Virve Pääkkönen

Cysteine Cathepsins in Human Dentin-Pulp Complex

INTRODUCTION Collagen-degrading matrix metalloproteinases (MMPs) are expressed by odontoblasts and present in dentin. We hypothesized that odontoblasts express other collagen-degrading enzymes such as cysteine cathepsins, and their activity would be present in dentin, because odontoblasts are known to express at least cathepsin D. Effect of transforming growth factor beta (TGF-beta) on cathepsin expression was also analyzed. METHODS Human odontoblasts and pulp tissue were cultured with and without TGF-beta, and cathepsin gene expression was analyzed with DNA microarrays. Dentin cathepsin and MMP activities were analyzed by degradation of respective specific fluorogenic substrates. RESULTS Both odontoblasts and pulp tissue demonstrated a wide range of cysteine cathepsin expression that gave minor responses to TGF-beta. Cathepsin and MMP activities were observed in all dentin samples, with significant negative correlations in their activities with tooth age. CONCLUSIONS These results demonstrate for the first time the presence of cysteine cathepsins in dentin and suggest their role, along with MMPs, in dentin modification with aging.

Unique microRNA profile in Dupuytren's contracture supports deregulation of β -catenin pathway

Dupuytrens contracture, a proliferative disease of unknown origin, is characterized by an abnormal fibroblast proliferation process. Evidence from numerous microRNA (miRNA) studies shows that miRNAs have a vital function in many biological processes, for instance, in cellular signaling networks, cell growth, tissue differentiation, and cell proliferation. Our aim was to characterize, to our knowledge for the first time, the miRNA-expression profile of Dupuytrens contracture. The miRNAs identified may have a function in the pathogenesis of Dupuytrens contracture by targeting and regulating important pathways. We compared the miRNA-expression profile of 29 Dupuytrens contracture patients with that of control samples (fibroblast cells and palmar fascia). Some of the miRNAs identified in our Dupuytrens contracture samples, including miR-29c, miR-130b, miR-101, miR-30b, and miR-140-3p, were found to regulate important genes related to the β-catenin pathway: WNT5A, ZIC1, and TGFB1. Expression profiles of these genes reanalyzed from published gene-expression data from similar patient material correlated with our miRNA results. Analysis was also performed for groups of patients with recurrent/non-recurrent and patients with hereditary/non-hereditary Dupuytrens contracture, but no significant differences appeared in miRNA-expression profiles of these groups. Identification of unique miRNA expression in Dupuytrens contracture may lead to the development of novel molecular therapy for its treatment.

Matrix Metalloproteinase-13 (MMP-13, Collagenase-3) is Highly Expressed in Human Tooth Pulp

Matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs) participate into extracellular matrix degradation in physiological and pathological conditions. We hypothesized that MMP expression in pulp tissue changes in response to caries attack and investigated the gene expression profiles of MMPs and TIMPs in pulp tissue of sound and carious teeth with cDNA microarray. cDNA microarray demonstrated an extremely high MMP-13 (collagenase-3) mRNA expression in pooled pulp samples of sound and carious teeth, with less pronounced expression of MMP-16 (MT3-MMP) and TIMP-1. Real-time quantitative polymerase chain reaction of individual pulp samples revealed a wide range of the MMP-13 expression level between pulp samples with possible downregulation of MMP-13 expression during caries progression. Western blot and immunohistochemical staining confirmed the presence of MMP-13 with no observable differences between sound and carious teeth pulp tissues. The results reveal that MMP-13 is expressed and synthesized in pulp tissue, an interesting feature considering the very limited expression of MMP-13 in normal adult tissues. Further studies with a larger sample size are needed to clarify the changes in MMP-13 expression during caries progression.

General expression profiles of human native odontoblasts and pulp-derived cultured odontoblast-like cells are similar but reveal differential neuropeptide expression levels

OBJECTIVES Odontoblasts play a central role during the dentin formation by organic matrix production and mineralisation. Recently, suitable in vitro techniques for studying mature primary odontoblasts and the newly differentiated odontoblasts have been developed. Firstly, the gene expression profiles of native and cultured odontoblasts were compared at large-scale to investigate the similarities and differences between the samples. Secondly, differential expression levels of the genes encoding neuronal proteins were analyzed to study odontoblasts sensory function. DESIGN Microarray analysis was performed to mature native and cultured pulp-derived odontoblast-like cells to compare their transcriptome. Then, the probes positive only in one sample were divided into gene ontology categories. Expression levels of selected neuronal proteins were further studied with quantitative PCR, and at the protein level by immunofluorescence of mature and newly differentiated odontoblasts in developing tooth. RESULTS Remarkable similarities between the general and neuronal protein gene expression profiles were observed. Higher cortistatin, galanin, somatostatin receptor 1 (SSTR1) and tyrosine phosphatase receptor type Z1 (PTPRZ1) expression was detected in native than in cultured odontoblast at the mRNA level. Pronociceptin was more abundantly expressed in cultured than in native odontoblasts. Immunofluorescence of mature and newly differentiated odontoblasts on human tooth germs confirmed the results. CONCLUSIONS Cultured odontoblasts used in this study have similar general gene expression pattern to native odontoblasts, and therefore offer a valuable tool for the in vitro odontoblast studies. The expression of PTPRZ1 and galanin, which participate in sensory signal transduction, supports the previously suggested role of odontoblasts as sensory cells.

