Thomas E. Lallier

Root-end Filling Materials Alter Fibroblast Differentiation

Root-end filling materials are commonly used following endodontic surgical procedures; however, their effect on adjacent soft tissues is poorly understood. We predict that, due to the differences in their chemical composition, these materials will have profoundly different effects on the survival and differentiation of fibroblasts. Many of the root-end filling materials examined were initially cytotoxic to both PDL and gingival fibroblasts in co-culture experiments; however, this was reduced after the materials were washed in either mineral trioxide aggregate (MTA) or hybrid ionomere composite resin (HICR) for 2 wks. Additionally, PDL fibroblasts displayed enhanced proliferation on MTA and survival on amalgam when compared with gingival fibroblasts. MTA preferentially induced alkaline phosphatase expression and activity in both PDL and gingival fibroblasts. In contrast, HICR inhibited alkaline phosphatase expression and activity. In addition, MTA and HICR repressed pleiotrophin in PDL fibroblasts, while HICR repressed periostin in both fibroblasts. Thus, root-end filling materials differentially affect periodontal fibroblast differentiation. Abbreviations: mineral trioxide aggregate (MTA), zinc-oxide eugenol cement (ZOEC), hybrid ionomer composite resin (HICR), reverse-transcriptase polymerase chain-reaction (RT-PCR).

Use of microarrays to find novel regulators of periodontal ligament fibroblast differentiation.

Periodontal regeneration requires the coordinated movement and differentiation of several cell types in order to re-establish the cementum, periodontal ligament (PDL), and alveolar bone. Cells in culture are often used as model systems for mature tissues, although they may represent expanded progenitor cell populations. Comparison of transcript expression between fresh PDL tissue and PDL cell isolates by MicroArray analysis has revealed numerous molecular differences. Several transcripts (including alkaline phosphatase, bone sialoprotein, periostin, and fibromodulin) are expressed at higher levels in fresh PDL than in cultured PDL cells. In contrast, PDL cells in culture selectively express a variety of growth factors. Several of these growth factors alter PDL fibroblast behavior. Two members of the transforming growth factor β family of growth factors, namely, bone morphogenic protein-7 (BMP7) and growth differentiation factor-5 (GDF5), reduce cell proliferation and Stro-1 expression (a bone marrow stromal stem cell marker), whereas only BMP7 induces alkaline phosphatase activity. In contrast, fibroblast growth factor-5 induces enhanced cell proliferation and Stro-1 expression, while repressing alkaline phosphatase activity. The stimulation of PDL cells to differentiate (either by BMP7 or GDF5) inhibits cell motility. Thus, PDL cells in culture are regulated by several factors that differentially stimulate a mineralized (cementoblast-like) fate, a non-mineralized fate (mature fibroblasts), or the propagation of a more naive phenotype (potential progenitors).

Adhesion of human fibroblasts to root-end-filling materials.

This study evaluated the attachment of cultured explants of human periodontal ligament fibroblasts and gingival fibroblasts to different root-end-filling materials. Although periodontal ligament and gingival fibroblasts initially attached avidly to Geristore, these same cells displayed no significant attachment to ProRoot, Tytin amalgam, or SuperEBA. With further incubation on Geristore, the attachment of both periodontal ligament and gingival fibroblasts improved and these cells proliferated. In contrast, no improvement in attachment or proliferation was observed for cells incubated for greater times with ProRoot, Tytin amalgam, or SuperEBA. Because the attachment characteristics of these two groups of fibroblasts were identical, we examined the potential role of the extracellular matrix family of receptors (integrins) on the attachment of gingival fibroblasts. Gingival fibroblast attachment to collagen type I was determined to be dependent on alpha1beta1 and alpha2beta1 integrins, whereas their attachment to the RGD-binding sequence of fibronectin and vitronectin was partially inhibited by antibodies to the beta1 and alphaV integrin subunits. However, attachment of gingival fibroblasts to Geristore was not reduced by the addition of any of the attachment-perturbing anti-integrin antibodies examined. Thus, gingival fibroblasts attach to Geristore, but this attachment was mediated by mechanisms other than integrins.

