabilize ACP to form nanocomplexes of CMC/ACP, which is able to be processed into scaffolds by lyophilization. In the single-layer collagen model, ACP nanoparticles are released from scaffolds of CMC/ACP nanocomplexes dissolved and then infiltrate into collagen fibrils via the gap zones (40 nm) to accomplish intrafibrillar mineralization of collagen. With this method, the completely demineralized dentine was partially remineralized in the tooth mode. This is a bottom-up remineralizing strategy based on non-classical crystallization theory. Since nanocomplexes of CMC/ACP show a promising effect of remineralization on demineralized dentine via biomimetic strategy, thereby preserving dentinal tissue to the maximum extent possible, it would be a potential indirect pulp capping (IPC) material for the management of deep caries during vital pulp therapy based on the concept of minimally invasive dentistry (MID).     

HGF synthesis in human lung fibroblasts is regulated by oncostatin M

Oncostatin M (OSM) is a IL-6 family cytokine locally produced in acute lung injury. Its profibrotic properties suggest a role in lung wound repair. Hepatocyte growth factor (HGF), produced by fibroblasts, is involved in pulmonary epithelial repair. We investigated the role of OSM in HGF synthesis by human lung fibroblasts. We showed that OSM upregulated HGF mRNA in MRC5 cells and in human lung fibroblasts, whereas IL-6 and leukemia inhibitory factor did not. OSM induced HGF secretion to a similar extent as IL-1beta in both a time- and dose-dependent manner. HGF was released in its cleaved mature form, and its secretion was completely inhibited in the presence of cycloheximide, indicating a de novo protein synthesis. OSM in combination with prostaglandin E(2), a powerful HGF inductor, led to an additive effect. OSM and indomethacin in combination further increased HGF secretion. This could be explained, at least in part, by a moderate upregulation of specific OSM receptor beta mRNA expression through cyclooxygenase inhibition. These results demonstrate that OSM-induced HGF synthesis did not involve a PGE(2) pathway. OSM-induced HGF secretion was inhibited by PD-98059 (a specific pharmacological inhibitor of ERK1/2), SB-203580 (a p38 MAPK inhibitor), and SP-600125 (a JNK inhibitor) by 70, 82, and 100%, respectively, whereas basal HGF secretion was only inhibited by SP-600125 by 30%. Our results demonstrate a specific upregulation of HGF synthesis by OSM, most likely through a MAPK pathway, and support the suggestion that OSM may participate in lung repair through HGF production.     

The hepatocyte growth factor (HGF)–MET receptor tyrosine kinase signaling pathway: Diverse roles in modulating immune cell functions

Hepatocyte growth factor (HGF) signaling via the MET receptor is essential for embryonic development and tissue repair. On the other hand, deregulated MET signaling promotes tumor progression in diverse types of cancers. Even though oncogenic MET signaling remains the major research focus, the HGF–MET axis has also been implicated in diverse aspects of immune cell development and functions. In the presence of other hematopoietic growth factors, HGF promotes the development of erythroid, myeloid and lymphoid lineage cells and thrombocytes. In monocytes and macrophages responding to inflammatory stimuli, induction of autocrine HGF–MET signaling can contribute to tissue repair via stimulating anti-inflammatory cytokine production. HGF–MET signaling can also modulate adaptive immune response by facilitating the migration of Langerhans cells and dendritic cells to draining lymph nodes. However, MET signaling has also been shown to induce tolerogenic dendritic cells in mouse models of graft-versus-host disease and experimental autoimmune encephalomyelitis. HGF–MET axis is also implicated in promoting thymopoiesis and the survival and migration of B lymphocytes. Recent studies have shown that MET signaling induces cardiotropism in activated T lymphocytes. Further understanding of the HGF–MET axis in the immune system would allow its therapeutic manipulation to improve immune cell reconstitution, restore immune homeostasis and to treat immuno-inflammatory diseases.