High-throughput Gene and Protein Expression Analysis in Pulp Biologic Research: Review

INTRODUCTION In recent years, the use of high-throughput transcriptomics and proteomics has expanded rapidly in molecular biology and biomedical science. These methods, including DNA microarray and suppression subtractive hybridization at the mRNA level and 2-dimensional electrophoresis and antibody arrays at the protein level, enable studying the expression levels of thousands of genes and proteins simultaneously and thus allow forming genome-wide expression profiles and evaluation of biologic signaling networks. METHODS This review discusses the most used high-throughput expression analysis methods and their use in pulp biologic research. RESULTS The use of these methods in pulp biology has been limited but is expanding. The methods have been used to compare pulp and bone marrow stem cells and to study the function of pulp tissue in vivo and in vitro. CONCLUSIONS Even though the adoption of the high-throughput transcriptomic and proteomic techniques in pulp biology has been fairly slow, their use is increasing and will significantly increase the understanding of pulp tissue physiology and pathology. The comprehensive data of the transcriptome and proteome of the pulp tissue and the odontoblasts will facilitate the understanding of their functions during health and disease and provide novel target molecules for diagnosis and treatment. Identification of the genes controlling odontoblast differentiation might lead to development of methods enabling induction of reparative dentin formation under carious lesions. Identification of the genes active during dentinogenesis might lead to recognition of regulatory factors, which would cause secondary dentinogenesis to proceed at the rate of primary dentinogenesis.

Macrophages modulate migration and invasion of human tongue squamous cell carcinoma.

Oral tongue squamous cell carcinoma (OTSCC) has a high mortality rate and the incidence is rising worldwide. Despite advances in treatment, the disease lacks specific prognostic markers and treatment modality. The spreading of OTSCC is dependent on the tumor microenvironment and involves tumor-associated macrophages (TAMs). Although the presence of TAMs is associated with poor prognosis in OTSCC, the specific mechanisms underlying this are still unknown. The aim here was to investigate the effect of macrophages (Mfs) on HSC-3 tongue carcinoma cells and NF-kappaB activity. We polarized THP-1 cells to M1 (inflammatory), M2 (TAM-like) and R848 (imidazoquinoline-treated) type Mfs. We then investigated the effect of Mfs on HSC-3 cell migration and NF-kappaB activity, cytokine production and invasion using several different in vitro migration models, a human 3D tissue invasion model, antibody arrays, confocal microscopy, immunohistochemistry and a mouse invasion model. We found that in co-culture studies all types of Mfs fused with HSC-3 cells, a process which was partially due to efferocytosis. HSC-3 cells induced expression of epidermal growth factor and transforming growth factor-beta in co-cultures with M2 Mfs. Direct cell-cell contact between M2 Mfs and HSC-3 cells induced migration and invasion of HSC-3 cells while M1 Mfs reduced HSC-3 cell invasion. M2 Mfs had an excess of NF-kappaB p50 subunit and a lack of p65 subunits both in the presence and absence of HSC-3 cells, indicating dysregulation and pro-tumorigenic NF-kappaB activation. TAM-like cells were abundantly present in close vicinity to carcinoma cells in OTSCC patient samples. We conclude that M2 Mfs/TAMs have an important role in OTSCC regulating adhesion, migration, invasion and cytokine production of carcinoma cells favouring tumor growth. These results demonstrate that OTSCC patients could benefit from therapies targeting TAMs, polarizing TAM-like M2 Mfs to inflammatory macrophages and modulating NF-kappaB activity.

Collagen degradation and preservation of MMP-8 activity in human dentine matrix after demineralization.

OBJECTIVE Dental caries is a process driven by acids produced by oral microorganisms followed by degradation of the dentine collagen matrix by proteolytic enzymes. Matrix metalloproteinases (MMPs) have been suggested to contribute to caries by degrading collagen. The aim of this study was to develop a method for generating demineralized dentine matrix substrate (DDM) maintaining MMP-8 bioactivity and no interference with later assays. Such a substrate would allow study of the effects of various treatments on MMP-8 activity and collagen degradation in demineralized dentine. DESIGN Human dentine was powderized in a tissue grinder and frozen (-80°C). The powder was demineralized in dialysis tubes, using EDTA or acetic acid. The demineralized dentine matrix (DDM) was harvested and analyzed for collagen content using SDS-PAGE. The DDM was subsequently suspended in PBS or TESCA buffer. Protein, MMP-8 (ELISA) and collagen (HYP) was analyzed directly or after 1 wk. RESULTS EDTA or acid demineralization of dentine using dialysis yielded a substrate rich in collagen coupled with preserved MMP-8 activity. Collagen degraded in room temperature, assessed by higher HYP amounts in the soluble fraction of DDM after one wk, indicating that the methods used preserved active DDM-components after the demineralization process. CONCLUSIONS The presented demineralization methods both provided insoluble DDM substrates suitable for further intervention studies. However, it was found that the substrates differed depending on the demineralization method and buffers used. This needs further study to find an optimal technique for generating DDM with retained proteins as well as enzymatic bioactivity.