Extracellular Matrix Molecules Improve Periodontal Ligament Cell Adhesion to Anorganic Bone Matrix

Bone replacement graft (BRG) materials are used in periodontal therapy to encourage new bone formation. Extracellular matrix proteins may improve periodontal ligament fibroblast (PDLF) attachment to these materials. We demonstrate that PDLFs adhere well to the extracellular matrix (ECM) proteins fibronectin, vitronectin, laminin, and collagen types I and IV. PDLFs express numerous ECM-receptor integrin subunit transcripts (α1, a2, a3, a4, a5, α11, (31, β5, and (38) at high levels, while others (a6, a9, aV, β3, (36, and β7) are expressed at reduced levels. Despite the fact that PDLFs adhere well to fibronectin and collagen type IV bound to plastic, and express integrins that recognize these ECM proteins, they do not attach well to anorganic bovine bone matrix (ABM) coated with these same proteins. However, the addition of vitronectin, laminin, or collagen type I to these same ABMs substantially increased PDL cell attachment. Thus, selective use of ECM proteins may be clinically useful in promoting cell attachment to ABM and bone regrowth. Abbreviations: extracellular matrix (ECM), periodontal ligament (PDL), bone replacement graft (BRG), anorganic bovine bone matrix (ABM), reverse transcriptase polymerase chain-reaction (RT-PCR).

Semaphorin Profiling of Periodontal Fibroblasts and Osteoblasts

Cells of the periodontal attachment (cementoblasts, osteoblasts, and periodontal ligament fibroblasts) are descended from a common progenitor (the cranial neural crest). During their differentiation into different cell types, these cells separate from one another to form a laminated structure. Semaphorins (and their neuropilins and plexin receptors) act as cell guidance molecules for other neural crest derivatives. It is predicted that the differential expression of these molecules will correlate with the ability of these cells to segregate. It is demonstrated that human pre-osteoblasts segregate from PDL and gingival fibroblasts in culture. In addition, these cells express different semaphorins and plexins. Semaphorins 3D and 7A were expressed preferentially in dermal fibroblasts, while semaphorin 6B was selectively expressed by pre-osteoblasts. Semaphorins 3B, 4C, 5B, and 6C and plexins B1 and C1 were expressed in reduced levels in pre-osteoblasts. Analysis of the data suggests that differential expression of semaphorins and plexins may be involved in regulating cell-sorting in the formation and regeneration of the periodontal attachment structure. Abbreviations: Periodontal Ligament (PDL), Reverse Transcriptase Polymerase Chain-reaction (RT-PCR).

A simple cell motility assay demonstrates differential motility of human periodontal ligament fibroblasts, gingival fibroblasts, and pre-osteoblasts.

During periodontal regeneration, multiple cell types can invade the wound site, thereby leading to repair. Cell motility requires interactions mediated by integrin receptors for the extracellular matrix (ECM), which might be useful in guiding specific cell populations into the periodontal defect. Our data demonstrate that fibroblasts exhibit differential motility when grown on ECM proteins. Specifically, gingival fibroblasts are twice as motile as periodontal ligament fibroblasts, whereas osteoblasts are essentially non-motile. Collagens promote the greatest motility of gingival fibroblasts in the following order: collagen III>collagen V>collagen I. Differences in motility do not correlate with cell proliferation or integrin expression. Osteoblasts display greater attachment to collagens than does either fibroblast population, but lower motility. Gingival fibroblast motility on collagen I is generally mediated by α2 integrins, whereas motility on collagen III involves α1 integrins. Other integrins (α10 or α11) may also contribute to gingival fibroblast motility. Thus, ECM proteins do indeed differentially promote the cell motility of periodontal cells. Because of their greater motility, gingival fibroblasts have more of a potential to invade periodontal wound sites and to contribute to regeneration. This finding may explain the formation of disorganized connective tissue masses rather than the occurrence of the true regeneration of the periodontium.

Effect of Essential Oil and Chlorhexidine Mouthwashes on Gingival Fibroblast Survival and Migration

BACKGROUND Chemical plaque control is the most commonly recommended means of oral hygiene after periodontal surgery. Commercially available mouthwashes contain a variety of active ingredients that have bactericidal properties but may potentially be toxic to the host cells. The goal of this in vitro study is to investigate the effect of commercially available mouthwashes on the survival and migratory capacity of human fibroblasts. METHODS Human gingival and periodontal ligament (PDL) fibroblasts were treated with commercially available mouthwashes that contained either chlorhexidine (CHX) or essential oils (EO) as the active ingredient. Each mouthwash was tested over a range of concentrations for its ability to affect fibroblast survival and migration, as well as long-term effects on cell viability. RESULTS Undiluted mouthwashes induced near-complete cell death 24 hours after only a 60-second treatment. Dilutions of 15% to 20% for both CHX and EO mouthwashes resulted in 50% cell death. When diluted to 10% to 15%, EO did not reduce cell migration, whereas similar dilutions of CHX resulted in reduced cell migration. Concentrations of 10% of both EO and CHX mouthwashes retained most of their antibacterial capacity. Treatment with EO did not result in gingival fibroblast death, whereas 5% CHX resulted in near-complete gingival fibroblast death 7 days after exposure. CONCLUSIONS The results of this in vitro study indicate that diluted EO displayed no detectable detrimental effects on human gingival and PDL fibroblasts, whereas diluted CHX reduced both cell migration and long-term survival. Both solutions retained their antimicrobial activity in lower concentrations.

Cigarette Smoke Extract Induces Select Matrix Metalloproteinases and Integrin Expression in Periodontal Ligament Fibroblasts

BACKGROUND The periodontal ligament (PDL) is the connective tissue that anchors the cementum of the teeth to the alveolar bone. PDL fibroblasts are responsible for the production of collagen and remodeling of the PDL. Periodontal disease is increased among smokers in both incidence and severity. This study examines the direct effect of smoking on PDL fibroblasts and their production of various matrix components and remodeling enzymes. METHODS PDL cells were plated for 1 day and then treated with various concentrations of cigarette smoke extract (CSE). Survival of PDL cells was quantified after exposure to CSE, and their ability to contract three-dimensional collagen gels was examined. Changes in transcript expression after CSE treatment was compared using reverse transcription-polymerase chain reaction analysis for matrix metalloproteinases (MMPs), collagens, and integrins. RESULTS Treatment with CSE-induced cell death at concentrations of ≥5%. PDL-cell-induced collagen gel contraction was reduced at concentrations of 1.5% CSE. Treatment with CSE selectively increased the expression of collagen Vα3 and decreased collagen XIα1. CSE increased the expression of MMP1 and MMP3 and, to a lesser extent, MMP2 and MMP8. CSE also increased the expression of integrins α1, α2, and α10 (collagen receptors) and α9 (a tenascin receptor). CONCLUSIONS This study shows that cigarette smoking has local effects on the cells of the PDL. CSE reduced survival of PDL cells and their ability to contract collagen matrices. CSE also altered the expression of molecules known to provide the structural integrity of the ligament by altering collagen synthesis and remodeling as well as cell adhesion.

Novel amelogenin-releasing hydrogel for remineralization of enamel artificial caries

Recently, the use of recombinant full-length amelogenin protein in combination with fluoride has shown promising results in the formation of densely packed enamel-like structures. In this study, amelogenin (rP172)-releasing hydrogels containing calcium, phosphate, and fluoride were investigated for remineralization efficacy using in vitro early enamel caries models. The hydrogels were applied to artificial caries lesions on extracted human third molars, and the remineralization efficacy was tested in different models: static gel remineralization in the presence of artificial saliva, pH cyclic treatment at pH 5.4 acetic buffer and pH 7.3 gel remineralization, and treatment with multispecies oral biofilms grown in a continuous flowing constant-depth film fermenter. The surface microhardness of remineralized enamel increased significantly when amelogenin was released from hydrogel. No cytotoxicity was observed when periodontal ligament cells were cultured with the mineralized hydrogels.

Differential Expression of Adenylyl Cyclase mRNAs in Lacrimal Glands of NZB/NZW and NOD Pre-Autoimmune Mice

Sjogren’s Syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration of the salivary and lacrimal glands. The disease is most prevalent in postmenopausal women who exhibit salivary and lacrimal insufficiencies resulting in dry mouth and dry eye. Diagnosis of SS is variously dependent upon the detection of anti-Ro (SS-A), anti-La (SS-B) and antinuclear (ANA) autoantibodies, as well as rheumatoid factor. Biopsies of the salivary and lacrimal glands of SS patients typically reveal periductal and perivascular lymphocytic infiltrates that are predominated by CD4+ T and B lymphocytes1–6. Emerging theories of the pathogenesis of SS postulate the disease proceeds in stages with the first stage characterized by an intrinsic failure of the secretory epithelial cells7–12. Successive stages include the autoimmune response in the lacrimal and salivary glands initiated by the target cells themselves. The final stage is seen as one in which immune-mediated destruction of the secretory architecture of the cell, as well as of the nerve fibers innervating the gland, occurs.