Mature human odontoblasts express virus-recognizing toll-like receptors.

AIM To study the expression of toll-like receptors (TLR) -3, -7, -8 and -9 as well as interferon receptors alpha and gamma (IFNAR1/IFNAR2 and IFNGR1/IFNGR2), which play important roles in the defence against viruses. METHODOLOGY DNA microarray and quantitative PCR analyses of TLR3, -7, -8 and -9 as well as IFNAR1/IFNAR2 and IFNGR1/IFNGR2 genes in mature native human odontoblasts and pulp were performed. Immunohistochemistry was used to confirm TLR8 protein in odontoblasts of healthy and carious human teeth. RESULTS TLR3, -7, -8 and -9 mRNAs were detected both in odontoblasts and in pulp, but TLR8 expression level was higher in the odontoblasts. IFNAR and IFNGR expression was observed in both tissues. Immunohistochemical analysis of healthy teeth revealed positive TLR8 staining in the pre-dentine and the dentine but varying staining patterns in the different portions of tooth. Lighter TLR8 staining was observed in dentine of mildly carious teeth. In teeth with carious lesions extending into the mid-dentine, only very weak staining was detected. CONCLUSIONS The finding of these virus-recognition-related genes in odontoblasts strengthens the view that odontoblasts participate in the immune response of the dentine-pulp complex.

Enzymatic isolation of viable human odontoblasts.

AIM To improve an enzymatic method previously used for isolation of rat odontoblasts to isolate viable mature human odontoblasts. METHODOLOGY Collagenase I, collagenase I/hyaluronidase mixture and hyaluronidase were used to extract mature human odontoblasts from the pulp chamber. Detachment of odontoblasts from dentine was determined with field emission scanning electron microscopy (FESEM) and to analyse the significance of differences in tubular diameter, and the t-test was used. MTT-reaction was used to analyse cell viability, and nonparametric Kruskal-Wallis and Mann-Whitney post hoc tests were used to analyse the data. Immunofluorescent staining of dentine sialoprotein (DSP), aquaporin-4 (AQP4) and matrix metalloproteinase-20 (MMP-20) and quantitative PCR (qPCR) of dentine sialophosphoprotein (DSPP) were used to confirm the odontoblastic nature of the cells. RESULTS MTT-reaction and FESEM demonstrated collagenase I/hyaluronidase resulted in more effective detachment and higher viability than collagenase I alone. Hyaluronidase alone was not able to detach odontoblasts. Immunofluorescence revealed the typical odontoblastic-morphology with one process, and DSP, AQP4 and MMP-20 were detected. Quantitative PCR of DSPP confirmed that the isolated cells expressed this odontoblast-specific gene. CONCLUSION The isolation of viable human odontoblasts was successful. The cells demonstrated morphology typical for odontoblasts and expressed characteristic odontoblast-type genes and proteins. This method will enable new approaches, such as apoptosis analysis, for studies using fully differentiated odontoblasts.

The expression and role of Lysyl oxidase (LOX) in dentinogenesis.

AIM To establish whether eliminating Lysyl oxidase (LOX) gene would affect dentine formation. METHODOLOGY Newborn wild-type (wt) and homo- and heterozygous LOX knock-out (Lox(-/-) and Lox(+/-) , respectively) mice were used to study developing tooth morphology and dentine formation. Collagen aggregation in the developing dentine was examined histochemically with picrosirius red (PSR) staining followed by polarized microscopy. Because Lox(-/-) die at birth, adult wt and Lox(+/-) mouse tooth morphologies were examined with FESEM. Human odontoblasts and pulp tissue were used to study the expression of LOX and its isoenzymes with Affymetrix cDNA microarray. RESULTS No differences between Lox(-/-) , Lox(+/-) and wt mice developing tooth morphology were seen by light microscopy. Histochemically, however, teeth in wt mice demonstrated yellow-orange and orange-red polarization colours with PSR staining, indicating thick and more densely packed collagen fibres, whilst in Lox(-/-) and Lox(+/-) mice, most of the polarization colours were green to green-yellow, indicating thinner, less aggregated collagen fibres. Fully developed teeth did not show any differences between Lox(+/-) and wt mice with FESEM. Human odontoblasts expressed LOX and three of four of its isoenzymes. CONCLUSIONS The data indicate that LOX is not essential in dentinogenesis, even though LOX deletion may affect dentine matrix collagen thickness and packing. The absence of functional LOX may be compensated by LOX isoenzymes.